ARTICLE 299
et et al.
A. pullulans being one of Myriangiales. 2009,al. et Key words: 18S 28S BenA black yeast Dothidiomycetes ITS RPB2 xerotolerant fungi Zalaria et al. 2000), bathrooms, food, . 2010, Nonneman et al. 2016). The morphospecies The morphospecies 2016). al. et 8(2): 299–315 (2017) 299–315 8(2): · FUNGUS IMA Aureobasidium et al. et 2014), it also makes definitive 2013, Schoch 2013, al. et closest relatives. by having only one to their . 2014). These studies showed the order et al. 2014). 2011), and polythermal glaciers (Zalar et al. 2011),
et al. 2013, Amend A 28S rDNA phylogeny resolved 28S rDNA A 1,2,4 Dothideaceae The class Dothideomycetes is the largest in Ascomycota , 3-7-2 Kajino-cho, Koganei, Tokyo, Japan Tokyo, , 3-7-2 Kajino-cho, Koganei, and with significant osmotic stress, such as hypersaline waters in salterns (Gunde-Cimerman and feeds (Samson et al. 2010), water-damaged wood (Andersen al. 2008). In surveys of the indoor environment, is one of the most abundant and widespread fungi reported (Adams Nieuwenhuijzen 2012, Van exhibits a high degree of phenotypic plasticity (Slepecky & Starmer 2009) and strains can have 2008). While 1995, Zalar et al. et al. pigmentation significantly(Yurlova different this high degree unique of variation may contribute to its adaptability (Gostinčar morphological identification challenging, and the many ITS variants identified as this species in GenBank are unlikely to represent one species. Reports of A. pullulans abundantthe most members in fungal communities based on near-neighbour next-generation sequencing analyses of (NGS) data may be skewed to some extent. and was recently examined and re-defined using multigene (Hyde phylogenetics Thambugala to sister monophyletic a be to Dothideales ) is ) media and produces much darker colonies alba than Z. , and a new family to accommodate them in accommodate them family to new and a , obscura and Z. w , and Cobus M. Visagie , and Cobus et al. 2016) 3 alba
(Flannigan & w species -like species for Aureobasidium Dothideales (Dothideales microscopically Hormonema closely resembles but were phylogenetically distinct. ) coupled with high w with families Aureobasidiaceae 2000). Studying theStudying 2000). al. et , Yuuri Hirooka , Yuuri 1,2 . 2014, Garber 2001) or allergensor 2001) Garber 2014, al. et
is a black yeast-like fungus, grows restrictedly and produces conidiogenousblack yeast-like fungus, grows restrictedly and produces is a cells with holoblastic grows faster on lower water activity (a (sexual morphs ?Sydowia), , Keith A. Seifert , Keith 1 . 2011, Tanno Tanno et al. 2009, Karvala et al. 2011, Zalaria . An international survey of house dust collected from eleven countries using a modified dilution-to-extinction modified a using countries eleven from collected dust house of survey international An
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). You Z. obscura Hormonema The black yeast Aureobasidium pullulans Department of Biology, University of Ottawa, 30 Marie-Curie, Ottawa, ON, Canada, K1N 6N5 University of Ottawa, 30 Marie-Curie, Ottawa, Department of Biology, University Plant Science, Faculty of Bioscience, Hosei Department of Clinical e-mail: author corresponding Africa; South Pretoria, 0121, Queenswood P/BagX134, Protection, and Health ARC-Plant Division, Biosystematics Biodiversity (Mycology), Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, method yielded 7904 isolates. Of these, six strains morphologically resembled the asexual morphs of Abstract: and clade in Dothideales distinct a strains as [email protected] Canada, K1A 0C6 Canada, K1A © 2017 International Mycological Association © 2017 International Mycological following conditions: distribute and transmit the work, under the are free to share - to copy, You Attribution: 2 3 4 1 Zoë Humphries A new family and genus in family and new A dust from house isolated Submitted: 5 July 2017; Accepted: 10 October 2017; Published: 26 October 2017. Article info: Submitted: 5 July 2017; after 7 d. Their sexual states, if extant, are unknown. Their sexual states, if extant, are after 7 d. Non-commercial: No derivative works: Any of the above conditions can be waived if you get 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. transform, or build upon this work. may not alter, You may not use this work for commercial purposes. You Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. Further analyses based on the ITS rDNA region, β-tubulin, 28S rDNA, and RNA polymerase II second largest subunit largest second II polymerase RNA and rDNA, 28S β-tubulin, region, rDNA ITS the on based analyses Further confirmed the distinct status of this cladenew species as Zalaria new genus and two introduce a and divided strains among two consistent subclades. As a result, we two loci per conidiogenous cell, but species of our new genus generally has more restricted growth. Comparing the two species, Dothideales synchronous or percurrent conidiation. Zalaria Miller 2011), while plastics like polyvinyl chloride (PVC, used(PVC, chloride polyvinyl like plastics while 2011), Miller little in the way of available carbon but in construction) offer (Webb fungi oligotrophic support still indoor mycobiota is therefore important to better understand us. these interactions and how they may affect recorded from a wide variety of sources, including environments INTRODUCTION doi:10.5598/imafungus.2017.08.02.05 (Aimanianda The average person in industrialized countries spendscountries industrialized in person average The (Höppe & Martinacapproximately 90 % of their time indoors mostthe of one environment indoor the makes This 1998). are constantly exposed important human-fungal interfaces. We impactand their and metabolites fragments, spores, fungal to Jesenská 1999) asranges from human health (Piecková & Hoog (De pathogens 2011), and building materials like temperatures (Zalar et al. 2011), drywall, and cement have very low a plaster, VOLUME 8 · NO. 2 to food spoilage (Pitt & Hocking 2009, Samson et al. 2010) or damage to building materials (Flannigan & Miller 2011). Although indoor environments are not generally recognized as extreme environments, microclimates such as dishwashers contain relatively high water activity (a Humphries et al.
Thambugala et al. (2014) reviewed and re-evaluated the Aureobasidium by the production of synchronous blastoconidia morphologically-based taxonomy of Dothideales known from from undifferentiated, hyaline cells, whereas Hormonema was culture, informed by a combined phylogeny of 28S rDNA, 18S said to produce conidia in basipetal succession from hyaline rDNA and ITS. They accepted two families, synonymising or dark hyphae. Wang & Zabel (1990) suggested that at least the often-accepted Dothioraceae (e.g. Barr 2001) with some conidiogenous cells of H. dematioides were phialidic or
ARTICLE Dothideaceae, as first proposed by Von Arx & Müller (1975), percurrent. In a later review, Aureobasidium was distinguished and introducing Aureobasidiaceae for Aureobasidium and based on its conidiogenous cells having multiple loci closely related genera. The polythetic morphological definitions (synchronous conidiogenesis), in contrast to one or two loci provided by Thambugala et al. (2014) did not identify unique in Hormonema (De Hoog & Yurlova 1994). These characters diagnostic characters among the sexual or asexual morphs in are difficult to detect, and are best observed along hyphae at either family. Both families include a poorly integrated mixture the growing margin of the colony. Colonies of both morphs of genera known from fungarium specimens with others that often begin as palely pigmented growths, which become are mostly known from culture. For both families, sexually and slimy and almost black as the colonies mature. The different asexually typified genera were keyed out separately. Sexually patterns and apparent plasticity of conidiogenesis, including typified genera were separated by characters of the stromata, yeast-like cells, young blastospores, swollen blastospores, number of ascospores per ascus, and ascospore characters chlamydospores, and septation and constrictions of hyphae such as septation and pigmentation. In Dothideales, the confound interpretations of homologous characters (Guterman characters normally used to classify asexual morphs were & Shabtai 1996, Zalar et al. 2008). clearly phylogenetically uninformative, and mixtures of black During our survey of fungi isolated from house dust yeast, hyphomycetous, coelomycetous, and intermediate using a dilution-to-extinction approach, many isolates asexual morphs are scattered over the various clades of morphologically resembled Aureobasidium and Hormonema, Aureobasidiaceae and Dothideaceae. Both families include a but six were phylogenetically distinct. Here we introduce these variety of asexual morphs, including sporodochial hyphomycete as two new species, in a new genus and family in Dothideales. forms usually observed in nature (e.g. Kabatiella, Kabatina), We present a 28S rDNA (nuclear large ribosomal subunit) coelomycete (e.g. Endoconidioma, Neocylindroseptoria, phylogeny of Dothidiomycetes to determine the strains’ Rhizosphaera), and black yeast-like forms usually observed phylogenetic position and subsequently present phylogenies in culture. of Dothideales based on BenA (β-tubulin), ITS rDNA, 28S Black yeasts have a confused taxonomic history that may rDNA, 18S rDNA (nuclear small ribosomal subunit), and RPB2 eventually be clarified with the single name classification (RNA polymerase II second largest subunit), to determine the system, but presently remains difficult to navigate. Black relationships within the order. Strains were characterized yeasts are a phylogenetically diverse morphogroup of morphologically and compared to morphologically similar asexual morphs, mostly in Dothideales or Chaetothyriales, species and genera. This work follows previous reports of which produce dark, slimy colonies and at least some new taxa of indoor fungi discovered by dilution-to-extinction, budding yeast-like cells in culture. Many species have one including species of Rasamsonia (Tanney & Seifert 2013), or more hyphal asexual morphs in addition to the yeast-like Aspergillus, Penicillium and Talaromyces (Visagie et al. forms. This pleiomorphy complicates their identification and 2014, 2017, Sklenář et al. 2017), Wallemia (Jancic et al. taxonomic interpretation (De Hoog & Hermanides-Nijhof 2015, Nguyen et al. 2015), Spiromastix, Pseudospiromastix, 1977). Two of the most frequently reported black yeast genera Sigleria (Hirooka et al. 2016), and Myrmecridium (Crous et are Aureobasidium (Aureobasidiaceae) and Hormonema al. 2016). (Dothideaceae). Aureobasidium was associated with several sexually typified genera inDothideales, and although no sexual morph is definitively known for the most frequently reported MATERIALS AND METHODS species A. pullulans, an ascospore-derived strain identified as Columnosphaeria fagi (CBS 171.93), has identical ITS and Isolations RPB2 sequences to the clade including the ex-type culture of Settled house dust was collected from twelve countries A. pullulans. The similar asexually typified genusHormonema, (Australia, Canada, Federated States of Micronesia, (H. dematioides type), generally considered the asexual morph Indonesia, Mexico, The Netherlands, New Zealand, of Sydowia polyspora, is also frequently reported. A single South Africa, Thailand, the United Kingdom, Uruguay, name solution for this clade is not yet proposed and we use the and USA) using sterilized Duststream® collectors (Indoor name Hormonema for comparisons of asexual morphotypes. Biotechnologies, Charlottesville, VA) attached to vacuum Despite the differences in associated sexual morphs, cleaners. Isolations were made from malt extract agar (MEA) Aureobasidium and Hormonema are difficult to distinguish and MEA with 20 % sucrose using a dilution-to-extinction morphologically when grown in culture. For example, neither method modified from Collado et al. (2007) as described Hermanides-Nijhof (1977) nor De Hoog & Yurlova (1994) in Visagie et al. (2014). More recent isolations targeting could find morphological differences among asexual morphs xerophilic fungi from Canadian and Hawaiian house dust in cultures of the sexually typified genera Pringsheimia, were made as described in Visagie et al. (2017). Dothidea, Dothiora or Sydowia, all of which they attributed Living strains of new species are deposited in the Canadian to Hormonema. Aureobasidium and Hormonema were Collection of Fungal Cultures (DAOMC, Ottawa, Canada), the considered distinct in the dual nomenclature era, because of Westerdijk Fungal Biodiversity Institute (CBS, Utrecht, The the different sexual morphs. Hermanides-Nijhof (1977) defined Netherlands) and dried specimens are accessioned in the
300 IMA FUNGUS A new family, genus, and species from house dust ARTICLE b E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange C.M. Visagie C.M. Visagie C.M. Visagie E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange C.M. Visagie E. Whitfield & K. Mwange E. Whitfield & K. Mwange C.M. Visagie C.M. Visagie C.M. Visagie C.M. Visagie C.M. Visagie C.M. Visagie C.M. Visagie C.M. Visagie E. Whitfield & K. Mwange Isolator Date collected 31 Jan. 2009 10 Feb. 2009 3 May 2009 20 Dec. 2014 May 2015 11 May 2015 11 unknown 2005 31 Mar. 2007 12 Mar. 2007 12 Mar. 2007 12 Mar. 2007 12 Mar. 21 Aug. 2007 21 Aug. 2007 21 Aug. 2007 10 Feb. 2009 10 Feb. 2009 24 Jul. 2009 27 Jan. 2015 2007 12 Mar. 2005 31 Mar. May 2015 11 May 2015 11 May 2015 11 May 2015 11 29 Jan. 2015 27 Jan. 2015 27 Jan. 2015 29 Jan. 2015 2007 12 Mar. a Collector Amend A. B. Horton Atkinson T. K.A. Seifert Amend A. Amend A. Amend A. Amend A. Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada B. Horton B. Horton K. Jacobs B. Kendrick Health Canada Amend A. Amend A. Amend A. Amend A. Amend A. A. Walker B. Kendrick B. Kendrick A. Walker Health Canada Australia, Tasmania, Hobart Australia, Tasmania, New Zealand, Wellington, Wellington Canada, Ontario, Stittsville USA, Hawaii, Kailua USA, Hawaii, Kailua USA USA, California, Berkeley Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Hobart Australia, Tasmania, Hobart Australia, Tasmania, Cape, Africa, Western South Kuilsrivier Canada, British Columbia, Victoria Canada, Saskatchewan, Regina USA, California, Berkeley USA, Hawaii, Kailua USA, Hawaii, Kailua USA, Hawaii, Kailua USA, Hawaii, Kailua Canada, New Brusnwick, Little Lepreau Canada, British Columbia, Victoria Canada, British Columbia, Victoria Canada, New Brusnwick, Little Lepreau Canada, Saskatchewan, Regina Origin Mexico, Nayarit, Sayulita MEA MEA MY50G MY1012 DG18 20%S-MEA 20%S-MEA n.a. n.a. 20%S-MEA 20%S-MEA 20%S-MEA 20%S-MEA n.a. 20%S-MEA 20%S-MEA MEA DG18 MEA 20%S-MEA MY50G MY50G MY1012 MY1012 MY50G DG18 DG18 DG18 20%S-MEA Isolation medium MEA SLOAN 1606 = BH02AU-110 TA10NZ-214a SLOAN 5623 = KAS 5840 KAS 7917 KAS 7956 AA04US-587 SLOAN 7256 = AA02US-306 SLOAN 7261 = SLOAN 203 = 7050035.79-65 SLOAN 207 = 7050035.79-71 SLOAN 48 = 7050035.79-127 SLOAN 62 = 7050035.79-148 SLOAN 349 = 7330009.33-45 SLOAN 359 = 7330009.33-60 SLOAN 7249 = 7330009.34-925 SLOAN 1652 = BH02AU-154a SLOAN 1653 = BH02AU-154b SLOAN 3214 = KJ09SA-65 KAS 5951 SLOAN 41 = 7050035.79-119 AA02US-332 SLOAN 7263 = KAS 7942 KAS 7947 KAS 7949 KAS 7953 DAOMC 251499 = KAS 6309 DAOMC 251470 = KAS 5918 DAOMC 251471 = KAS 5919 DAOMC 251469 = KAS 5999 DAOMC 250847 = SLOAN 52 7050035-79-132 Strain number AA07MX-884 SLOAN 1260 = species Aureobasidium melanogenum Aureobasidium melanogenum Aureobasidium melanogenum Aureobasidium melanogenum Aureobasidium melanogenum Aureobasidium melanogenum Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium pullulans Aureobasidium Aureobasidium subglaciale Hortaea werneckii Hortaea werneckii Hortaea werneckii Hortaea werneckii Rhizosphaera pini Sydowia polyspora Sydowia polyspora Sydowia polyspora Zalaria alba Table 1. Information on house dust isolates included in this study. Table Species Aureobasidium melanogenum
VOLUME 8 · NO. 2 301 Humphries et al.
Canadian National Mycological Herbarium (DAOM, Ottawa, Canada). Strains used in this study are summarized in Table 1.
Morphology The strains considered here were suspected to be xerophilic and were thus ARTICLE b characterized from colonies grown on a wide range of media, including MEA, potato-dextrose agar (PDA; Oxoid CM139), oatmeal agar (OA), dichloran 18 % glycerol agar (DG18; Hocking & Pitt 1980), starch-nitrate E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange E. Whitfield & K. Mwange Isolator agar (SNA; Dodman & Reinke 1982), malt extract yeast extract with 50 % glucose agar (MY50G), malt extract yeast extract 10 % glucose 12 % NaCl agar (MY10-12; Pitt & Hocking 2009), and MEA with the addition of 5–24 % NaCl (MEA5NaCl, MEA10NaCl, MEA15NaCl, MEA24NaCl). The malt extract used for media was always BD BactoTM (Mississauga, ON). Date collected 21 Aug. 2007 2005 31 Mar. 2005 31 Mar. 21 Aug. 2007 21 Aug. 2007 Plates were incubated for 7 and 14 d in the dark at 25 °C. Additional MEA plates were incubated at 10 and 30 °C. Colour names and codes used in descriptions refer to Kornerup & Wanscher (1967). Microscope preparations
a were made from colonies grown on MEA and DG18, using lactic acid as mounting fluid. An Olympus BX50 compound microscope attached with an InfinityX camera powered by Infinity Analyze v. 6.5.1 software (Lumenera, Collector Health Canada Amend A. Amend A. Health Canada Health Canada Ottawa, ON) was used for microscopic observations, capturing images and making measurements. Photographic plates were prepared in Affinity Photo v. 1.5.2 (https://affinity.serif.com).
DNA extraction, sequencing, and analysis DNA was extracted from 7–10-day-old cultures grown on Blakeslee’s malt extract agar (MEA; (Blakeslee 1915)) using the UltraClean™ Microbial DNA isolation Kit (MoBio Laboratories, Solano Beach, CA) with extracts stored at -20 °C. Loci were amplified using the following primer pairs: 28S rDNA with LR0R & LR5 (Vilgalys & Hester 1990); ITS with V9G/LS266 (Gerrits van den Ende & De Hoog 1999, Masclaux et al. 1995); RPB2 with fRPB2-5F/ fRPB2- USA, California, Berkeley USA, California, Berkeley Canada, Saskatchewan, Regina Canada, Saskatchewan, Regina Origin Canada, Saskatchewan, Regina 7cR (Liu et al. 1999); 18S rDNA with NS1/NS4 (White et al. 1990); and BenA with T10/Bt2b (Glass & Donaldson 1995, O’Donnell & Cigelnik 1997). An annealing temperature of 55 °C was used for all reactions. PCR amplification was performed in 10 µL volume reactions, containing 0.5 µL template DNA, 1 µL Titanium Taq buffer (Takara Bio USA, Mountain View, CA), 0.5 µL (2 mM) dNTP’s, 0.04 µL (3.2 mM) of each primer, 0.1 µL Titanium Taq polymerase 20%S-MEA 20%S-MEA n.a. n.a. Isolation medium MEA (Takara Bio USA), and 7.82 µL MilliQ water. Sequencing reactions were set up using the BigDye™ Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA) and the same primer pairs used for PCR amplification, with additional sequence reactions set up for 28S rDNA with primers LR3/LR3R (Vilgalys & Hester 1990). Sequence contigs were assembled in Geneious v. 8.1.8 (BioMatters, Auckland, NZ) and are deposited in GenBank (KX579092–KX579121, KY654326, KY659498, KY659500–KY659528). Accession numbers are also displayed on phylogenetic trees. BLAST searches were performed using NCBI to determine closest sequence matches and whether our species were detected in previous studies. DAOMC 250851 = SLOAN 7266 AA02US-340 DAOMC 250852 = SLOAN 7674 AA02US-351 DAOMC 250849 = SLOAN 7244 7330009.34-884 DAOMC 250850 = SLOAN 7246 7330009.34-921 Strain number DAOMC 250848 = SLOAN 352 7330009.33-5 Phylogenetic analyses isolator of strain using the modified dilution to exctinction method. b The phylogenetic position of our strains within Dothideomycetes was determined using a 28S rDNA phylogeny compared to reference sequences obtained from Schoch et al. (2009) and Hyde et al. (2013). Secondly, phylogenies of BenA, ITS, 28S rDNA, RPB2 and 18S rDNA were used to determine the relationships among our strains within the new genus, and the relationship of the genus and family with close relatives in Dothideales and Myriangiales. Newly generated sequences obtained from dust isolates belonging to Aureobasidium, Hortaea, Rhizosphaera, and Sydowia are also included in the phylogenies. Reference sequences for comparisons were obtained from GenBank and accession numbers are Collector of house dust sample; Zalaria obscurum Zalaria obscurum Zalaria obscurum Zalaria obscurum Table 1. (Continued). Table Species Zalaria alba a included on trees.
302 IMA FUNGUS A new family, genus, and species from house dust
Datasets were aligned using MAFFT v. 7.017 (Katoh & Standley ARTICLE 2013) with the L-INS-i algorithm. For the BenA dataset, G-INS-i was used. Alignments were manually trimmed in Geneious. The
et al . (2003) Dothideomycetes phylogeny was calculated using RAxML v. 8.0.0 (Stamatakis 2014) and support at nodes calculated using a bootstrap analysis of 1000 replicates. Additional phylogenies were calculated based on Maximum Likelihood done in RAxML and Bayesian tree interference (BI) using MrBayes v. 3.2 (Ronquist et al. 2012). For BI, the most suitable model for each dataset was Publication Unpublished Unpublished Rittenour et al . (2014) Unpublished Unpublished Unpublished Unpublished Álvarez-Rodríguez Unpublished Unpublished et al . (2013) Turchetti determined using MrModeltest v. 2.3 (Nylander 2004) based on the lowest Akaike information criteria (Akaike 1974) value. Trees were visualized in Figtree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/ figtree) and prepared for publication in Adobe® Illustrator® CS6. Aligned datasets and trees were uploaded to TreeBASE (www. treebase.org) with submission ID 19764.
RESULTS
Phylogeny Dothideomycetes 28S rDNA phylogeny (Fig. 1): The aligned 28S rDNA dataset contained 515 taxa and was 2448 bp long. Schismatomma decolorans was selected as outgroup based on Schoch et al. (2009). In general, our phylogeny shared similar topologies to those observed in Schoch et al. (2009) and Hyde et al. (2013). The phylogeny placed our isolates within the monophyletic order Dothideales, although there was poor support on the branch separating it from Myriangiales. Furthermore, our isolates resolved in a clade distinct from the two families currently recognized in Origin and source USA; Red-cockaded woodpecker excavation in living pine Area; Baotianman Natural Reserve China, Henan, Nanyang City, forest soil USA, Missouri, Kansas City; House dust Greece; hospital incubator Antarctica; wood The Netherlands; Soil Thailand, Prachuap Khiri Khan, Pran Buri Forest Park; sediment in mangrove forest Spain?; cork samples Area; Baotianman Natural Reserve China, Henan, Nanyang City, forest soil Portugal; Sound grapes Mont Blanc, Miage glacier; supraglacial sediments Italy, Dothideales, i.e. Aureobasidiaceae and Dothideaceae. Therefore, we introduce the new genus Zalaria and classify it in a new family named Zalariaceae below.
Dothideales phylogenies (Figs 2–3): To examine relationships and the phylogenetic species concept within Zalaria and its relationship with related families more closely, we calculated
Strain/clone Rcw 47 C 21 CMH 267 UOA/HCPF ENV57A AB 6 16628 ATCC WB 27 11965 CECT C 21 SP 9 DBVPG 5996 focused phylogenies based on 18S rDNA, 28S rDNA, BenA, ITS, and RPB2, including only closely related species and genera from Zalaria. Dothideaceae and Aureobasidiaceae, as well as species in the order Myriangiales. All loci consistently resolved the Zalaria strains in clades distinct from Aureobasidiaceae and Dothideaceae. In the ITS phylogeny, they resolved as a close relative of Myriangiales, but this tree had poor backbone support. All loci, except for the highly conserved 18S rDNA, resolved strains into two clades, Current identification Zalaria cf sp2 Zalaria cf sp2 Zalaria cf sp2 Zalaria sp1 Zalaria sp2 Zalaria cf sp2 Zalaria cf sp2 Zalaria cf sp2 Zalaria cf sp2 Zalaria cf sp2 Zalaria cf sp2 which were strongly supported in 28S rDNA, BenA, ITS, and RPB2. They are described below as Zalaria alba and Z. obscura spp. nov. Furthermore, the 28S rDNA phylogeny revealed several
sp. sp. sp. sp. sp. sp. strains that we consider identical to Z. obscura. These strains (CBS 122350; CBS 122359; EXF-922; EXF-1934; EXF-1936) originated from Norwegian arctic ice and were published in Zalar et al. (2008), but were never given a formal name. Remaining house dust isolates were identified here as Aureobasidium pullulans, A. Original identification Uncultured fungus Aureobasidium Uncultured fungus Aureobasidium pullulans Fungal sp. Aureobasidium pullulans Aureobasidium Aureobasidium Aureobasidium Aureobasidium Aureobasidium melanogenum, A. subglaciale, an undescribed Aureobasidium species (DTO 285-D8), Rhizosphaera pini, Sydowia polyspora, and Hortaea werneckii (Capnodiales).
GenBank nr KM103983 KJ130063 KF800358 KC253970 FJ235939 AF121282 AB456556 AY167611 KJ130062 JN004186 KC433818 NCBI-BLAST (Table 2): BLAST searches resulted in several hits similar to Zalaria ITS and 28S rDNA sequences. These sequences were from a diverse range of studies and originate from the USA, BLAST results showing origin of strains or clones belonging to 2. BLAST Table Gene ITS ITS ITS ITS ITS ITS LSU LSU LSU LSU LSU China, Greece, The Netherlands, Portugal, Spain, Thailand, and
VOLUME 8 · NO. 2 303 Humphries et al.
x2 Tubeufiales | Venturiales clade 1 Acrospermales Botryosphaeriales Catinella olivacea UAMH10679 (EF622212)
* Zalaria alba DAOMC250848 (KX579103) T ARTICLE Zalaria alba DAOMC250847 (KX579099) * Zalaria obscura DAOMC250849T (KX579100) Zalaria obscura DAOMC250850 (KX579098) * Zalaria obscura DAOMC250852 (KX579102) Zalaria obscura DAOMC250851 (KX579101) Zalariaceae
98 Dothiora elliptica CBS736.71 (GU301811) Dothiora cannabinae CBS737.71 (DQ470984) 98 Dothidea insculpta CBS189.58 (DQ247802)
98 Stylodothis puccinioides CBS193.58 (AY004342) 86 93 Dothidea hippophaes CBS188.58 (DQ678048) Dothidea sambuci DAOM231303 (AY544681) Phaeocryptopus nudus CBS268.37 (GU301856) 97 * Sydowia polyspora CBS116.29 (DQ678058) 88 Delphinella strobiligena CBS735.71 (DQ470977) Dothideaceae Aureobasidium caulivorum CBS242.64 (EU167576) *
* Columnosphaeria fagi CBS171.93 (AY016359)
Aureobasidium pullulans CBS584.75 (DQ470956) Aureobasideaceae Dothideales Phaeosclera dematioides CBS157.81 (GU301858) 83
97 Endosporium aviarium UAMH10530 (EU304351) 98 Endosporium populi-tremuloidis UAMH10529 (EU304348) insertae sedis * 88 Elsinoe centrolobi CBS222.50 (DQ678094) 86 Elsinoe phaseoli CBS165.31 (DQ678095)
97 Elsinoe veneta CBS150.27 (DQ767658) Elsinoaceae
* Myriangium duriaei CBS260.36 (DQ678059)
Myriangium hispanicum CBS247.33 (GU301854) Myriangiaceae Myriangiales * Capnodiales 87 Pleosporales Hysteriales | Strigulales 98 Mytilinidiales | Gloniaceae Micropeltis zingiberacicola IFRDCC2264 (JQ036227) * Phaeotrichales Neomicrothyrium siamense IFRDCC2194 (JQ036228) * Kirschteiniotheliaceae * Natipusillales Microthyrium microscopicum CBS115976 (GU301846) Venturiales clade 2 * Asterinales * Jahnulales 99 Lichenoconium * Monoblastiales
x2 Trypetheliales | Lichenotheliaceae Schismatomma decolorans DUKE47570 (AY548815)
0.2
Fig.1. Phylogenetic tree of Dothideomycetes based on 28S rDNA showing the distinct nature of the new family Zalariaceae within Dothideales. Schismatomma decolorans was selected as outgroup. Bootstrap support values higher than 80 % are indicated above branches (* indicates 100 % bootstrap support). House dust isolates are shown in bold text.
Antarctica and from habitats including house dust, cork Morphology samples, grapes, a hospital incubator, sediment, soil, wood, Strains were characterized morphologically on several and woodpecker excavations. Our Zalaria isolates were agar media and shared several characters with species of obtained from house dust collected in the USA (CA) and Aureobasidium and Hormonema. As noted in the Introduction, Canada (Regina, SK). the only way to reliably distinguish between these groups
304 IMA FUNGUS A new family, genus, and species from house dust
/86 Dothidea ribesia MFL 14.004 (KM388552) 0.99/81 Hormonema dematioides D817.03.98 (AJ278929) 0.97/- Dothidea ribesia MFL CC13.0670 (KM388553) Rhizosphaera kalkhoffii HAF27 (KM435342) 0.97/- -/
Dothidea berberidis C S187.58 (KC800752) Rhizosphaera kalkhoffii HM F-CHN1 (J 353721) ARTICLE /87 Dothidea hippophaeos C S188.58 (D 678048) Rhizosphaera kalkhoffii HM F-CHN2 (J 353722) Stylodothis puccinioides C S193.58 Dothideales (AY004342) Sydowia polyspora C S128.64 (AJ244262) /99 Dothidea ribesia C S195.58 (AY016360) Hormonema dematioides C107.07.98 (AJ278927) Dothidea sambuci C S198.58 (AF382387) Hormonema dematioides D106.07.98 (AJ278928) Dothidea sambuci DA M231303 (AY544681) Hormonema dematioides E419.05.98 (AJ278930) Dothidea insculpta C S189.58 (D 247802) Rhizosphaera kalkhoffii C S280.38 (E 700375) / Coniozyma leucospermi C S111289 (E 552113) Rhizosphaera kalkhoffii C S114656 (E 700376) Endoconidioma populi AMH10902 (HM185488) Atramixtia arboricola AMH11211 (HM347778) Dothiora bupleuricola C S112.75 (K 728538) 0.99/93 Hormonema dematioides E99156 (AY253451) / 0.99/- Hormonema viticola L9D.11 (KF201297) do ia ol s ora DAOMC251470 (K 659502) Hormonema viticola FMR13040 (KF201298) do ia ol s ora DAOMC251471 (K 659503) Dothiora viburnicola C S274.72 (K 728554) 0.95/- Hormonema macrosporum C S536.94 (AJ244247) Dothiora maculans C S299.76 (K 728543) Hormonema dematioides 106 07 98 (AJ278926) Dothiora maculans C S301.76 (K 728544) /99 Sydowia polyspora C S544.95 (AY152548) Dothiora maculans C S686.70 (K 728546) do ia ol s ora DAOMC251469 (K 659505) Dothiora elliptica C S736.71 (G 301811) Sydowia polyspora C S248.93 (G 412724) Dothiora phaeosperma C S870.71 (K 728550) Hormonema dematioides HDDFMI (AF462439) Dothiora prunorum C S933.72 (K 728551) Sydowia polyspora C S750.71 (KT693747) Dothiora cannabinae C S737.71 (D 470984) Sydowia aponica FFPRI411088 (J 814699) Dothiora laureolae C S744.71 (K 728542) / Sydowia aponica FFPRI411084 (J 814700) -/89 Dothiora ceratoniae C S477.69 (KF251655) Sydowia aponica FFPRI411085 (J 814701) Dothiora ceratoniae C S290.72 (K 728539) Sydowia aponica FFPRI411087 (J 814702) Dothiora ceratoniae C S441.72 (K 728540) Endoconidioma populi AMH10297 (AY604526) Dothiora phillyreae C S473.69 (E 754146) Endoconidioma populi AMH10298 (AY604527) Coleophoma oleae C S615.72 (E 754148) Endoconidioma populi AMH10299 (HM185486) Pringsheimia smilacis C S873.71 (FJ150970) Hormonema carpetanum TRN201 (AY616204) Dothiora agapanthi CPC20600 (K 728537) Hormonema carpetanum TRN25 (AY616205) Dothiora oleae C S152.71 (K 728547) /80 Hormonema carpetanum TRN31 (AY616206) Dothiora oleae C S235.57 (K 728548) Endoconidioma populi AMH10902 (HM185487) Dothiora oleae C S472.69 (K 728549) /87 Endoconidioma populi AMH10903 (HM185489) Dothiora sorbi C S742.71 (K 728552) Hormonema carpetanum ATCC74360 (AF182375) Sydowia polyspora C S116.29 (D 678058) Hormonema carpetanum TRN278 (AY616199) Sydowia polyspora C S750.71 (FJ150912) Hormonema carpetanum TRN40 (AY616201) / Dothichiza pityophila C S215.50 (FJ150968) 0.96/- Hormonema carpetanum TRN24 (AY616202) /91 Atramixtia arboricola AMH11211 (HM347777) Coniozyma leucospermi C S111289 (E 552113) Delphinella strobiligena C S735.71 (D 470977) / Dothidea sambuci C S198.58 (AY930108) Phaeocryptopus nudus C S268.37 (EF114700) 0.99/- Dothidea sambuci DA M231303 (D 491505) 0.98/81 hi os haera i i DAOMC251499 (K 654326) / Dothidea ribesia MFL 14.004 (KM388544) Plowrightia abietis ATCC24339 (EF114703) / Dothidea ribesia MFL CC13.0670 (KM388545) Plowrightia periclymeni 178096 (FJ215702) Dothidea insculpta C S189.58 (AF027764) Neocylindroseptoria pistaciae C S471.69 (KF251656) Dothidea hippophaeos C S186.58 (AF027763) Columnosphaeria fagi C S171.93 (AY016359) /82 Kabatina uniperi C S239.66 (AY616211) Aureobasidium pullulans C S146.30 (FJ150916) Kabatina uniperi C S466.66 (AY616212) Aureobasidium pullulans C S100280 (FJ150941) 0.99/- Kabatina thu ae C S238.66 (AF013226) /96 Aureobasidium pullulans C S584.75 (FJ150942) Dothiora oleae C S152.71 (K 728508) Aureobasidium microstictum C S342.66 (FJ150945) Dothiora oleae C S235.57 (K 728509) Aureobasidium lini C S125.21 (FJ150946) 0.97/- Dothiora oleae C S472.69 (K 728510) Aureobasidium pullulans C S701.76 (FJ150951) /- Dothiora bupleuricola C S112.75 (K 728499) Aureobasidium proteae C S114273 (JN712557) 0.95/- Dothiora phillyreae C S473.69 (K 728513) /86 Aureobasidium proteae C S111973 (JN712558) Dothiora ceratoniae C S477.69 (KF251151) Aureobasidium namibiae C S147.97 (FJ150937) Dothiora ceratoniae C S290.72 (K 728500) Aureobasidium leucospermi C S130593 (JN712555) Dothiora ceratoniae C S441.72 (K 728501) Aureobasidium subglaciale C S123387 (FJ150913) /96 Dothiora agapanthi CPC20600 (K 728498) Aureobasidium subglaciale C S123388 (FJ150935) Dothiora viburnicola C S274.72 (K 728515) 0.98/- Aureobasidium melanogenum C S123.37 (FJ150917) 0.99/- Kabatiella harpospora C S122914 (KT693741) Aureobasidium melanogenum C S105.22 (FJ150926) x2 0.99/98 Dothiora phaeosperma C S870.71 (K 728512) 0.99/90 Aureobasidium melanogenum C S110374 (FJ150929) 0.99/89 Dothiora elliptica C S736.71 (K 728502) Selenophoma mahoniae C S388.92 (E 754213) Dothiora prunorum C S933.72 (AJ244248) Aureobasidium caulivorum C S242.64 (FJ150944) Hormonema prunorum C S933.72 (AY616213) Aureobasidium microstictum C S114.64 (FJ150940) Dothiora rhamni-alpinae C S745.71 (AJ244245) /98 Selenophoma linicola C S468.48 (E 754212) Dothiora sorbi C S742.71 (K 728514) Pseudoseptoria collariana C S135104 (KF251721) /90 Pseudoseptoria obscura C S135103 (KF251722) Dothiora cannabinae C S737.71 (AJ244243) Saccothecium sepincola MFL CC14.0276 (KM388554) Dothiora laureolae C S744.71 (K 728503) /98 / Pseudosydowia eucalypti CPC14028 (G 303327) Dothiora europaea C S739.71 (AJ244244) Pseudosydowia eucalypti CPC14927 (G 303328) Dothiora maculans C S299.76 (K 728504) Saccothecium species MFL CC14.1171 (K 290336) Dothiora maculans C S301.76 (K 728505) Selenophoma australiensis C S124776 (G 303324) Dothiora maculans C S686.70 (K 728507) Celosporium larixicol L3.1 (FJ997288) Rhizosphaera macrospora C S467.82 (E 700368) -/93 Rhizosphaera pseudotsugae C S101222 (E 700369) Zalarium obscura C S122350 (FJ150961) Rhizosphaera macrospora ATCC4636 (AF462431) Zalarium obscura E F1934 (FJ150962) 0.98/- Zalarium obscura C S122362 (FJ150963) Rhizosphaera macrospora C S208.79 (G ) Zalarium obscura E F922 (FJ150964) /85 Rhizosphaera pini ATCC46387 (AY183365) /99 Zalarium obscura E F1936 (FJ150965) / Rhizosphaera pini C S206.79 (E 700370) Zalarium obscura C S122359 (FJ150966) /- hi os haera i i DAOMC251499 (K 659500) Phaeocryptopus nudus C S268.37 (E 700371) alaria obs ura DAOMC250850 (K 579098) /96 / alaria obs ura DAOMC250849 (K 579100) /85 Rhizosphaera oudemansii C S226.83 (E 700366) alaria obs ura DAOMC250851 (K 579101) Rhizosphaera oudemansii C S427.82 (E 700367) alaria obs ura DAOMC250852 (K 579102) Scleroconidioma sphagnicola AMH9731 (AY220610) / alaria alba DAOMC250847 (K 579099) / Hormonema viticola L9D.11 (KF201300) alaria alba DAOMC250848 (K 579103) Hormonema viticola FMR13040 (KP641179) 0.99/- Elsinoe phaseoli C S165.31 (D 678095) Hormonema viticola L9D.17 (KP641179) Sphaceloma erythrinae CPC18530 (JN940392) Neocylindroseptoria pistaciae C S471.69 (KF251152) /94 /89 Sphaceloma arachidis CPC18533 (JN940374) Celosporium larixicol L3.1 (FJ997287) x4 Rhizosphaera kobayashii ATCC46389 (AF462432) /86 Sphaceloma bidentis CPC18526 (JN940379) Sphaceloma asclepiadis CPC18532 (JN940380) Aureobasidium pullulans C S146.30 (FJ150902) / Aureobasidium pullulans C S701.76 (FJ150907) /82 Myriangium duriaei C S260.36 (D 678059) x4 Myriangium hispanicum C S247.33 (G 301854) Aureobasidium ullula s KAS5951 (K 659504) /93 Endosporium populitremuloidis AMH10529 (E 304348) Aureobasidium ullula s SLOAN1652 (K 659514) Endosporium aviarium AMH10530 (E 304351) Aureobasidium ullula s SLOAN1653 (K 659515) x4 Phaeosclera dematioides AMH4265 (E 981288) Aureobasidium ullula s SLOAN203 (K 659516) / Saccothecium sepincola C S278.32 (G 301870) Aureobasidium ullula s SLOAN207 (K 659517) x4 Aureobasidium ullula s SLOAN3214 (K 659518) x2 Metasphaeria species M11.1 (KF590163) x4 Lineolata rhizophorae C S641.66 (G 479792) Aureobasidium ullula s SLOAN349 (K 659519) / Hortaea werneckii C S107.67 (E 019270) Aureobasidium ullula s SLOAN359 (K 659520) Hortaea thailandica CPC16651 (G 214429) Aureobasidium ullula s SLOAN48 (K 659522) Neohortaea acidophila C S113389 (G 323202) Aureobasidium ullula s SLOAN62 (K 659524) Phaeocryptopus gaeumannii C S267.37 (EF114698) Aureobasidium ullula s SLOAN7249 (K 659525) Aureobasidium ullula s SLOAN7261 (K 659527) 0.04 Aureobasidium proteae C S114273 (KT693731) Aureobasidium proteae C S111973 (KT693732) Aureobasidium pullulans C S584.75 (KT693733) Aureobasidium pullulans C S100280 (KT693734) Columnosphaeria fagi C S171.93 (KT693737) Aureobasidium lini C S125.21 (KT693742) Aureobasidium microstictum C S342.66 (KT693743) Kabatiella microsticta C S342.66 (KT693743) 0.99/- Aureobasidium namibiae C S147.97 (KT693730) 0.97/84 Aureobasidium mela o e um KAS7917 (K 659506) Aureobasidium mela o e um KAS7956 (K 659511) Aureobasidium melanogenum C S123.37 (FJ150881) Aureobasidium mela o e um KAS5840 (K 659501) Aureobasidium mela o e um SLOAN1260 (K 659512) Aureobasidium mela o e um SLOAN1606 (K 659513) Aureobasidium mela o e um SLOAN5623 (K 659523) Aureobasidium mela o e um SLOAN7256 (K 659526) Aureobasidium melanogenum C S110374 (KT693728) Aureobasidium melanogenum C S105.22 (KT693729) /96 Aureobasidium sub la iale SLOAN7263 (K 659528) Aureobasidium subglaciale C S123387 (KT693735) /- Aureobasidium subglaciale C S123388 (KT693736) Aureobasidium species DT 302H2 (KT693725) Aureobasidium leucospermi C S130593 (KT693727) Aureobasidium species DT 301G9 (KT693694) Aureobasidium species SLOAN41 (K 659521) 0.93/87 -/99 Aureobasidium species DT 285D8 (KT693673) Selenophoma mahoniae C S388.92 (KT693746) Kabatiella bupleuri C S131304 (KT693738) /- Kabatiella bupleuri C S131303 (KT693739) /- Aureobasidium caulivorum C S242.64 (KT693740) Aureobasidium microstictum C S114.64 (KT693744) /- / Aureobasidium thailandense NRRL58539 (J 462674) Aureobasidium thailandense NRRL58543 (J 462675) /98 Pseudoseptoria collariana C S135104 (KF251218) Pseudoseptoria obscura C S135103 (KF251219) /- /- Dothichiza pityophila C S215.50 (AJ244242) Pringsheimia smilacis C S873.71 Dothideales (AJ244257) /84 Saccothecium species MFL CC14.1171 (K 290338) / Selenophoma eucalypti STE 659 (AY293059) /- Pseudosydowia eucalypti CPC14028 (G 303296) Pseudosydowia eucalypti CPC14927 (G 303297) Selenophoma australiensis C S124776 (G 303293) 0.99/ alaria alba DAOMC250847 (K 579093) /99 alaria alba DAOMC250848 (K 579097) /98 alaria obs ura DAOMC250850 (K 579092) alaria obs ura DAOMC250849 (K 579094) alaria obs ura DAOMC250851 (K 579095) alaria obs ura DAOMC250852 (K 579096) x4 Pringsheimia euphorbiae C S747.71 (AJ244276) /88 Endosporium populitremuloidis AMH10529 Myriangiales (E 304347) Endosporium aviarium AMH10530 Myriangiales (E 304350) Sphaceloma bidentis CPC18526 Myriangiales (JN943491) / /99 Sphaceloma asclepiadis CPC18532 Myriangiales (JN943493) / Sphaceloma arachidis CPC18533 Myriangiales (JN943485) Sphaceloma erythrinae CPC18530 Myriangiales (JN943502) Hortaea werneckii C S107.67 (AJ238468) Hortaea werneckii C S111.31 (AJ238679) / ortaea er e ii KAS7942 (K 659507) ortaea er e ii KAS7947 (K 659508) ortaea er e ii KAS7949 (K 659509) 0.98/- ortaea er e ii KAS7953 (K 659510) Hortaea thailandica CPC16651 (G 214637) Neohortaea acidophila C S113389 (G 214636) / Phaeocryptopus gaeumannii C S267.37 (EF114685) Phaeocryptopus gaeumannii C S244.38 (E 700364) 0.1
Fig. 2. Phylogenies based on 28S rDNA and ITS showing the relationship of Zalaria (green text and branches) with other closely related genera from families Dothideaceae (orange branches), Aureobasidiaceae (blue branches) and order Myriangiales (maroon branches). Bootstrap support values or Bayesian posterior probabilities higher than 79 % or 0.94 are indicated above thickened branches (* indicates 100 % or 1.00; – indicates lack of support). House dust isolates from this study are indicated by bold text. GenBank accession numbers are provided between brackets.
VOLUME 8 · NO. 2 305 Humphries et al.
alaria obs ura DAOMC250850 (K 579110) alaria obs ura DAOMC250852 (K 579114) /99 alaria obs ura DAOMC250849 (K 579112) alaria obs ura DAOMC250851 (K 579113) alaria alba DAOMC250847 (K 579111) alaria alba DAOMC250848 (K 579115) Columnosphaeria fagi C S171.93 (AY016342) Aureobasidium caulivorum C S242.64 (E 167576) Aureobasidium pullulans C S584.75 (E 682922) Aureobasidium lini C S125.21 (E 707925) Selenophoma linicola C S468.48 (E 754113) Selenophoma mahoniae C S388.92 (E 754114) Saccothecium species MFL CC14.1171 (K 290337) Stylodothis puccinioides C S193.58 (AY016353) ARTICLE Dothidea sambuci DA M231303 (AY544722) 0.96/- Dothidea insculpta C S189.58 (D 247810) Dothidea hippophaeos C S186.58 (E 167601) 0.98/- Dothidea ribesia MFL 14.004 (KM388549) Dothidea ribesia MFL CC13.0670 (KM388550) /- Dothidea ribesia C S195.58 (AY538346) Dothiora phillyreae C S473.69 (E 754047) Coleophoma oleae C S615.72 (E 754049) Dothiora cannabinae C S737.71 (D 479933) Hormonema prunorum C S933.72 (E 707926) Dothiora prunorum C S933.72 (K 728551) 0.96/- Phaeocryptopus nudus C S268.37 (EF114724) 0.97/- /82 Plowrightia periclymeni 178096 (FJ215709) Plowrightia abietis ATCC24339 (EF114727) 0.95/94 Delphinella strobiligena C S735.71 (D 471029) Sydowia polyspora C S116.29 (D 678005)
/91 x4 Saccothecium sepincola C S278.32 (G 296195)
x4 Lineolata rhizophorae C S641.66 (G 479758) /90 Myriangium duriaei C S260.36 (AY016347) Myriangium hispanicum C S247.33 (G 296180) Sphaceloma erythrinae CPC18530 (JN940566) Sphaceloma bidentis CPC18526 (JN940555) /98 Elsinoe phaseoli C S165.31 (D 678042) /90 Sphaceloma arachidis CPC18533 (JN940548) Sphaceloma asclepiadis CPC18532 (JN940556) /85 Endosporium populitremuloidis AMH10529 (E 304346) Endosporium aviarium AMH10530 (E 304349) /98 Hortaea thailandica CPC16651 (JN938703) Hortaea werneckii C S107.67 (Y18693) Phaeocryptopus gaeumannii C S267.37 (EF114722) 0.005 Aureobasidium pullulans C S584.75 (D 470906) Aureobasidium pullulans C S100280 (KT693977) Columnosphaeria fagi C S171.93 (KT693980) / Aureobasidium microstictum C S342.66 (KT693985) Aureobasidium proteae C S114273 (KT693974) /- /95 Aureobasidium proteae C S111973 (KT693975) Lorem ipsum Aureobasidium caulivorum C S242.64 (KT693983) Aureobasidium lini C S125.21 (KT693984) Selenophoma mahoniae C S388.92 (KT693987) / Kabatiella bupleuri C S131304 (KT693981) 0.98/- Kabatiella bupleuri C S131303 (KT693982) Aureobasidium microstictum C S114.64 (KT693986) / Aureobasidium subglaciale C S123387 (KT693978) Aureobasidium subglaciale C S123388 (KT693979) /98 Aureobasidium leucospermi C S130593 (KT693970) Aureobasidium namibiae C S147.97 (KT693973) /99 Aureobasidium melanogenum C S110374 (KT693971) Aureobasidium melanogenum C S105.22 (KT693972) Aureobasidium thailandense NRRL58539 (E 719566) / /- Dothidea sambuci DA M231303 (D 522854) /99 Dothidea sambuci C S198.58 (KT216559) 0.99/- Dothidea insculpta C S189.58 (D 247792) Dothidea hippophaeos C S188.58 (D 677942) / Dothiora cannabinae C S737.71 (D 470936) / /80 Sydowia polyspora C S116.29 (D 677953) / Sydowia polyspora C S750.71 (KT693988) Delphinella strobiligena C S735.71 (D 677951) alaria obs ura DAOMC250850 (K 579104) / alaria obs ura DAOMC250849 (K 579106) alaria obs ura DAOMC250851 (K 579107) / alaria obs ura DAOMC250852 (K 579108) alaria alba DAOMC250847 (K 579105) /89 /96 alaria alba DAOMC250848 (K 579109)
0.99/- x4 Saccothecium sepincola C S278.32 (G 371745)
x4 Lineolata rhizophorae C S641.66 (G 479828) / Myriangium hispanicum C S247.33 (G 371744) /89 Myriangium duriaei C S260.36 (KT216528) Elsinoe phaseoli C S165.31 (KT216560) Neohortaea acidophila C S113389 (KT216521) Hortaea thailandica CPC16651 (KF902206) Phaeocryptopus gaeumannii C S244.38 (G 371740) 0.2 / Dothiora ceratoniae C S290.72 (K 728619) /90 Dothiora ceratoniae C S441.72 (K 728620) Dothiora phillyreae C S473.69 (K 728629) Dothiora oleae C S152.71 (K 728625) / Dothiora oleae C S472.69 (K 728627) /- Dothiora oleae C S235.57 (K 728626) Dothiora maculans C S299.76 (K 728621) / Dothiora maculans C S301.76 (K 728622) Dothiora maculans C S686.70 (K 728624) Dothiora agapanthi CPC20600 (K 728617) Rhizosphaera oudemansii C S427.82 (E 747279) 0.99/90 Rhizosphaera oudemansii C S226.83 (E 747278) /87 Rhizosphaera macrospora C S467.82 (E 747280) /97 Rhizosphaera pseudotsugae C S101222 (E 747281) -/85 hi os haera i i DAOMC251499 (K 659498) /91 Phaeocryptopus nudus C S268.37 (E 747283) Rhizosphaera pini C S206.79 (E 747282) / Rhizosphaera kalkhoffii C S280.38 (E 747273) Rhizosphaera kalkhoffii C S114656 (E 747274) Aureobasidium pullulans C S584.75 (FJ157869) 0.99/96 0.95/- Aureobasidium pullulans C S100280 (FJ157864) Aureobasidium pullulans C S701.76 (FJ157865) 0.98/- Aureobasidium pullulans C S146.30 (FJ157871) Aureobasidium microstictum C S342.66 (FJ157872) Aureobasidium lini C S125.21 (FJ157873) Selenophoma mahoniae C S388.92 (FJ157874) 0.98/- /96 Aureobasidium melanogenum C S123.37 (FJ157852) Aureobasidium melanogenum C S105.22 (FJ157858) 0.99/- Aureobasidium namibiae C S147.97 (FJ157863) / Aureobasidium subglaciale C S123387 (FJ157878) Aureobasidium subglaciale C S123388 (FJ157877) Aureobasidium thailandense NRRL58539 (E 719407) alaria obs ura DAOMC250850 (K 579116) -/94 alaria obs ura DAOMC250849 (K 579118) /99 alaria obs ura DAOMC250851 (K 579119) alaria obs ura DAOMC250852 (K 579120) / alaria alba DAOMC250847 (K 579117) alaria alba DAOMC250848 (K 579121) Phaeocryptopus gaeumannii C S244.38 (E 747284) 0.08
Fig. 3. Phylogenies based on 18S rDNA, RPB2 and BenA showing the relationship of Zalaria (green text and branches) with other closely related genera from families Dothideaceae (orange branches), Aureobasidiaceae (blue branches) and order Myriangiales (maroon branches). Bootstrap support values or Bayesian posterior probabilities higher than 79 % or 0.94 are indicated above thickened branches (* indicates 100 % or 1.00; – indicates lack of support). House dust isolates from this study are indicated by bold text. GenBank accession numbers are provided between brackets.
306 IMA FUNGUS A new family, genus, and species from house dust
are the 1–2 conidiogenous loci per cell in Hormonema (and is indicated in bold text (5’-AGCTCAAATTTGAAA TCTGGCC- ARTICLE asexual morphs of many other Dothideaceae species), and CTTTC-AGGGTCCGAGTTGTAATTTGTAGAGG-3’). up to 14 loci in Aureobasidium (De Hoog & Yurlova 1994, Yurlova et al. 1999). The new genus is more similar in this Zalaria Visagie, Z. Humphries & Seifert, gen. nov. regard to Hormonema, with only 1–2 loci per conidiogenous MycoBank MB821628 cell. In general, growth of Zalaria species is more restricted than in any of these other genera. Etymology: Named in honour of Polona Zalar, mycologist at Three types of conidiogenesis were observed in the the University of Ljubljana, Slovenia, in recognition of her Zalaria strains. Their yeast forms are very common, especially studies on extremophilic fungi, including her 2008 study that in younger colonies when cells reproduce by budding. After included strains from Norwegian arctic regions that belong to prolonged incubation, these yeast cells are often covered this genus. by melanized hyphal growth; microscopic observations suggest that hyphae from germinating yeast cells eventually Diagnosis: Differs from Aureobasidium by blastic conidiogen- give rise to this dark melanized growth (Figs 4C–D, 5C–D). esis occurring from one to two loci per conidiogenous cell. With age, these hyphae may develop into dark brown, thick- Morphologically Zalaria is indistinguishable from Hormonema walled chlamydospores (Figs 4E, I, 5E, I). Further, intercalary (often reported as asexual morphs of Sydowia), leaving DNA conidiogenous cells develop mostly at margins of young sequences the only diagnostic character (see Diagnosis for colonies. family Zalariaceae above). The strains resolved as distinct clades observed in the phylogenies were also distinct morphologically, with Z. alba Type species: Zalaria obscura Visagie et al. 2017 compared to Z. obscura growing more restrictedly on most media. Zalaria obscura is also capable of growth at low aw Description: Sexual morph unknown. Colonies often covered media such as MEA-10%-NaCl, MY-1012 and MY50G, in slimy masses of conidia or yeast-like cells, becoming with Z. alba not growing on these media. Generally, Z. alba dark and often leathery with time, occasionally with sparse colonies are also more yeast-like and take up to 3 wk to aerial mycelium; cream-colored, red-brown, olive-brown, darken, whereas Z. obscura colonies darken in 7 d and are dark brown, or black; margins entire or fimbriate. Hyphae often covered by a leathery layer within 14 d. This character transversely and longitudinally septate, hyaline and thin- was originally used for distinguishing the two varieties of walled when young, frequently becoming melanized and A. pullulans var. pullulans and var. melanogenum, later thick-walled with age, may develop into chlamydospores. recognized as two distinct species (Gostinčar et al 2014). Conidiogenous cells undifferentiated, intercalary, terminal Based on both phylogenetic and morphological results uncommon, cylindrical, with blastic conidiogenesis occurring we introduce a new family, genus and two new species to from one to two loci per cell. Chlamydospores dark brown, accommodate our unique black yeast-like fungi. smooth to lightly rough-walled, globose to ellipsoidal, septate. Morphological examinations confirmed sequence-based Conidia often yeast-like, hyaline, aseptate, smooth-walled, identifications of the remaining house dust isolates (Figs ellipsoidal to lemon-shaped, variable in size, indistinct hilum, 6–7). Aureobasidium strains produced the typical conidiog- budding common, polar, bipolar and multilateral. enous cells with multiple loci (Fig. 6A–G), while only 1–2 conidiogenous loci were observed in strains identified as Zalaria alba Visagie, Z. Humphries & Seifert, sp. nov. Sydowia polyspora (Fig. 6H–K). Hortaea werneckii was com- MycoBank MB821629 mon in Hawaiian dust samples and was only isolated from (Fig. 4) the halophilic MY10-12 medium. The typical pigmented hy- phae, yeast-like growth, sympodial and percurrent conidio- Etymology: Latin, alba, meaning white, in reference to colony genesis were observed in newly isolated strains (Fig. 7A–E). appearance after 7 d of growth. The strain identified as Rhizosphaera pini produced colonies with pycnidium-like structures and a Hormonema-like morph Diagnosis: Differs from Z. obscura in the inability to grow producing very large conidia, all characteristic of that species at lowered aw. Colonies remain yellowish to orange-white (Fig. 7F–I). until it darkens after about 3 wk. Conidia appearing more slender than Z. obscura. ITS barcode: KX579093. Alternative Taxonomy identification markers: 28S rDNA: KX579099, RPB2: KX579105, 18S rDNA: KX579111, BenA: KX579117. Zalariaceae Visagie, Z. Humphries & Seifert, fam. nov. MycoBank MB821627 Type: Canada: Saskatchewan: Regina, isol. ex house dust, 12 Mar. 2007, E. Whitfield & K. Mwange (DAOM 734001 – Type genus: Zalaria Visagie et al. 2017. holotype; DAOMC 250847 – ex-type culture).
Diagnosis: Distinguished from other families classified in Description: Colony diameters (mm after 7 d (14 d at 25 °C)): Dothideales and Myriangiales based on a short unique 28S MEA 5–6 (8–9); MEA 5 °C microcolonies, 10 °C microcolonies, rDNA sequence flanked by two conserved regions. The 30 °C 2–5, 35 °C 1–2, 40 °C microcolonies; MEA-5 %-NaCl section defining Zalariaceae in our alignment (Treebase ID no growth, MEA-10 %-NaCl no growth, MEA-15 %-NaCl no 19764) are found between nucleotide positions 39 to 62 and growth, MEA-24 %-NaCl no growth; OA 6–7 (11–14), PDA
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Fig. 4. Morphological characters of Zalaria alba (DAOMC 250847 in A–C, E, F; DAOMC 250848 in D, G–K). A. Colonies on MEA after 2 wk. B. Close-up colony on MEA after 4 wk. C. Germinating conidia. D. Germinating conidia with age. E. Melanized hyphae developing into arthrospores and chlamydospores. F–H. Intercalary conidiogenous cells. I. Chlamydospores. J–K. Conidia with some yeast-like budding occurring. Bars = 10 µm.
308 IMA FUNGUS A new family, genus, and species from house dust
7–8 (10–11), DG18 3–4 (5–7), MY1012 no growth, MY50G Description: Colony diameters (mm after 7 d, (14 d) at 25 ARTICLE no growth, SNA 3–5 (7–8). °C)): MEA 25 °C 7–10 (12–14); MEA 5 °C microcolonies, 10 °C microcolonies, 30 °C 3–9, 35 °C 3–8, 40 °C 1–2 mm; Cultural characters: Colonies on MEA at 25 °C after 7 d MEA-5 %-NaCl 2–4 (3–5), MEA-10 %-NaCl microcolonies, yeast-like, smooth and slimy, obverse yellowish white to MEA-15 %-NaCl no growth, MEA-24 %-NaCl no growth; OA orange white (4A2–5A2), reverse greenish grey to pale 7–8 (15–16), PDA 7–9 (10–14), DG18 3–5 (6–8), MY1012 no orange (1B2–5A3), generally becoming dark within 3 wk, with growth, sometimes microcolonies after prolonged incubation, dark areas sometimes present after 7 d, olive-yellow to dark MY50G no growth (microcolonies), SNA 4–5 (8–9). brown (3D6-7F5), some aerial mycelium developing after longer incubation. Cultural characteristics: Colonies on MEA at 25 °C after 7 d yeast-like, smooth and slimy, obverse dark brown (7F5) to Microscopic characters: Young somatic hyphae at colony black with some yellowish white to orange-white (4A2–5A2), periphery mostly hyaline, smooth, thin-walled, branched, olive-yellow (3D6), and olive (1E5) areas, surface leathery transversely septate, 1.5–5 µm diam; older hyphae after 14 d; some aerial mycelium developing after prolonged towards colony centre melanized, dark brown, smooth to incubation. lightly roughened, thick-walled, branched, transversely and longi-septate, 2–6.5 µm diam, often developing into Microscopic characters: Young somatic hyphae at colony chlamydospores. Conidiogenous cells undifferentiated, periphery mostly hyaline, smooth, thin-walled, branched, intercalary, rarely terminal, mostly on hyaline hyphae, transversely septate, 1.5–4.5 µm diam; older hyphae producing conidia percurrently from short lateral denticles towards colony centre melanized, dark brown, smooth to not exceeding 2 µm long. Chlamydospores dark brown, lightly roughened, thick-walled, branched, transversely smooth to lightly rough-walled, globose to ellipsoidal, septate and longi-septate, 2–11 µm diam, often developing into to aseptate, one-septate spores sometimes constricted at chlamydospores. Conidiogenous cells undifferentiated, septum, 5.5–10 × 3–7.5 µm (x̄ = 8 ± 0.99 µm; 5.5 ± 0.71 µm). intercalary, rarely terminal, mostly on hyaline hyphae, Conidia often yeast-like, hyaline, aseptate, smooth walled, producing conidia percurrently from short lateral denticles ellipsoidal to lemon-shaped, variable in size, 2.5–10 × 1.5–5 not exceeding 2 µm long. Chlamydospores dark brown, µm (x̄ = 5.5 ± 1.53 µm, 3 ± 0.73 µm), with an indistinct hilum, smooth to lightly rough walled, globose to ellipsoidal, septate budding common, polar, bipolar and multilateral. to aseptate, one-septate spores sometimes constricted at septum, 5–17 µm × 3.5–8 µm (x̄ = 8 ± 2.11 µm; 6 ± 0.89 µm).
Notes: Growth on media with lowered aw distinguishes between Conidia often yeast-like, hyaline, aseptate, smooth-walled, the two Zalaria species. Zalaria alba does not grow on MEA-5 ellipsoidal to lemon-shaped, variable in size, 2.5–10 × 1.5– %-NaCl, MY10-12 or MY50G after 14 d. In contrast, Z. obscura 5.5 µm (x̄ = 5 ± 1.4 µm; 3.5 ± 0.77 µm), with an indistinct produces at least microcolonies on these media. Colony size hilum, budding common, polar, bipolar and multilateral. varies significantly after 14 d on MEA at 25 °C, with Z. alba colonies (8–9 mm) more restricted than those of Z. obscura Notes: See notes under Z. alba. (12–14 mm). Also, Z. obscura colonies darken much faster than those of Z. alba. Microscopically these species are very similar. Additional material examined: Canada: Saskatchewan: Regina, isol. In general, however, spores of Z. alba seem more slender. exhouse dust, 21 Aug. 2007, E. Whitfield & K. Mwange (DAOMC 250850). – USA: California: Berkeley, isol. ex house dust, 31 Mar. Additional material examined: Canada: Saskatchewan: Regina, isol. 2005, A. Amend, E. Whitfield & K. Mwange (DAOMC 250851, Ex house dust, 21 Aug. 2007, E. Whitfield & K. Mwange (DAOMC 250852). 250848 – culture).
Zalaria obscura Visagie, Z. Humphries & Seifert, sp. DISCUSSION nov. MycoBank MB821630 In this paper, we describe a novel lineage comprising six (Fig. 5) black yeast-like strains isolated from house dust collected in Canada and the USA as a new genus of Dothideales, Etymology: Latin obscura, dark, in reference to colony Zalaria. Mature agar colonies become dark and leathery, but appearance after 7 d of growth. are covered with slimy masses of conidia or yeast-like cells. The conidiogenous cells are undifferentiated and usually Diagnosis: Differs from Z. alba in the ability to grow at lowered intercalary, with blastic conidiogenesis occurring on 1–2 loci aw. Colonies dark brown to black after about 7 d. Conidia per cell, giving rise to aseptate, smooth-walled, ellipsoidal appearing less slender than Z. alba. ITS barcode: KX579094. to lemon-shaped conidia, that commonly bud in a polar, Alternative identification markers: 28S rDNA: KX579100, bipolar or multilateral pattern; dark brown, rough-walled RPB2: KX579106, 18S rDNA: KX579112, BenA: KX579118 chlamydospores are often seen. Strains were resolved at the species level into two clades, described as Z. alba and Type: Canada: Saskatchewan: Regina, isol. ex house dust, Z. obscura, with strains of the latter growing faster and with 21 Aug. 2007, E. Whitfield & K. Mwange (DAOM 734002 – colonies darkening within 7 d. No sexual morph is known for holotype; DAOMC 250849 – ex-type culture). either species.
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Fig. 5. Morphological characters of Zalaria obscura (DAOMC 250849 in A, B, D, F, G, J, K; DAOMC 250852 in C, H, I; DAOMC 250850 in E). A. Colonies on MEA after 2 wk. B. Close-up colony on MEA after 4 wk. C. Germinating conidia. D. Germinating conidia with age. E. Melanized hyphae developing into chlamydospores. F–H. Intercalary conidiogenous cells. I. Chlamydospores. J–K. Conidia with some yeast-like budding occurring. Bars = 10 µm.
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Fig. 6. A–C. Aureobasidium pullulans (KAS 5840). A. Melanized hyphae/chlamydospores. B. Conidiogenous cells with multiple loci. C. Conidia. D–G. Aureobasidium melanogenum (KAS 1951). D. Dark brown conidia. E–F. Conidiogenous cells with multiple loci. G. Conidia. H–K. Sydowia polyspora (DAOMC 251471). H. Melanized hyphae/chlamydospores. I–J. Hormonema-like conidiogenous cells with 1–2 loci. K. Conidia. Bars = 10 µm except A and H = 50 µm.
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Fig. 7. A–E. Hortaea werneckii (DAOMC 251499). A. Yeast-like cells with sympodial producing daughter cells. B–C. Yeast-like cell with annellations. D–E. Conidiogenous apparatus. F–I. Rhizosphaera pini (DAOMC 251499). F–H. Hormonema-like conidiogenous cells. I. Conidia. Bars = 10 µm.
312 IMA FUNGUS A new family, genus, and species from house dust
Because of the black yeast asexual morphology, Zalaria is glacier, it could be hard to imagine how an organism so ARTICLE difficult to distinguish from Aureobasidium and Hormonema. specifically adapted could out-compete other life-forms in Conidiogenesis has been used to differentiate the latter more hospitable climates. However, arctic fungal species two genera, but does not consistently provide accurate seem to have very effective dispersal strategies over long identification unless combined with growth rates and distances (Geml 2011). Combined with Zalaria’s phenotypic physiological characters such as carbohydrate assimilation plasticity, melanisation and the halotolerance of Z. obscura (De Hoog & Yurlova 1994, Loncaric et al. 2009, Yurlova et (similar to that in Aureobasidium), these species may be al. 1996). The most reliable morphological character for capable of widespread dispersal and also survive in or on distinguishing these genera is the number of loci present on many substrates. conidiogenous cells. Species of Zalaria and Hormonema have Before our study, the only way to identify and communicate only 1–2 loci per cell, whereas Aureobasidium has up to 14 information on strains, clones or Zalaria OTU’s was by means (De Hoog &Yurlova 1994, Yurlova et al. 1999). Distinguishing of UNITE’s species hypotheses (Kõljalg et al. 2013), i.e. Zalaria and Hormonema based only on conidiogenesis, based on 0 % threshold, SH377734.07FU represents Zalaria however, is nearly impossible. Sexual morph characters are alba and SH377739.07FU represents Z. obscurum. With used to distinguish Dothidea, Pringsheimia and Sydowia formal names now available to these species hypotheses, (Thambugala et al. 2014) with similar asexual morphs, but no communicating information about these fungi and studying sexual morph has been observed in Zalaria. the extent of their distribution, habitats and possible ecological Although the two species described here can be confidently roles will be much easier. interpreted as representing a new genus, our decision to propose a new family Zalariaceae is less satisfying. Our phylogenetic analyses (Figs 1–3) consistently resolved Zalaria ACKNOWLEDGEMENTS strains as distinct from Aureobasidiaceae and Dothideaceae. Working within the framework and concepts adopted for the This research was supported by grants from the Alfred P. Sloan order (Thambugala et al. 2014), we could either synonymise Foundation Program on the Microbiology of the Built Environment. all current families or introduce a new family; we chose the We would like to acknowledge Ed Whitfield and Kalima Mwange latter approach. Our proposal of a new family on primarily who made all isolations from house dust samples. We also thank phylogenetic grounds, in the absence of presumably more everybody who collected house dust used during this project. informative characters of so-far unknown sexual morphs, perpetuates but does not add to the vagueness of phenotypic characters underlying the phylogeny. However, we hope REFERENCES that increased sampling, especially of sexually reproducing species across Dothideomycetes, of unsequenced but Abdel-Hafez S, Mohawed S, El-Said A (1989) Seasonal fluctuations known and unknown taxa, will reveal morphological or other of soil fungi of Wadi Qena at eastern desert of Egypt. 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