A new family and genus in Dothideales for Aureobasidium -like species isolated from house dust

Abstract

An international survey of house dust collected from eleven countries using a modified dilution-to-extinction method yielded 7904 isolates. Of these, six strains morphologically resembled the asexual morphs of Aureobasidium and Hormonema (sexual morphs ?Sydowia), but were phylogenetically distinct. A 28S rDNA phylogeny resolved strains as a distinct clade in Dothideales with families Aureobasidiaceae and Dothideaceae their closest relatives. Further analyses based on the ITS rDNA region, β-tubulin, 28S rDNA, and RNA polymerase II second largest subunit confirmed the distinct status of this clade and divided strains among two consistent subclades. As a result, we introduce a new genus and two new species as Zalariaalba and Z. obscura, and a new family to accommodate them in Dothideales. Zalaria is a black yeast-like fungus, grows restrictedly and produces conidiogenous cells with holoblastic synchronous or percurrent conidiation. Zalaria microscopically closely resembles Hormonema by having only one to two loci per conidiogenous cell, but species of our new genus generally has more restricted growth. Comparing the two species, Z. obscura grows faster on lower water activity (aw) media and produces much darker colonies than Z. alba after 7 d. Their sexual states, if extant, are unknown.

Full text

ARTICLE
299
© 2017 International Mycological Association
You are free to share - to copy, distribute and transmit the work, under the following conditions:
Attribution:  Youmustattributetheworkinthemannerspeciedbytheauthororlicensor(butnotinanywaythatsuggeststhattheyendorseyouoryouruseofthework).
Non-commercial: Youmaynotusethisworkforcommercialpurposes.
No derivative works: Youmaynotalter,transform,orbuilduponthiswork.
For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get
permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights.
VOLUME 8 · NO. 2
INTRODUCTION
The average person in industrialized countries spends
approximately 90 % of their time indoors (Höppe & Martinac
1998). This makes the indoor environment one of the most
important human-fungal interfaces. We are constantly exposed
to fungal spores, fragments, and metabolites and their impact
ranges from human health (Piecková & Jesenská 1999) as
pathogens (De Hoog et al. 2014, Garber 2001) or allergens
(Aimanianda et al. 2009, Karvala et al. 2011, Tanno et al. 2016)
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 (aw) coupled with high
temperatures (Zalar et al. 2011), and building materials like
plaster, drywall, and cement have very low aw (Flannigan &
Miller 2011), while plastics like polyvinyl chloride (PVC, used
in construction) offer little in the way of available carbon but
still support oligotrophic fungi (Webb et al. 2000). Studying the
indoor mycobiota is therefore important to better understand
these interactions and how they may affect us.
The black yeast Aureobasidium pullulans (Dothideales) is
recorded from a wide variety of sources, including environments
with signicant osmotic stress, such as hypersaline waters
in salterns (Gunde-Cimerman et al. 2000), bathrooms, food,
and feeds (Samson et al. 2010), water-damaged wood
(Andersen et al. 2011), and polythermal glaciers (Zalar et
al. 2008). In surveys of the indoor environment, A. pullulans
is one of the most abundant and widespread fungi reported
(Adams et al. 2013, Amend et al. 2010, Nonneman et al.
2012, Van Nieuwenhuijzen et al. 2016). The morphospecies
exhibits a high degree of phenotypic plasticity (Slepecky &
Starmer 2009) and strains can have signicantly different
pigmentation (Yurlova et al. 1995, Zalar et al. 2008). While
this high degree of variation may contribute to its unique
adaptability (Gostinčar et al. 2014), it also makes denitive
morphological identication challenging, and the many ITS
variants identied as this species in GenBank are unlikely to
represent one species. Reports of A. pullulans being one of
the most abundant members in fungal communities based
on near-neighbour analyses of next-generation sequencing
(NGS) data may be skewed to some extent.
The class Dothideomycetes is the largest in Ascomycota
and was recently examined and re-dened using multigene
phylogenetics (Hyde et al. 2013, Schoch et al. 2009,
Thambugala et al. 2014). These studies showed the order
Dothideales to be a monophyletic sister to Myriangiales.
doi:10.5598/imafungus.2017.08.02.05 IMA FUNGUS · 8(2): 299–315 (2017)
A new family and genus in Dothideales for Aureobasidium-like species
isolated from house dust
Zoë Humphries1, Keith A. Seifert1,2, Yuuri Hirooka3, and Cobus M. Visagie1,2,4
1Biodiversity (Mycology), Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON,
Canada, K1A 0C6
2Department of Biology, University of Ottawa, 30 Marie-Curie, Ottawa, ON, Canada, K1N 6N5
3Department of Clinical Plant Science, Faculty of Bioscience, Hosei University, 3-7-2 Kajino-cho, Koganei, Tokyo, Japan
4Biosystematics Division, ARC-Plant Health and Protection, P/BagX134, Queenswood 0121, Pretoria, South Africa; corresponding author e-mail:
visagiec@arc.agric.za
Abstract: An international survey of house dust collected from eleven countries using a modied dilution-to-extinction
method yielded 7904 isolates. Of these, six strains morphologically resembled the asexual morphs of Aureobasidium
and Hormonema (sexual morphs ?Sydowia), but were phylogenetically distinct. A 28S rDNA phylogeny resolved
strains as a distinct clade in Dothideales with families Aureobasidiaceae and Dothideaceae their closest relatives.
Further analyses based on the ITS rDNA region, β-tubulin, 28S rDNA, and RNA polymerase II second largest subunit
conrmed the distinct status of this clade and divided strains among two consistent subclades. As a result, we
introduce a new genus and two new species as Zalaria alba and Z. obscura, and a new family to accommodate them in
Dothideales. Zalaria is a black yeast-like fungus, grows restrictedly and produces conidiogenous cells with holoblastic
synchronous or percurrent conidiation. Zalaria microscopically closely resembles Hormonema by having only one to
two loci per conidiogenous cell, but species of our new genus generally has more restricted growth. Comparing the two
species, Z. obscura grows faster on lower water activity (aw) media and produces much darker colonies than Z. alba
after 7 d. Their sexual states, if extant, are unknown.
Article info: Submitted: 5 July 2017; Accepted: 10 October 2017; Published: 26 October 2017.
Key words:
18S
28S
BenA
black yeast
Dothidiomycetes
ITS
RPB2
xerotolerant fungi
Zalaria

Humphries et al.
ARTICLE
300 IMA FUNGUS
Thambugala et al. (2014) reviewed and re-evaluated the
morphologically-based taxonomy of Dothideales known from
culture, informed by a combined phylogeny of 28S rDNA, 18S
rDNA and ITS. They accepted two families, synonymising
the often-accepted Dothioraceae (e.g. Barr 2001) with
Dothideaceae, as rst proposed by Von Arx & Müller (1975),
and introducing Aureobasidiaceae for Aureobasidium and
closely related genera. The polythetic morphological denitions
provided by Thambugala et al. (2014) did not identify unique
diagnostic characters among the sexual or asexual morphs in
either family. Both families include a poorly integrated mixture
of genera known from fungarium specimens with others that
are mostly known from culture. For both families, sexually and
asexually typied genera were keyed out separately. Sexually
typied genera were separated by characters of the stromata,
number of ascospores per ascus, and ascospore characters
such as septation and pigmentation. In Dothideales, the
characters normally used to classify asexual morphs were
clearly phylogenetically uninformative, and mixtures of black
yeast, hyphomycetous, coelomycetous, and intermediate
asexual morphs are scattered over the various clades of
Aureobasidiaceae and Dothideaceae. Both families include a
variety of asexual morphs, including sporodochial hyphomycete
forms usually observed in nature (e.g. Kabatiella, Kabatina),
coelomycete (e.g. Endoconidioma, Neocylindroseptoria,
Rhizosphaera), and black yeast-like forms usually observed
in culture.
Black yeasts have a confused taxonomic history that may
eventually be claried with the single name classication
system, but presently remains difcult to navigate. Black
yeasts are a phylogenetically diverse morphogroup of
asexual morphs, mostly in Dothideales or Chaetothyriales,
which produce dark, slimy colonies and at least some
budding yeast-like cells in culture. Many species have one
or more hyphal asexual morphs in addition to the yeast-like
forms. This pleiomorphy complicates their identication and
taxonomic interpretation (De Hoog & Hermanides-Nijhof
1977). Two of the most frequently reported black yeast genera
are Aureobasidium (Aureobasidiaceae) and Hormonema
(Dothideaceae). Aureobasidium was associated with several
sexually typied genera in Dothideales, and although no sexual
morph is denitively known for the most frequently reported
species A. pullulans, an ascospore-derived strain identied
as Columnosphaeria fagi (CBS 171.93), has identical ITS and
RPB2 sequences to the clade including the ex-type culture of
A. pullulans. The similar asexually typied genus Hormonema,
(H. dematioides type), generally considered the asexual morph
of Sydowia polyspora, is also frequently reported. A single
name solution for this clade is not yet proposed and we use the
name Hormonema for comparisons of asexual morphotypes.
Despite the differences in associated sexual morphs,
Aureobasidium and Hormonema are difcult to distinguish
morphologically when grown in culture. For example, neither
Hermanides-Nijhof (1977) nor De Hoog & Yurlova (1994)
could nd morphological differences among asexual morphs
in cultures of the sexually typied genera Pringsheimia,
Dothidea, Dothiora or Sydowia, all of which they attributed
to Hormonema. Aureobasidium and Hormonema were
considered distinct in the dual nomenclature era, because of
the different sexual morphs. Hermanides-Nijhof (1977) dened
Aureobasidium by the production of synchronous blastoconidia
from undifferentiated, hyaline cells, whereas Hormonema was
said to produce conidia in basipetal succession from hyaline
or dark hyphae. Wang & Zabel (1990) suggested that at least
some conidiogenous cells of H. dematioides were phialidic or
percurrent. In a later review, Aureobasidium was distinguished
based on its conidiogenous cells having multiple loci
(synchronous conidiogenesis), in contrast to one or two loci
in Hormonema (De Hoog & Yurlova 1994). These characters
are difcult to detect, and are best observed along hyphae at
the growing margin of the colony. Colonies of both morphs
often begin as palely pigmented growths, which become
slimy and almost black as the colonies mature. The different
patterns and apparent plasticity of conidiogenesis, including
yeast-like cells, young blastospores, swollen blastospores,
chlamydospores, and septation and constrictions of hyphae
confound interpretations of homologous characters (Guterman
& Shabtai 1996, Zalar et al. 2008).
During our survey of fungi isolated from house dust
using a dilution-to-extinction approach, many isolates
morphologically resembled Aureobasidium and Hormonema,
but six were phylogenetically distinct. Here we introduce these
as two new species, in a new genus and family in Dothideales.
We present a 28S rDNA (nuclear large ribosomal subunit)
phylogeny of Dothidiomycetes to determine the strains’
phylogenetic position and subsequently present phylogenies
of Dothideales based on BenA (β-tubulin), ITS rDNA, 28S
rDNA, 18S rDNA (nuclear small ribosomal subunit), and RPB2
(RNA polymerase II second largest subunit), to determine the
relationships within the order. Strains were characterized
morphologically and compared to morphologically similar
species and genera. This work follows previous reports of
new taxa of indoor fungi discovered by dilution-to-extinction,
including species of Rasamsonia (Tanney & Seifert 2013),
Aspergillus, Penicillium and Talaromyces (Visagie et al.
2014, 2017, Sklenář et al. 2017), Wallemia (Jancic et al.
2015, Nguyen et al. 2015), Spiromastix, Pseudospiromastix,
Sigleria (Hirooka et al. 2016), and Myrmecridium (Crous et
al. 2016).
MATERIALS AND METHODS
Isolations
Settled house dust was collected from twelve countries
(Australia, Canada, Federated States of Micronesia,
Indonesia, Mexico, The Netherlands, New Zealand,
South Africa, Thailand, the United Kingdom, Uruguay,
and USA) using sterilized Duststream® collectors (Indoor
Biotechnologies, Charlottesville, VA) attached to vacuum
cleaners. Isolations were made from malt extract agar (MEA)
and MEA with 20 % sucrose using a dilution-to-extinction
method modied from Collado et al. (2007) as described
in Visagie et al. (2014). More recent isolations targeting
xerophilic fungi from Canadian and Hawaiian house dust
were made as described in Visagie et al. (2017).
Living strains of new species are deposited in the Canadian
Collection of Fungal Cultures (DAOMC, Ottawa, Canada), the
Westerdijk Fungal Biodiversity Institute (CBS, Utrecht, The
Netherlands) and dried specimens are accessioned in the

A new family, genus, and species from house dust
ARTICLE
301
VOLUME 8 · NO. 2
Table 1. Information on house dust isolates included in this study.
Species Strain number Isolation medium Origin CollectoraDate collected Isolatorb
Aureobasidium melanogenum SLOAN 1260 = AA07MX-884 MEA Mexico, Nayarit, Sayulita A. Amend 31 Jan. 2009 E. Whiteld & K. Mwange
Aureobasidium melanogenum SLOAN 1606 = BH02AU-110 MEA Australia, Tasmania, Hobart B. Horton 10 Feb. 2009 E. Whiteld & K. Mwange
Aureobasidium melanogenum SLOAN 5623 = TA10NZ-214a MEA New Zealand, Wellington,
Wellington
T. Atkinson 3 May 2009 E. Whiteld & K. Mwange
Aureobasidium melanogenum KAS 5840 MY50G Canada, Ontario, Stittsville K.A. Seifert 20 Dec. 2014 C.M. Visagie
Aureobasidium melanogenum KAS 7917 MY1012 USA, Hawaii, Kailua A. Amend 11 May 2015 C.M. Visagie
Aureobasidium melanogenum KAS 7956 DG18 USA, Hawaii, Kailua A. Amend 11 May 2015 C.M. Visagie
Aureobasidium melanogenum SLOAN 7256 = AA04US-587 20%S-MEA USA A. Amend unknown E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 7261 = AA02US-306 20%S-MEA USA, California, Berkeley A. Amend 31 Mar. 2005 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 203 = 7050035.79-65 n.a. Canada, Saskatchewan, Regina Health Canada 12 Mar. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 207 = 7050035.79-71 n.a. Canada, Saskatchewan, Regina Health Canada 12 Mar. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 48 = 7050035.79-127 20%S-MEA Canada, Saskatchewan, Regina Health Canada 12 Mar. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 62 = 7050035.79-148 20%S-MEA Canada, Saskatchewan, Regina Health Canada 12 Mar. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 349 = 7330009.33-45 20%S-MEA Canada, Saskatchewan, Regina Health Canada 21 Aug. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 359 = 7330009.33-60 20%S-MEA Canada, Saskatchewan, Regina Health Canada 21 Aug. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 7249 = 7330009.34-925 n.a. Canada, Saskatchewan, Regina Health Canada 21 Aug. 2007 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 1652 = BH02AU-154a 20%S-MEA Australia, Tasmania, Hobart B. Horton 10 Feb. 2009 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 1653 = BH02AU-154b 20%S-MEA Australia, Tasmania, Hobart B. Horton 10 Feb. 2009 E. Whiteld & K. Mwange
Aureobasidium pullulans SLOAN 3214 = KJ09SA-65 MEA South Africa, Western Cape,
Kuilsrivier
K. Jacobs 24 Jul. 2009 E. Whiteld & K. Mwange
Aureobasidium pullulans KAS 5951 DG18 Canada, British Columbia, Victoria B. Kendrick 27 Jan. 2015 C.M. Visagie
Aureobasidium species SLOAN 41 = 7050035.79-119 MEA Canada, Saskatchewan, Regina Health Canada 12 Mar. 2007 E. Whiteld & K. Mwange
Aureobasidium subglaciale SLOAN 7263 = AA02US-332 20%S-MEA USA, California, Berkeley A. Amend 31 Mar. 2005 E. Whiteld & K. Mwange
Hortaea werneckii KAS 7942 MY50G USA, Hawaii, Kailua A. Amend 11 May 2015 C.M. Visagie
Hortaea werneckii KAS 7947 MY50G USA, Hawaii, Kailua A. Amend 11 May 2015 C.M. Visagie
Hortaea werneckii KAS 7949 MY1012 USA, Hawaii, Kailua A. Amend 11 May 2015 C.M. Visagie
Hortaea werneckii KAS 7953 MY1012 USA, Hawaii, Kailua A. Amend 11 May 2015 C.M. Visagie
Rhizosphaera pini DAOMC 251499 = KAS 6309 MY50G Canada, New Brusnwick, Little
Lepreau
A. Walker 29 Jan. 2015 C.M. Visagie
Sydowia polyspora DAOMC 251470 = KAS 5918 DG18 Canada, British Columbia, Victoria B. Kendrick 27 Jan. 2015 C.M. Visagie
Sydowia polyspora DAOMC 251471 = KAS 5919 DG18 Canada, British Columbia, Victoria B. Kendrick 27 Jan. 2015 C.M. Visagie
Sydowia polyspora DAOMC 251469 = KAS 5999 DG18 Canada, New Brusnwick, Little
Lepreau
A. Walker 29 Jan. 2015 C.M. Visagie
Zalaria alba DAOMC 250847 = SLOAN 52 =
7050035-79-132
20%S-MEA Canada, Saskatchewan, Regina Health Canada 12 Mar. 2007 E. Whiteld & K. Mwange

Humphries et al.
ARTICLE
302 IMA FUNGUS
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
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
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).
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
were made from colonies grown on MEA and DG18, using lactic acid as
mounting uid. An Olympus BX50 compound microscope attached with an
InnityX camera powered by Innity Analyze v. 6.5.1 software (Lumenera,
Ottawa, ON) was used for microscopic observations, capturing images and
making measurements. Photographic plates were prepared in Afnity Photo
v. 1.5.2 (https://afnity.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 amplied 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-
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 amplication
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
(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 amplication, 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.
Phylogenetic analyses
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
included on trees.
Table 1. (Continued).
Species Strain number Isolation medium Origin CollectoraDate collected Isolatorb
Zalaria alba DAOMC 250848 = SLOAN 352 =
7330009.33-5
MEA Canada, Saskatchewan, Regina Health Canada 21 Aug. 2007 E. Whiteld & K. Mwange
Zalaria obscurum DAOMC 250851 = SLOAN 7266 =
AA02US-340
20%S-MEA USA, California, Berkeley A. Amend 31 Mar. 2005 E. Whiteld & K. Mwange
Zalaria obscurum DAOMC 250852 = SLOAN 7674 =
AA02US-351
20%S-MEA USA, California, Berkeley A. Amend 31 Mar. 2005 E. Whiteld & K. Mwange
Zalaria obscurum DAOMC 250849 = SLOAN 7244 =
7330009.34-884
n.a. Canada, Saskatchewan, Regina Health Canada 21 Aug. 2007 E. Whiteld & K. Mwange
Zalaria obscurum DAOMC 250850 = SLOAN 7246 =
7330009.34-921
n.a. Canada, Saskatchewan, Regina Health Canada 21 Aug. 2007 E. Whiteld & K. Mwange
a Collector of house dust sample; b isolator of strain using the modied dilution to exctinction method.

A new family, genus, and species from house dust
ARTICLE
303
VOLUME 8 · NO. 2
Datasets were aligned using MAFFT v. 7.017 (Katoh & Standley
2013) with the L-INS-i algorithm. For the BenA dataset, G-INS-i
was used. Alignments were manually trimmed in Geneious. The
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
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/
gtree) 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
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
focused phylogenies based on 18S rDNA, 28S rDNA, BenA, ITS,
and RPB2, including only closely related species and genera from
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,
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
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 identied here as Aureobasidium pullulans, A.
melanogenum, A. subglaciale, an undescribed Aureobasidium
species (DTO 285-D8), Rhizosphaera pini, Sydowia polyspora,
and Hortaea werneckii (Capnodiales).
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,
China, Greece, The Netherlands, Portugal, Spain, Thailand, and
Table 2. BLAST results showing origin of strains or clones belonging to Zalaria.
Gene GenBank nr Original identication Current identication Strain/clone Origin and source Publication
ITS KM103983 Uncultured fungus Zalaria cf sp2 Rcw 47 USA; Red-cockaded woodpecker excavation in living pine Unpublished
ITS KJ130063 Aureobasidium sp. Zalaria cf sp2 C 21 China, Henan, Nanyang City, Baotianman Natural Reserve Area;
forest soil
Unpublished
ITS KF800358 Uncultured fungus Zalaria cf sp2 CMH 267 USA, Missouri, Kansas City; House dust Rittenour et al. (2014)
ITS KC253970 Aureobasidium pullulans Zalaria cf sp2 UOA/HCPF ENV57A Greece; hospital incubator Unpublished
ITS FJ235939 Fungal sp. Zalaria sp1 AB 6 Antarctica; wood Unpublished
ITS AF121282 Aureobasidium pullulans Zalaria sp2 ATCC 16628 The Netherlands; Soil Unpublished
LSU AB456556 Aureobasidium sp. Zalaria cf sp2 WB 27 Thailand, Prachuap Khiri Khan, Pran Buri Forest Park; sediment
in mangrove forest
Unpublished
LSU AY167611 Aureobasidium sp. Zalaria cf sp2 CECT 11965 Spain?; cork samples Álvarez-Rodríguez et al. (2003)
LSU KJ130062 Aureobasidium sp. Zalaria cf sp2 C 21 China, Henan, Nanyang City, Baotianman Natural Reserve Area;
forest soil
Unpublished
LSU JN004186 Aureobasidium sp. Zalaria cf sp2 SP 9 Portugal; Sound grapes Unpublished
LSU KC433818 Aureobasidium sp. Zalaria cf sp2 DBVPG 5996 Italy, Mont Blanc, Miage glacier; supraglacial sediments Turchetti et al. (2013)

Humphries et al.
ARTICLE
304 IMA FUNGUS
Antarctica and from habitats including house dust, cork
samples, grapes, a hospital incubator, sediment, soil, wood,
and woodpecker excavations. Our Zalaria isolates were
obtained from house dust collected in the USA (CA) and
Canada (Regina, SK).
Morphology
Strains were characterized morphologically on several
agar media and shared several characters with species of
Aureobasidium and Hormonema. As noted in the Introduction,
the only way to reliably distinguish between these groups
Myriangiales
Myriangiaceae
Elsinoaceae
insertae sedis
Zalariaceae
Dothideaceae
Dothideales
Aureobasideaceae
Natipusillales
Microthyrium microscopicum CBS115976 (GU301846)
Delphinella strobiligena CBS735.71 (DQ470977)
Schismatomma decolorans DUKE47570 (AY548815)
Myriangium hispanicum CBS247.33 (GU301854)
Venturiales clade 2
Zalaria obscura DAOMC250849T (KX579100)
Zalaria alba DAOMC250848 (KX579103)
Endosporium aviarium UAMH10530 (EU304351)
Kirschteiniotheliaceae
Aureobasidium caulivorum CBS242.64 (EU167576)
Elsinoe phaseoli CBS165.31 (DQ678095)
Zalaria alba DAOMC250847T (KX579099)
Capnodiales
Zalaria obscura DAOMC250850 (KX579098)
Elsinoe centrolobi CBS222.50 (DQ678094)
Endosporium populi-tremuloidis UAMH10529 (EU304348)
Aureobasidium pullulans CBS584.75 (DQ470956)
Dothidea sambuci DAOM231303 (AY544681)
Catinella olivacea UAMH10679 (EF622212)
Neomicrothyrium siamense IFRDCC2194 (JQ036228)
Lichenoconium
Tubeufiales | Venturiales clade 1
Zalaria obscura DAOMC250851 (KX579101)
Phaeocryptopus nudus CBS268.37 (GU301856)
Acrospermales
Dothidea insculpta CBS189.58 (DQ247802)
Stylodothis puccinioides CBS193.58 (AY004342)
Columnosphaeria fagi CBS171.93 (AY016359)
Jahnulales
Asterinales
Trypetheliales | Lichenotheliaceae
Zalaria obscura DAOMC250852 (KX579102)
Hysteriales | Strigulales
Dothidea hippophaes CBS188.58 (DQ678048)
Dothiora cannabinae CBS737.71 (DQ470984)
Phaeosclera dematioides CBS157.81 (GU301858)
Mytilinidiales | Gloniaceae
Micropeltis zingiberacicola IFRDCC2264 (JQ036227)
Monoblastiales
Elsinoe veneta CBS150.27 (DQ767658)
Dothiora elliptica CBS736.71 (GU301811)
Phaeotrichales
Myriangium duriaei CBS260.36 (DQ678059)
Sydowia polyspora CBS116.29 (DQ678058)
Pleosporales
Botryosphaeriales
0.2
*
88
*
98
*
98
*
83
99
*
*
98
*
97
*
87
93
*
86
*
97
88
*
*
97
*
98
98
*
*
86
x2
x2
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.

A new family, genus, and species from house dust
ARTICLE
305
VOLUME 8 · NO. 2
0.04
Dothiora ceratoniae CBS477.69 (KF251655)
Hormonema viticola FMR13040 (KF201298)
Dothichiza pityophila CBS215.50 (FJ150968)
Dothiora oleae CBS235.57 (KU728548)
Endosporium aviarium UAMH10530 (EU304351)
Saccothecium species MFLUCC14.1171 (KU290336)
Zalarium obscura CBS122359 (FJ150966)
Dothiora sorbi CBS742.71 (KU728552)
Aureobasidium caulivorum CBS242.64 (FJ150944)
Dothiora oleae CBS152.71 (KU728547)
Dothidea ribesia CBS195.58 (AY016360)
Lineolata rhizophorae CBS641.66 (GU479792)
Hormonema viticola L9D.11 (KF201297)
Zalaria obscura DAOMC250852 (KX579102)
Aureobasidium melanogenum CBS105.22 (FJ150926)
Aureobasidium pullulans CBS146.30 (FJ150916)
Coniozyma leucospermi CBS111289 (EU552113)
Elsinoe phaseoli CBS165.31 (DQ678095)
Hortaea werneckii CBS107.67 (EU019270)
Aureobasidium microstictum CBS342.66 (FJ150945)
Dothiora oleae CBS472.69 (KU728549)
Pringsheimia smilacis CBS873.71 (FJ150970)
Dothiora laureolae CBS744.71 (KU728542)
Aureobasidium subglaciale CBS123388 (FJ150935)
Dothiora phillyreae CBS473.69 (EU754146)
Aureobasidium subglaciale CBS123387 (FJ150913)
Aureobasidium melanogenum CBS110374 (FJ150929)
Sydowia polyspora CBS116.29 (DQ678058)
Neocylindroseptoria pistaciae CBS471.69 (KF251656)
Dothiora maculans CBS299.76 (KU728543)
Endosporium populitremuloidis UAMH10529 (EU304348)
Myriangium hispanicum CBS247.33 (GU301854)
Zalaria obscura DAOMC250851 (KX579101)
Atramixtia arboricola UAMH11211 (HM347777)
Dothidea berberidis CBS187.58 (KC800752)
Pseudoseptoria collariana CBS135104 (KF251721)
Zalarium obscura EXF922 (FJ150964)
Dothidea sambuci CBS198.58 (AF382387)
Sphaceloma asclepiadis CPC18532 (JN940380)
Dothidea ribesia MFLU14.004 (KM388552)
Endoconidioma populi UAMH10902 (HM185488)
Dothiora ceratoniae CBS441.72 (KU728540)
Dothiora agapanthi CPC20600 (KU728537)
Myriangium duriaei CBS260.36 (DQ678059)
Neohortaea acidophila CBS113389 (GU323202)
Dothidea ribesia MFLUCC13.0670 (KM388553)
Dothidea insculpta CBS189.58 (DQ247802)
Dothidea sambuci DAOM231303 (AY544681)
Dothiora cannabinae CBS737.71 (DQ470984)
Aureobasidium pullulans CBS701.76 (FJ150951)
Dothiora prunorum CBS933.72 (KU728551)
Dothiora ceratoniae CBS290.72 (KU728539)
Dothiora bupleuricola CBS112.75 (KU728538)
Stylodothis puccinioides CBS193.58 Dothideales (AY004342)
Dothiora maculans CBS686.70 (KU728546)
Zalaria obscura DAOMC250849 (KX579100)
Selenophoma linicola CBS468.48 (EU754212)
Aureobasidium microstictum CBS114.64 (FJ150940)
Selenophoma australiensis CBS124776 (GQ303324)
Celosporium larixicol L3.1 (FJ997288)
Aureobasidium proteae CBS114273 (JN712557)
Rhizosphaera pini DAOMC251499 (KY654326)
Plowrightia periclymeni 178096 (FJ215702)
Dothiora maculans CBS301.76 (KU728544)
Dothiora viburnicola CBS274.72 (KU728554)
Zalarium obscura EXF1936 (FJ150965)
Zalaria alba DAOMC250847 (KX579099)
Zalarium obscura CBS122362 (FJ150963)
Aureobasidium pullulans CBS584.75 (FJ150942)
Sphaceloma arachidis CPC18533 (JN940374)
Plowrightia abietis ATCC24339 (EF114703)
Aureobasidium namibiae CBS147.97 (FJ150937)
Aureobasidium leucospermi CBS130593 (JN712555)
Phaeocryptopus gaeumannii CBS267.37 (EF114698)
Saccothecium sepincola CBS278.32 (GU301870)
Dothidea hippophaeos CBS188.58 (DQ678048)
Pseudoseptoria obscura CBS135103 (KF251722)
Zalarium obscura CBS122350 (FJ150961)
Delphinella strobiligena CBS735.71 (DQ470977)
Zalaria alba DAOMC250848 (KX579103)
Pseudosydowia eucalypti CPC14927 (GQ303328)
Aureobasidium lini CBS125.21 (FJ150946)
Sphaceloma bidentis CPC18526 (JN940379)
Selenophoma mahoniae CBS388.92 (EU754213)
Zalarium obscura EXF1934 (FJ150962)
Phaeocryptopus nudus CBS268.37 (EF114700)
Aureobasidium proteae CBS111973 (JN712558)
Aureobasidium pullulans CBS100280 (FJ150941)
Saccothecium sepincola MFLUCC14.0276 (KM388554)
Phaeosclera dematioides UAMH4265 (EU981288)
Coleophoma oleae CBS615.72 (EU754148)
Pseudosydowia eucalypti CPC14028 (GQ303327)
Sydowia polyspora CBS750.71 (FJ150912)
Dothiora phaeosperma CBS870.71 (KU728550)
Hortaea thailandica CPC16651 (GU214429)
Sphaceloma erythrinae CPC18530 (JN940392)
Zalaria obscura DAOMC250850 (KX579098)
Dothiora elliptica CBS736.71 (GU301811)
Aureobasidium melanogenum CBS123.37 (FJ150917)
Columnosphaeria fagi CBS171.93 (AY016359)
Metasphaeria species M11.1 (KF590163)
0.99/-
*/94
*/82
-/89
*/96
0.99/-
*/99
*/98
*/*
*/86
0.98/81
*/87
*/86
*/*
*/*
*/86
*/*
*/*
*/99
*/*
*/96
*/87
*/98 */*
*/89
0.98/-
*/*
0.99/98
*/93
*/91
0.99/90
0.97/-
*/*
0.97/-
x4
x4
x4
x4
x4
x2
0.1
Sydowia japonica FFPRI411088 (JQ814699)
Aureobasidium pullulans SLOAN7249 (KY659525)
Aureobasidium melanogenum KAS7956 (KY659511)
Dothidea ribesia MFLU14.004 (KM388544)
Rhizosphaera kalkhoffii HMBF-CHN2 (JQ353722)
Aureobasidium pullulans SLOAN207 (KY659517)
Aureobasidium subglaciale CBS123388 (KT693736)
Hortaea werneckii KAS7947 (KY659508)
Aureobasidium pullulans CBS584.75 (KT693733)
Endosporium aviarium UAMH10530 Myriangiales (EU304350)
Neohortaea acidophila CBS113389 (GU214636)
Kabatina juniperi CBS239.66 (AY616211)
Kabatina thujae CBS238.66 (AF013226)
Hormonema prunorum CBS933.72 (AY616213)
Aureobasidium melanogenum SLOAN1260 (KY659512)
Rhizosphaera kalkhoffii HMBF-CHN1 (JQ353721)
Dothiora oleae CBS472.69 (KU728510)
Aureobasidium pullulans KAS5951 (KY659504)
Pringsheimia euphorbiae CBS747.71 (AJ244276)
Hortaea werneckii CBS107.67 (AJ238468)
Hormonema carpetanum TRN25 (AY616205)
Hormonema carpetanum TRN278 (AY616199)
Hortaea werneckii KAS7949 (KY659509)
Rhizosphaera macrospora CBS208.79 (GB)
Sphaceloma erythrinae CPC18530 Myriangiales (JN943502)
Aureobasidium melanogenum KAS7917 (KY659506)
Endoconidioma populi UAMH10902 (HM185487)
Aureobasidium melanogenum CBS105.22 (KT693729)
Aureobasidium pullulans SLOAN62 (KY659524)
Rhizosphaera pini CBS206.79 (EU700370)
Dothidea ribesia MFLUCC13.0670 (KM388545)
Dothiora maculans CBS301.76 (KU728505)
Pseudosydowia eucalypti CPC14028 (GQ303296)
Rhizosphaera pini ATCC46387 (AY183365)
Kabatina juniperi CBS466.66 (AY616212)
Dothiora europaea CBS739.71 (AJ244244)
Atramixtia arboricola UAMH11211 (HM347778)
Sydowia polyspora DAOMC251470 (KY659502)
Hormonema dematioides D817.03.98 (AJ278929)
Aureobasidium namibiae CBS147.97 (KT693730)
Dothiora oleae CBS235.57 (KU728509)
Dothiora ceratoniae CBS441.72 (KU728501)
Dothiora sorbi CBS742.71 (KU728514)
Dothidea insculpta CBS189.58 (AF027764)
Aureobasidium melanogenum CBS110374 (KT693728)
Zalaria obscura DAOMC250850 (KX579092)
Aureobasidium pullulans SLOAN48 (KY659522)
Hormonema macrosporum CBS536.94 (AJ244247)
Dothidea sambuci DAOM231303 (DQ491505)
Aureobasidium microstictum CBS114.64 (KT693744)
Dothiora phaeosperma CBS870.71 (KU728512)
Zalaria alba DAOMC250848 (KX579097)
Aureobasidium species DTO285D8 (KT693673)
Pseudosydowia eucalypti CPC14927 (GQ303297)
Celosporium larixicol L3.1 (FJ997287)
Coniozyma leucospermi CBS111289 (EU552113)
Zalaria obscura DAOMC250851 (KX579095)
Aureobasidium pullulans CBS701.76 (FJ150907)
Sydowia polyspora CBS248.93 (GQ412724)
Dothiora prunorum CBS933.72 (AJ244248)
Dothichiza pityophila CBS215.50 (AJ244242)
Aureobasidium species DTO301G9 (KT693694)
Sydowia japonica FFPRI411085 (JQ814701)
Hortaea werneckii KAS7953 (KY659510)
Rhizosphaera kobayashii ATCC46389 (AF462432)
Aureobasidium microstictum CBS342.66 (KT693743)
Sydowia polyspora CBS128.64 (AJ244262)
Rhizosphaera oudemansii CBS226.83 (EU700366)
Dothiora cannabinae CBS737.71 (AJ244243)
Sphaceloma bidentis CPC18526 Myriangiales (JN943491)
Endoconidioma populi UAMH10297 (AY604526)
Rhizosphaera pini DAOMC251499 (KY659500)
Rhizosphaera kalkhoffii CBS280.38 (EU700375)
Endoconidioma populi UAMH10298 (AY604527)
Dothiora viburnicola CBS274.72 (KU728515)
Sphaceloma arachidis CPC18533 Myriangiales (JN943485)
Hormonema dematioides E419.05.98 (AJ278930)
Hormonema carpetanum TRN40 (AY616201)
Sydowia polyspora CBS544.95 (AY152548)
Sydowia japonica FFPRI411087 (JQ814702)
Hormonema carpetanum TRN31 (AY616206)
Zalaria obscura DAOMC250852 (KX579096)
Aureobasidium pullulans SLOAN1652 (KY659514)
Phaeocryptopus nudus CBS268.37 (EU700371)
Aureobasidium subglaciale
SLOAN7263 (KY659528)
Selenophoma eucalypti STEU659 (AY293059)
Saccothecium species MFLUCC14.1171 (KU290338)
Rhizosphaera pseudotsugae CBS101222 (EU700369)
Rhizosphaera oudemansii CBS427.82 (EU700367)
Aureobasidium melanogenum CBS123.37 (FJ150881)
Dothiora agapanthi CPC20600 (KU728498)
Rhizosphaera macrospora ATCC4636 (AF462431)
Hormonema viticola FMR13040 (KP641179)
Sydowia polyspora CBS750.71 (KT693747)
Hortaea werneckii KAS7942 (KY659507)
Dothiora maculans CBS686.70 (KU728507)
Aureobasidium melanogenum SLOAN5623 (KY659523)
Aureobasidium caulivorum CBS242.64 (KT693740)
Kabatiella harpospora CBS122914 (KT693741)
Aureobasidium pullulans CBS100280 (KT693734)
Dothiora ceratoniae CBS477.69 (KF251151)
Hormonema carpetanum TRN201 (AY616204)
Zalaria obscura DAOMC250849 (KX579094)
Phaeocryptopus gaeumannii CBS267.37 (EF114685)
Rhizosphaera kalkhoffii HAF27 (KM435342)
Rhizosphaera macrospora CBS467.82 (EU700368)
Aureobasidium species DTO302H2 (KT693725)
Dothiora rhamni-alpinae CBS745.71 (AJ244245)
Sphaceloma asclepiadis CPC18532 Myriangiales (JN943493)
Dothidea hippophaeos CBS186.58 (AF027763)
Aureobasidium pullulans SLOAN203 (KY659516)
Sydowia japonica FFPRI411084 (JQ814700)
Aureobasidium pullulans SLOAN7261 (KY659527)
Endoconidioma populi UAMH10903 (HM185489)
Dothiora phillyreae CBS473.69 (KU728513)
Aureobasidium pullulans SLOAN349 (KY659519)
Aureobasidium thailandense NRRL58543 (JX462675)
Rhizosphaera kalkhoffii CBS114656 (EU700376)
Aureobasidium pullulans CBS146.30 (FJ150902)
Aureobasidium proteae CBS111973 (KT693732)
Hormonema dematioides C107.07.98 (AJ278927)
Dothiora elliptica CBS736.71 (KU728502)
Endosporium populitremuloidis UAMH10529 Myriangiales (EU304347)
Hormonema dematioides B106 07 98 (AJ278926)
Dothiora oleae CBS152.71 (KU728508)
Aureobasidium thailandense NRRL58539 (JX462674)
Phaeocryptopus gaeumannii CBS244.38 (EU700364)
Sydowia polyspora DAOMC251471 (KY659503)
Dothiora bupleuricola CBS112.75 (KU728499)
Pringsheimia smilacis CBS873.71 Dothideales (AJ244257)
Hormonema carpetanum TRN24 (AY616202)
Kabatiella microsticta CBS342.66 (KT693743)
Aureobasidium subglaciale CBS123387 (KT693735)
Aureobasidium proteae CBS114273 (KT693731)
Aureobasidium species SLOAN41 (KY659521)
Dothidea sambuci CBS198.58 (AY930108)
Dothiora ceratoniae CBS290.72 (KU728500)
Pseudoseptoria obscura CBS135103 (KF251219)
Dothiora maculans CBS299.76 (KU728504)
Scleroconidioma sphagnicola UAMH9731 (AY220610)
Neocylindroseptoria pistaciae CBS471.69 (KF251152)
Aureobasidium pullulans SLOAN1653 (KY659515)
Aureobasidium pullulans SLOAN359 (KY659520)
Dothiora laureolae CBS744.71 (KU728503)
Selenophoma mahoniae CBS388.92 (KT693746)
Hortaea thailandica CPC16651 (GU214637)
Aureobasidium melanogenum SLOAN1606 (KY659513)
Hortaea werneckii CBS111.31 (AJ238679)
Aureobasidium melanogenum SLOAN7256 (KY659526)
Hormonema carpetanum ATCC74360 (AF182375)
Hormonema dematioides E99156 (AY253451)
Hormonema viticola L9D.11 (KF201300)
Aureobasidium leucospermi CBS130593 (KT693727)
Zalaria alba DAOMC250847 (KX579093)
Pseudoseptoria collariana CBS135104 (KF251218)
Columnosphaeria fagi CBS171.93 (KT693737)
Aureobasidium melanogenum KAS5840 (KY659501)
Aureobasidium pullulans SLOAN3214 (KY659518)
Aureobasidium lini CBS125.21 (KT693742)
Hormonema dematioides HDDFMI (AF462439)
Kabatiella bupleuri CBS131303 (KT693739)
Kabatiella bupleuri CBS131304 (KT693738)
Sydowia polyspora DAOMC251469 (KY659505)
Selenophoma australiensis CBS124776 (GQ303293)
Endoconidioma populi UAMH10299 (HM185486)
Hormonema viticola L9D.17 (KP641179)
Hormonema dematioides D106.07.98 (AJ278928)
0.95/-
*/-
*/*
*/-
0.99/-
0.99/93
-/93
*/-
*/80
*/-
0.99/-
*/90
0.99/-
*/-
*/99
0.96/-
0.99/-
*/82
*/99
*/98
*/85
*/85
0.99/81
0.93/87
*/*
*/*
*/96
-/*
0.95/-
0.98/-
*/-
*/98
*/*
*/-
*/-
*/*
0.98/-
0.99/*
0.99/89
*/96
*/*
*/*
*/*
*/99
*/*
*/*
*/*
*/*
*/-
*/88
-/99
0.97/84
*/84
0.97/-
x4
x2
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.

Humphries et al.
ARTICLE
306 IMA FUNGUS
0.08
Dothiora phillyreae CBS473.69 (KU728629)
Phaeocryptopus nudus CBS268.37 (EU747283)
Dothiora maculans CBS301.76 (KU728622)
Rhizosphaera kalkhoffii CBS280.38 (EU747273)
Rhizosphaera oudemansii CBS427.82 (EU747279)
Aureobasidium subglaciale CBS123387 (FJ157878)
Aureobasidium melanogenum CBS123.37 (FJ157852)
Zalaria obscura DAOMC250849 (KX579118)
Rhizosphaera kalkhoffii CBS114656 (EU747274)
Dothiora oleae CBS152.71 (KU728625)
Aureobasidium microstictum CBS342.66 (FJ157872)
Rhizosphaera pini DAOMC251499 (KY659498)
Rhizosphaera pini CBS206.79 (EU747282)
Dothiora ceratoniae CBS290.72 (KU728619)
Rhizosphaera pseudotsugae CBS101222 (EU747281)
Aureobasidium lini CBS125.21 (FJ157873)
Dothiora ceratoniae CBS441.72 (KU728620)
Dothiora maculans CBS686.70 (KU728624)
Aureobasidium melanogenum CBS105.22 (FJ157858)
Zalaria obscura DAOMC250850 (KX579116)
Phaeocryptopus gaeumannii CBS244.38 (EU747284)
Dothiora oleae CBS235.57 (KU728626)
Dothiora agapanthi CPC20600 (KU728617)
Rhizosphaera macrospora CBS467.82 (EU747280)
Rhizosphaera oudemansii CBS226.83 (EU747278)
Aureobasidium namibiae CBS147.97 (FJ157863)
Dothiora maculans CBS299.76 (KU728621)
Aureobasidium pullulans CBS100280 (FJ157864)
Aureobasidium pullulans CBS146.30 (FJ157871)
Selenophoma mahoniae CBS388.92 (FJ157874)
Aureobasidium thailandense NRRL58539 (EU719407)
Zalaria alba DAOMC250848 (KX579121)
Zalaria obscura DAOMC250851 (KX579119)
Zalaria alba DAOMC250847 (KX579117)
Zalaria obscura DAOMC250852 (KX579120)
Dothiora oleae CBS472.69 (KU728627)
Aureobasidium pullulans CBS584.75 (FJ157869)
Aureobasidium pullulans CBS701.76 (FJ157865)
Aureobasidium subglaciale CBS123388 (FJ157877)
*/-
0.98/-
-/94
*/*
*/99
0.95/-
-/85
*/90
0.98/-
0.99/-
*/97
0.99/96
*/96
*/*
*/91
*/*
*/*
*/*
0.99/90
*/87
*/*
0.005
Dothidea hippophaeos CBS186.58 (EU167601)
Phaeocryptopus nudus CBS268.37 (EF114724)
Stylodothis puccinioides CBS193.58 (AY016353)
Columnosphaeria fagi CBS171.93 (AY016342)
Plowrightia abietis ATCC24339 (EF114727)
Hortaea werneckii CBS107.67 (Y18693)
Dothidea sambuci DAOM231303 (AY544722)
Dothiora phillyreae CBS473.69 (EU754047)
Saccothecium sepincola CBS278.32 (GU296195)
Zalaria obscura DAOMC250851 (KX579113)
Dothidea ribesia MFLUCC13.0670 (KM388550)
Sphaceloma bidentis CPC18526 (JN940555)
Zalaria alba DAOMC250848 (KX579115)
Sphaceloma erythrinae CPC18530 (JN940566)
Zalaria obscura DAOMC250852 (KX579114)
Zalaria obscura DAOMC250849 (KX579112)
Selenophoma mahoniae CBS388.92 (EU754114)
Sydowia polyspora CBS116.29 (DQ678005)
Dothidea insculpta CBS189.58 (DQ247810)
Myriangium hispanicum
CBS247.33 (GU296180)
Selenophoma linicola CBS468.48 (EU754113)
Hortaea thailandica CPC16651 (JN938703)
Phaeocryptopus gaeumannii CBS267.37 (EF114722)
Zalaria obscura DAOMC250850 (KX579110)
Aureobasidium caulivorum CBS242.64 (EU167576)
Lineolata rhizophorae CBS641.66 (GU479758)
Delphinella strobiligena CBS735.71 (DQ471029)
Zalaria alba DAOMC250847 (KX579111)
Aureobasidium lini CBS125.21 (EU707925)
Saccothecium species MFLUCC14.1171 (KU290337)
Sphaceloma asclepiadis CPC18532 (JN940556)
Myriangium duriaei CBS260.36 (AY016347)
Endosporium aviarium UAMH10530 (EU304349)
Dothidea ribesia MFLU14.004 (KM388549)
Dothidea ribesia CBS195.58 (AY538346)
Coleophoma oleae CBS615.72 (EU754049)
Dothiora prunorum CBS933.72 (KU728551)
Aureobasidium pullulans CBS584.75 (EU682922)
Dothiora cannabinae CBS737.71 (DQ479933)
Hormonema prunorum CBS933.72 (EU707926)
Plowrightia periclymeni 178096 (FJ215709)
Sphaceloma arachidis CPC18533 (JN940548)
Elsinoe phaseoli CBS165.31 (DQ678042)
Endosporium populitremuloidis UAMH10529 (EU304346)
*/91
*/85
*/98
*/82
*/90
*/90
0.98/-
*/-
0.96/-
*/99
0.96/-
0.97/-
0.95/94
*/98
x4
x4
0.2
Dothiora cannabinae CBS737.71 (DQ470936)
Sydowia polyspora CBS750.71 (KT693988)
Myriangium hispanicum CBS247.33 (GU371744)
Zalaria alba DAOMC250848 (KX579109)
Aureobasidium melanogenum CBS110374 (KT693971)
Aureobasidium lini CBS125.21 (KT693984)
Selenophoma mahoniae CBS388.92 (KT693987)
Aureobasidium microstictum CBS114.64 (KT693986)
Saccothecium sepincola CBS278.32 (GU371745)
Aureobasidium proteae CBS114273 (KT693974)
Zalaria obscura DAOMC250852 (KX579108)
Zalaria obscura DAOMC250851 (KX579107)
Aureobasidium subglaciale CBS123388 (KT693979)
Dothidea sambuci CBS198.58 (KT216559)
Kabatiella bupleuri CBS131304 (KT693981)
Kabatiella bupleuri CBS131303 (KT693982)
Elsinoe phaseoli CBS165.31 (KT216560)
Aureobasidium microstictum CBS342.66 (KT693985)
Hortaea thailandica CPC16651 (KF902206)
Aureobasidium pullulans CBS100280 (KT693977)
Columnosphaeria fagi CBS171.93 (KT693980)
Aureobasidium leucospermi CBS130593 (KT693970)
Delphinella strobiligena CBS735.71 (DQ677951)
Myriangium duriaei CBS260.36 (KT216528)
Lineolata rhizophorae CBS641.66 (GU479828)
Dothidea sambuci DAOM231303 (DQ522854)
Aureobasidium caulivorum CBS242.64 (KT693983)
Aureobasidium namibiae CBS147.97 (KT693973)
Aureobasidium subglaciale CBS123387 (KT693978)
Aureobasidium pullulans CBS584.75 (DQ470906)
Sydowia polyspora CBS116.29 (DQ677953)
Aureobasidium proteae CBS111973 (KT693975)
Zalaria alba DAOMC250847 (KX579105)
Aureobasidium melanogenum CBS105.22 (KT693972)
Phaeocryptopus gaeumannii CBS244.38 (GU371740)
Aureobasidium thailandense NRRL58539 (EU719566)
Dothidea insculpta CBS189.58 (DQ247792)
Dothidea hippophaeos CBS188.58 (DQ677942)
Zalaria obscura DAOMC250850 (KX579104)
Zalaria obscura DAOMC250849 (KX579106)
Neohortaea acidophila CBS113389 (KT216521)
*/89
*/*
*/98
0.99/-
*/*
*/*
*/*
*/*
*/95
0.99/-
*/99
*/96
*/*
*/*
0.98/-
*/89
*/-
*/-
*/80
*/*
*/*
*/99
*/*
x4
x4
Lorem ipsum
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.

A new family, genus, and species from house dust
ARTICLE
307
VOLUME 8 · NO. 2
are the 1–2 conidiogenous loci per cell in Hormonema (and
asexual morphs of many other Dothideaceae species), and
up to 14 loci in Aureobasidium (De Hoog & Yurlova 1994,
Yurlova et al. 1999). The new genus is more similar in this
regard to Hormonema, with only 1–2 loci per conidiogenous
cell. In general, growth of Zalaria species is more restricted
than in any of these other genera.
Three types of conidiogenesis were observed in the
Zalaria strains. Their yeast forms are very common, especially
in younger colonies when cells reproduce by budding. After
prolonged incubation, these yeast cells are often covered
by melanized hyphal growth; microscopic observations
suggest that hyphae from germinating yeast cells eventually
give rise to this dark melanized growth (Figs 4C–D, 5C–D).
With age, these hyphae may develop into dark brown, thick-
walled chlamydospores (Figs 4E, I, 5E, I). Further, intercalary
conidiogenous cells develop mostly at margins of young
colonies.
The strains resolved as distinct clades observed in the
phylogenies were also distinct morphologically, with Z. alba
compared to Z. obscura growing more restrictedly on most
media. Zalaria obscura is also capable of growth at low aw
media such as MEA-10%-NaCl, MY-1012 and MY50G,
with Z. alba not growing on these media. Generally, Z. alba
colonies are also more yeast-like and take up to 3 wk to
darken, whereas Z. obscura colonies darken in 7 d and are
often covered by a leathery layer within 14 d. This character
was originally used for distinguishing the two varieties of
A. pullulans var. pullulans and var. melanogenum, later
recognized as two distinct species (Gostinčar et al 2014).
Based on both phylogenetic and morphological results
we introduce a new family, genus and two new species to
accommodate our unique black yeast-like fungi.
Morphological examinations conrmed sequence-based
identications of the remaining house dust isolates (Figs
6–7). Aureobasidium strains produced the typical conidiog-
enous cells with multiple loci (Fig. 6A–G), while only 1–2
conidiogenous loci were observed in strains identied as
Sydowia polyspora (Fig. 6H–K). Hortaea werneckii was com-
mon in Hawaiian dust samples and was only isolated from
the halophilic MY10-12 medium. The typical pigmented hy-
phae, yeast-like growth, sympodial and percurrent conidio-
genesis were observed in newly isolated strains (Fig. 7A–E).
The strain identied as Rhizosphaera pini produced colonies
with pycnidium-like structures and a Hormonema-like morph
producing very large conidia, all characteristic of that species
(Fig. 7F–I).
Taxonomy
Zalariaceae Visagie, Z. Humphries & Seifert, fam. nov.
MycoBank MB821627
Type genus: Zalaria Visagie et al. 2017.
Diagnosis: Distinguished from other families classied in
Dothideales and Myriangiales based on a short unique 28S
rDNA sequence anked by two conserved regions. The
section dening Zalariaceae in our alignment (Treebase ID
19764) are found between nucleotide positions 39 to 62 and
is indicated in bold text (5’-AGCTCAAATTTGAAATCTGGCC-
CTTTC-AGGGTCCGAGTTGTAATTTGTAGAGG-3’).
Zalaria Visagie, Z. Humphries & Seifert, gen. nov.
MycoBank MB821628
Etymology: Named in honour of Polona Zalar, mycologist at
the University of Ljubljana, Slovenia, in recognition of her
studies on extremophilic fungi, including her 2008 study that
included strains from Norwegian arctic regions that belong to
this genus.
Diagnosis: Differs from Aureobasidium by blastic conidiogen-
esis occurring from one to two loci per conidiogenous cell.
Morphologically Zalaria is indistinguishable from Hormonema
(often reported as asexual morphs of Sydowia), leaving DNA
sequences the only diagnostic character (see Diagnosis for
family Zalariaceae above).
Type species: Zalaria obscura Visagie et al. 2017
Description: Sexual morph unknown. Colonies often covered
in slimy masses of conidia or yeast-like cells, becoming
dark and often leathery with time, occasionally with sparse
aerial mycelium; cream-colored, red-brown, olive-brown,
dark brown, or black; margins entire or mbriate. Hyphae
transversely and longitudinally septate, hyaline and thin-
walled when young, frequently becoming melanized and
thick-walled with age, may develop into chlamydospores.
Conidiogenous cells undifferentiated, intercalary, terminal
uncommon, cylindrical, with blastic conidiogenesis occurring
from one to two loci per cell. Chlamydospores dark brown,
smooth to lightly rough-walled, globose to ellipsoidal, septate.
Conidia often yeast-like, hyaline, aseptate, smooth-walled,
ellipsoidal to lemon-shaped, variable in size, indistinct hilum,
budding common, polar, bipolar and multilateral.
Zalaria alba Visagie, Z. Humphries & Seifert, sp. nov.
MycoBank MB821629
(Fig. 4)
Etymology: Latin, alba, meaning white, in reference to colony
appearance after 7 d of growth.
Diagnosis: Differs from Z. obscura in the inability to grow
at lowered aw. Colonies remain yellowish to orange-white
until it darkens after about 3 wk. Conidia appearing more
slender than Z. obscura. ITS barcode: KX579093. Alternative
identication markers: 28S rDNA: KX579099, RPB2:
KX579105, 18S rDNA: KX579111, BenA: KX579117.
Type: Canada: Saskatchewan: Regina, isol. ex house dust,
12 Mar. 2007, E. Whiteld & K. Mwange (DAOM 734001 –
holotype; DAOMC 250847 – ex-type culture).
Description: Colony diameters (mm after 7 d (14 d at 25 °C)):
MEA 5–6 (8–9); MEA 5 °C microcolonies, 10 °C microcolonies,
30 °C 2–5, 35 °C 1–2, 40 °C microcolonies; MEA-5 %-NaCl
no growth, MEA-10 %-NaCl no growth, MEA-15 %-NaCl no
growth, MEA-24 %-NaCl no growth; OA 6–7 (11–14), PDA

Humphries et al.
ARTICLE
308 IMA FUNGUS
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.

A new family, genus, and species from house dust
ARTICLE
309
VOLUME 8 · NO. 2
7–8 (10–11), DG18 3–4 (5–7), MY1012 no growth, MY50G
no growth, SNA 3–5 (7–8).
Cultural characters: Colonies on MEA at 25 °C after 7 d
yeast-like, smooth and slimy, obverse yellowish white to
orange white (4A2–5A2), reverse greenish grey to pale
orange (1B2–5A3), generally becoming dark within 3 wk, with
dark areas sometimes present after 7 d, olive-yellow to dark
brown (3D6-7F5), some aerial mycelium developing after
longer incubation.
Microscopic characters: Young somatic hyphae at colony
periphery mostly hyaline, smooth, thin-walled, branched,
transversely septate, 1.5–5 µm diam; older hyphae
towards colony centre melanized, dark brown, smooth to
lightly roughened, thick-walled, branched, transversely
and longi-septate, 2–6.5 µm diam, often developing into
chlamydospores. Conidiogenous cells undifferentiated,
intercalary, rarely terminal, mostly on hyaline hyphae,
producing conidia percurrently from short lateral denticles
not exceeding 2 µm long. Chlamydospores dark brown,
smooth to lightly rough-walled, globose to ellipsoidal, septate
to aseptate, one-septate spores sometimes constricted at
septum, 5.5–10 × 3–7.5 µm (x
̄ = 8 ± 0.99 µm; 5.5 ± 0.71 µm).
Conidia often yeast-like, hyaline, aseptate, smooth walled,
ellipsoidal to lemon-shaped, variable in size, 2.5–10 × 1.5–5
µm (x
̄ = 5.5 ± 1.53 µm, 3 ± 0.73 µm), with an indistinct hilum,
budding common, polar, bipolar and multilateral.
Notes: Growth on media with lowered aw distinguishes between
the two Zalaria species. Zalaria alba does not grow on MEA-5
%-NaCl, MY10-12 or MY50G after 14 d. In contrast, Z. obscura
produces at least microcolonies on these media. Colony size
varies signicantly after 14 d on MEA at 25 °C, with Z. alba
colonies (8–9 mm) more restricted than those of Z. obscura
(12–14 mm). Also, Z. obscura colonies darken much faster than
those of Z. alba. Microscopically these species are very similar.
In general, however, spores of Z. alba seem more slender.
Additional material examined: Canada: Saskatchewan: Regina, isol.
Ex house dust, 21 Aug. 2007, E. Whiteld & K. Mwange (DAOMC
250848 – culture).
Zalaria obscura Visagie, Z. Humphries & Seifert, sp.
nov.
MycoBank MB821630
(Fig. 5)
Etymology: Latin obscura, dark, in reference to colony
appearance after 7 d of growth.
Diagnosis: Differs from Z. alba in the ability to grow at lowered
aw. Colonies dark brown to black after about 7 d. Conidia
appearing less slender than Z. alba. ITS barcode: KX579094.
Alternative identication markers: 28S rDNA: KX579100,
RPB2: KX579106, 18S rDNA: KX579112, BenA: KX579118
Type: Canada: Saskatchewan: Regina, isol. ex house dust,
21 Aug. 2007, E. Whiteld & K. Mwange (DAOM 734002 –
holotype; DAOMC 250849 – ex-type culture).
Description: Colony diameters (mm after 7 d, (14 d) at 25
°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;
MEA-5 %-NaCl 2–4 (3–5), MEA-10 %-NaCl microcolonies,
MEA-15 %-NaCl no growth, MEA-24 %-NaCl no growth; OA
7–8 (15–16), PDA 7–9 (10–14), DG18 3–5 (6–8), MY1012 no
growth, sometimes microcolonies after prolonged incubation,
MY50G no growth (microcolonies), SNA 4–5 (8–9).
Cultural characteristics: Colonies on MEA at 25 °C after 7 d
yeast-like, smooth and slimy, obverse dark brown (7F5) to
black with some yellowish white to orange-white (4A2–5A2),
olive-yellow (3D6), and olive (1E5) areas, surface leathery
after 14 d; some aerial mycelium developing after prolonged
incubation.
Microscopic characters: Young somatic hyphae at colony
periphery mostly hyaline, smooth, thin-walled, branched,
transversely septate, 1.5–4.5 µm diam; older hyphae
towards colony centre melanized, dark brown, smooth to
lightly roughened, thick-walled, branched, transversely
and longi-septate, 2–11 µm diam, often developing into
chlamydospores. Conidiogenous cells undifferentiated,
intercalary, rarely terminal, mostly on hyaline hyphae,
producing conidia percurrently from short lateral denticles
not exceeding 2 µm long. Chlamydospores dark brown,
smooth to lightly rough walled, globose to ellipsoidal, septate
to aseptate, one-septate spores sometimes constricted at
septum, 5–17 µm × 3.5–8 µm (x
̄ = 8 ± 2.11 µm; 6 ± 0.89 µm).
Conidia often yeast-like, hyaline, aseptate, smooth-walled,
ellipsoidal to lemon-shaped, variable in size, 2.5–10 × 1.5–
5.5 µm (x
̄ = 5 ± 1.4 µm; 3.5 ± 0.77 µm), with an indistinct
hilum, budding common, polar, bipolar and multilateral.
Notes: See notes under Z. alba.
Additional material examined: Canada: Saskatchewan: Regina, isol.
exhouse dust, 21 Aug. 2007, E. Whiteld & K. Mwange (DAOMC
250850). – USA: California: Berkeley, isol. ex house dust, 31 Mar.
2005, A. Amend, E. Whiteld & K. Mwange (DAOMC 250851,
250852).
DISCUSSION
In this paper, we describe a novel lineage comprising six
black yeast-like strains isolated from house dust collected
in Canada and the USA as a new genus of Dothideales,
Zalaria. Mature agar colonies become dark and leathery, but
are covered with slimy masses of conidia or yeast-like cells.
The conidiogenous cells are undifferentiated and usually
intercalary, with blastic conidiogenesis occurring on 1–2 loci
per cell, giving rise to aseptate, smooth-walled, ellipsoidal
to lemon-shaped conidia, that commonly bud in a polar,
bipolar or multilateral pattern; dark brown, rough-walled
chlamydospores are often seen. Strains were resolved at
the species level into two clades, described as Z. alba and
Z. obscura, with strains of the latter growing faster and with
colonies darkening within 7 d. No sexual morph is known for
either species.

Humphries et al.
ARTICLE
310 IMA FUNGUS
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.

A new family, genus, and species from house dust
ARTICLE
311
VOLUME 8 · NO. 2
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.

Humphries et al.
ARTICLE
312 IMA FUNGUS
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.

A new family, genus, and species from house dust
ARTICLE
313
VOLUME 8 · NO. 2
Because of the black yeast asexual morphology, Zalaria is
difcult to distinguish from Aureobasidium and Hormonema.
Conidiogenesis has been used to differentiate the latter
two genera, but does not consistently provide accurate
identication unless combined with growth rates and
physiological characters such as carbohydrate assimilation
(De Hoog & Yurlova 1994, Loncaric et al. 2009, Yurlova et
al. 1996). The most reliable morphological character for
distinguishing these genera is the number of loci present on
conidiogenous cells. Species of Zalaria and Hormonema have
only 1–2 loci per cell, whereas Aureobasidium has up to 14
(De Hoog &Yurlova 1994, Yurlova et al. 1999). Distinguishing
Zalaria and Hormonema based only on conidiogenesis,
however, is nearly impossible. Sexual morph characters are
used to distinguish Dothidea, Pringsheimia and Sydowia
(Thambugala et al. 2014) with similar asexual morphs, but no
sexual morph has been observed in Zalaria.
Although the two species described here can be condently
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
strains as distinct from Aureobasidiaceae and Dothideaceae.
Working within the framework and concepts adopted for the
order (Thambugala et al. 2014), we could either synonymise
all current families or introduce a new family; we chose the
latter approach. Our proposal of a new family on primarily
phylogenetic grounds, in the absence of presumably more
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
that increased sampling, especially of sexually reproducing
species across Dothideomycetes, of unsequenced but
known and unknown taxa, will reveal morphological or other
phenotypic characters that are predictive of phylogeny,
resulting in stable family and genus concepts in Dothideales.
BLAST results with Zalaria strains (Table 2) recovered 11
sequences that we consider belong to Zalaria, but that were
originally identied as Aureobasidium sp. or A. pullulans.
These strains or clones originated from the USA, China,
Thailand, Greece, The Netherlands, Portugal, Spain, and
Antarctica. Strains placed by Zalar et al. (2008) in their group
5 are identied here as Z. obscura and originate from the
Norwegian arctic region. Furthermore, a BLAST search of
454 pyrosequencing data generated during our house dust
project (Amend et al. 2010), revealed 21 sequences belonging
to Zalaria in dust collected from Australia, Canada, New
Zealand, South Africa, and the USA. Zalaria seems to have a
truly worldwide distribution, and occurs on many substrates,
from wood, soil, dust, sediments, cork, and subglacial ice.
Understanding its ecology will be very challenging. Zalar et al.
(2008) suggested that, given the highly selective conditions
of the environment, their then new group 5 might be restricted
to areas like Kongsfjorden in Norway. Arctic environments
possess low aw because ice formation removes most of the
available water, while aw is lowered further as solute ions are
expelled during the freezing process (Gunde-Cimerman et
al. 2003). This lack of available water is a dominating factor
in microbial life in arctic regions (Gunde-Cimerman et al.
2003) and favours the growth of xerotolerant and xerophilic
fungi. Given the extreme environment of a polythermal
glacier, it could be hard to imagine how an organism so
specically adapted could out-compete other life-forms in
more hospitable climates. However, arctic fungal species
seem to have very effective dispersal strategies over long
distances (Geml 2011). Combined with Zalaria’s phenotypic
plasticity, melanisation and the halotolerance of Z. obscura
(similar to that in Aureobasidium), these species may be
capable of widespread dispersal and also survive in or on
many substrates.
Before our study, the only way to identify and communicate
information on strains, clones or Zalaria OTU’s was by means
of UNITE’s species hypotheses (Kõljalg et al. 2013), i.e.
based on 0 % threshold, SH377734.07FU represents Zalaria
alba and SH377739.07FU represents Z. obscurum. With
formal names now available to these species hypotheses,
communicating information about these fungi and studying
the extent of their distribution, habitats and possible ecological
roles will be much easier.
ACKNOWLEDGEMENTS
This research was supported by grants from the Alfred P. Sloan
Foundation Program on the Microbiology of the Built Environment.
We would like to acknowledge Ed Whiteld and Kalima Mwange
who made all isolations from house dust samples. We also thank
everybody who collected house dust used during this project.
REFERENCES
Abdel-Hafez S, Mohawed S, El-Said A (1989) Seasonal uctuations
of soil fungi of Wadi Qena at eastern desert of Egypt. Acta
Mycologica 25: 113–125.
Adams RI, Miletto M, Taylor JW, Bruns TD (2013) Dispersal in
microbes: fungi in indoor air are dominated by outdoor air and
show dispersal limitation at short distances. The ISME Journal
7: 1262–1273.
Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, et al.
(2009) Surface hydrophobin prevents immune recognition of
airborne fungal spores. Nature 460: 1117–1121.
Akaike H (1974) A new look at the statistical model identication.
IEEE Transactions on Automatic Control 19: 716–723.
Álvarez-Rodríguez ML, Belloch C, Villa M, Uruburu F, Larriba G,
Coque J-JR (2003) Degradation of vanillic acid and production of
guaiacol by microorganisms isolated from cork samples. FEMS
Microbiology Letters 220: 49–55.
Amend AS, Seifert KA, Samson RA, Bruns TD (2010) Indoor fungal
composition is geographically patterned and more diverse in
temperate zones than in the tropics. Proceedings of the National
Academy of Sciences, USA 107: 13748–13753.
Andersen B, Frisvad JC, Søndergaard I, Rasmussen IS, Larsen LS
(2011) Associations between fungal species and water-damaged
building materials. Applied and Environmental Microbiology 77:
4180–4188.
Arenz BE (2010) Fungi in Antarctica: a circumpolar study of
biodiversity in soils and historic structures. PhD, thesis, University
of Minnesota.
Barata A, Santos SC, Malfeito-Ferreira M, Loureiro V (2012) New
insights into the ecological interaction between grape berry

Humphries et al.
ARTICLE
314 IMA FUNGUS
microorganisms and Drosophila ies during the development of
sour rot. Microbial Ecology 64: 416–430.
Barr ME (2001) Revisionary studies on the Dothioraceae. Harvard
Papers in Botany 6: 25–34.
Blakeslee AF (1915) Lindner’s roll tube method of separation
cultures. Phytopathology 5: 68–69.
Boonmak C (2013) Diversity of yeast in water and sediment from
mangrove forest in the upper coast of the gulf of Thailand. MSc
this, Kasetsart University, Thailand.
Collado J, Platas G, Paulus B, Bills GF (2007) High-throughput
culturing of fungi from plant litter by a dilution-to-extinction
technique. FEMS Microbiology Ecology 60: 521–533.
Crous PW, Wingeld MJ, Burgess TI, Hardy GESJ, Crane C, et al.
(2016) Fungal Planet description sheets: 469-557. Persoonia 37:
218–403.
De Hoog GS, Guarro J, Gene J, Figueras MJ (2014) Atlas of Clinical
Fungi. 4th edn. Utrecht: CBS-KNAW Fungal Biodiversity Centre.
De Hoog GS, Hermanides-Nijhof EJ (1977) The black yeasts and
allied hyphomycetes. Studies in Mycology 15: 1–140.
De Hoog GS, Yurlova NA (1994) Conidiogenesis, nutritional
physiology and taxonomy of Aureobasidium and Hormonema.
Antonie van Leeuwenhoek 65: 41–54.
Dmitriev V, Gilichinski D, Faizutdinova R, Shershunov I, Golubev W,
Duda V (1997) Occurrence of viable yeasts in 3-million-year-old
permafrost in Siberia. Mikrobiologiya 66: 655–660.
Dodman RL, Reinke JR (1982) A selective medium for determining
the population of viable conidia of Cochliobolus sativus in soil.
Australian Journal of Agricultural Research 33: 287–291.
Flannigan B, Miller JD (2011) Microbial growth in indoor environments.
In: Microorganisms in Home and Indoor Work Environments:
diversity, health impacts, investigation and control (Flannigan B,
Samson RA, Miller JD, eds): 57–108. London: CRC Press.
Garber G (2001) An overview of fungal infections. Drugs 61 (Suppl.
1): 1–12.
Geml J (2011) Coalescent analyses reveal contrasting patterns of
intercontinental gene ow in arctic-alpine and boreal-temperate
fungi. In: Biogeography of Microscopic Organisms: is everything
small everywhere? (Fontaneto D, ed.): 177–190. Cambridge:
Cambridge University Press.
Gerrits van den Ende AH, De Hoog GS (1999) Variability and
molecular diagnostics of the neurotropic species. Studies in
Mycology 43: 151–162.
Glass NL, Donaldson GC (1995) Development of premier sets
designed for use with the PCR to amplify conserved genes
from lamentous ascomycetes. Applied and Environmental
Microbiology 61: 1323–1330.
Gostinčar C, Ohm RA, Kogej T, Sonjak S, Turk M, et al. (2014)
Genome sequencing of four Aureobasidium pullulans varieties:
biotechnological potential, stress tolerance, and description of
new species. BMC Genomics 15: 549.
Gunde-Cimerman N, Sonjak S, Zalar P, Frisvad J, Diderichsen
B, Plemenitaš A (2003) Extremophilic fungi in arctic ice: a
relationship between adaptation to low temperature and water
activity. Physics and Chemistry of the Earth 28: 1273–1278.
Gunde-Cimerman N, Zalar P, De Hoog S, Plemenitaš A (2000)
Hypersaline waters in salterns - natural ecological niches for
halophilic black yeasts. FEMS Microbiology Ecology 32: 235–
240.
Guterman H, Shabtai Y (1996) A self-tuning vision system for
monitoring biotechnological processes: I. Application to
production of pullulan by Aureobasidium pullulans. Biotechnology
and Bioengineering 51: 501–510.
Hermanides-Nijhof EJ (1977) Aureobasidium and allied genera.
Studies in Mycology 15: 141–177.
Hirooka Y, Tanney JB, Nguyen HD, Seifert KA (2016) Xerotolerant
fungi in house dust: taxonomy of Spiromastix, Pseudospiromastix
and Sigleria gen. nov. in Spiromastigaceae (Onygenales,
Eurotiomycetes). Mycologia 108: 135–156.
Hocking AD, Pitt JI (1980) Dichloran-glycerol medium for
enumeration of xerophilic fungi from low-moisture foods. Applied
and Environmental Microbiology 39: 488–492.
Höppe P, Martinac I (1998) Indoor climate and air quality. Review
of current and future topics in the eld of ISB study group 10.
International Journal of Biometeorology 42: 1–7.
Hyde KD, Jones EBG, Liu J-K, Ariyawansa, Hiran, et al. (2013)
Families of Dothideomycetes. Fungal Diversity 63: 1–313.
Jancic S, Nguyen HD, Frisvad JC, Zalar P, Schroers HJ, et al. (2015)
A taxonomic revision of the Wallemia sebi species complex.
PLoS One 10: e0125933.
Johnson D (1998) Biodiversity and ecology of acidophilic
microorganisms. FEMS Microbiology Ecology 27: 307–317.
Jusino MA (2014) The fungal communities associated with Red-
cockaded Woodpeckers and their excavations: descriptive
and experimental evidence of symbiosis. PhD thesis, Virginia
Polytechnic Institute and State University.
Karvala K, Toskala E, Luukkonen R, Uitti J, Lappalainen S, Nordman
H (2011) Prolonged exposure to damp and moldy workplaces
and new-onset asthma. International Archives of Occupational
and Environmental Health 84: 713–721.
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment
software version 7: improvements in performance and usability.
Molecular Biology and Evolution 30: 772–780.
Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, et
al. (2013) Towards a unied paradigm for sequence-based
identication of fungi. Molecular Ecology Resources 22: 5271–
5277.
Kornerup A, Wanscher JH (1967) Methuen Handbook of Colour.
London: Methuen.
Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among
ascomycetes: evidence from an RNA polymerse II subunit.
Molecular Biology and Evolution 16: 1799–1808.
Loncaric I, Oberlerchner JT, Heiss B (2009) Phenotypic and
genotypic diversity among strains of Aureobasidium pullulans in
comparison with related species. Antonie van Leeuwenhoek 95:
165–178.
Lopez-Garcia P, Rodriguez-Valera F, Pedros-Alio C, Moreira D (2001)
Unexpected diversity of small eukaryotes in deep-sea Antarctic
plankton. Nature 409: 603–606.
Masclaux F, Guého E, De Hoog GS, Christen R (1995) Phylogenetic
relationships of human-pathogenic Cladosporium (Xylohypha)
species inferred from partial LS rRNA sequences. Journal of
Medical and Veterinary Mycology 33: 327–338.
Nguyen HD, Jancic S, Meijer M, Tanney JB, Zalar P, et al. (2015)
Application of the phylogenetic species concept to Wallemia
sebi from house dust and indoor air revealed by multi-locus
genealogical concordance. PLoS One 10: e0120894.
Nonneman MW, Coronado G, Thempson B, Grifth WC, Hanson
JD, et al. (2012) Utilizing pyrosequencing and quantitative PCR
to characterize fungal populations among house dust samples.
Journal of Environmental Monitoring 14: 2038–2043.

A new family, genus, and species from house dust
ARTICLE
315
VOLUME 8 · NO. 2
Nylander AJJ (2004) MrModeltest v2. [Program distributed by
the author.] Uppsala: Evolutionary Biology Centre, Uppsala
University.
O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA
ITS2 types within a monophyletic lineage of the fungus Fusarium
are nonorthologous. Molecular Phylogenetics and Evolution 7:
103–116.
Piecková E, Jesenská Z (1999) Microscopic fungi in dwellings and
their health implications in humans. Annals of Agricultural and
Environmental Medicine 6: 1–11.
Pitt JI, Hocking AD (2009) Fungi and Food Spoilage. 3rd edn.
Dordrecht: Springer.
Rittenour WR, Ciaccio CE, Barnes CS, Kashon ML, Lemons AR, et
al. (2014) Internal transcribed spacer rRNA gene sequencing
analysis of fungal diversity in Kansas City indoor environments.
Environmental Science Processes & Impacts 16: 33–43.
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, et al.
(2012) Mrbayes 3.2: Efcient bayesian phylogenetic inference
and model choice across a large model space. Systematic
Biology 61: 539–542.
Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen B (2010)
Food and Indoor Fungi. [CBS Laboratory Manual no. 2.] Utrecht:
CBS-KNAW Fungal Biodiversity Institute.
Schoch CL, Crous PW, Groenewald JZ, Boehm EW, Burgess
TI, et al. (2009) A class-wide phylogenetic assessment of
Dothideomycetes. Studies in Mycology 64: 1–15.
Sklenár F, Jurjević Ž, Zalar P, Frisvad JC, Visagie CM, et al. (2017)
Phylogeny of xerophilic aspergilli (subgenus Aspergillus) and
taxonomic revision of section Restricti. Studies in Mycology (in
press, https://doi.org/10.1016/j.simyco.2017.09.002).
Slepecky RA, Starmer WT (2009) Phenotypic plasticity in fungi: a
review with observations on Aureobasidium pullulans. Mycologia
101: 823–832.
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic
analysis and post-analysis of large phylogenies. Bioinformatics
30: 1312–1313.
Tanney JB, Seifert KA (2013) Rasamsonia pulvericola sp. nov.,
isolated from house dust. IMA Fungus 4: 205–212.
Tanno LK, Calderon MA, Li J, Casale T, Demoly P, Joint Allergy A
(2016) Updating Allergy and/or Hypersensitivity Diagnostic
Procedures in the WHO ICD-11 Revision. Journal of Allergy and
Clinical Immunology: In Practice 4: 650–657.
Thambugala KM, Ariyawansa HA, Li Y-M, Boonmee S, Hongsanan
S, et al. (2014) Dothideales. Fungal Diversity 68: 105–158.
Turchetti B, Goretti M, Branda E, Diolaiuti G, D’Agata C, et al. (2013)
Inuence of abiotic variables on culturable yeast diversity in two
distinct Alpine glaciers. FEMS Microbiology Ecology 86: 327–
340.
Van Nieuwenhuijzen EJ, Houbraken JA, Meijer M, Adan OC,
Samson RA (2016) Aureobasidium melanogenum: a native of
dark bionishes on oil treated wood. Antonie van Leeuwenhoek
109: 661–683.
Vilgalys R, Hester M (1990) Rapid genetic identication and
mapping of enzymatically amplied ribosomal DNA from several
Cryptococcus species. Journal of Bacteriology 172: 4238–4246.
Visagie CM, Hirooka Y, Tanney JB, Whiteld E, Mwange K, et al.
(2014) Aspergillus, Penicillium and Talaromyces isolated from
house dust samples collected around the world. Studies in
Mycology 78: 63–139.
Visagie CM, Yilmaz N, Renaud JB, Sumarah MW, Hubka V, et al.
(2017) A survey of xerophilic Aspergillus from indoor environment,
including descriptions of two new section Aspergillus species
producing eurotium-like sexual states. MycoKeys 19: 1–30.
Von Arx JA, Müller E (1975) A re-evaluation of the bitunicate
ascomycetes with keys to families and genera. Studies in
Mycology 9: 1–159.
Wang CJ, Zabel RA (1990) Identication Manual for Fungi from Utility
Poles in the Eastern United States. Rockville, MD: American
Type Culture Collection.
Webb JS, Nixon M, Eastwood IM, Greenhalgh M, Robson GD,
Handley PS (2000) Fungal colonization and biodeterioration
of plasticized polyvinyl chloride. Applied and Environmental
Microbiology 66: 3194–3200.
White TJ, Bruns T, Lee S, Taylor J (1990) Amplication and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
PCR Protocols: a guide to methods and applications (Innis MA,
Gelfand DH, Sninsky JS, White TJ, eds): 315–322. San Diego:
Academic Press.
Yurlova NA, De Hoog GS, Gerrits van den AH (1999) Taxonomy of
Aureobasidium and allied genera. Studies in Mycology 43: 63–
69.
Yurlova NA, Mokrousov IV, De Hoog GS (1995) Intraspecic
variability and exopolysaccharide production in Aureobasidium
pullulans. Antonie van Leeuwenhoek 68: 57–63.
Yurlova NA, Uijthof JMJ, De Hoog GS (1996) Distinction of species in
Aureobasidium and related genera by PCR-ribotyping. Antonie
van Leeuwenhoek 69: 323–329.
Zalar P, Gostinčar C, De Hoog G, Uršič V, Sudhadham M, Gunde-
Cimerman N (2008) Redenition of Aureobasidium pullulans and
its varieties. Studies in Mycology 61: 21–38.
Zalar P, Novak M, De Hoog GS, Gunde-Cimerman N (2011)
Dishwashers – A man-made ecological niche accommodating
human opportunistic fungal pathogens. Fungal Biology 115:
997–1007.