a new family and genus in dothideales for aureobasidium ... · pdf filea new family, genus,...
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ARTIC
LE
299
© 2017 International Mycological Association
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V O L U M E 8 · N O . 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 significant 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 significantly 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 definitive morphological identification challenging, and the many ITS variants identified 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-defined 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.2017.08.02.05IMA 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: [email protected]
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 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 28SBenAblack yeastDothidiomycetesITSRPB2xerotolerant fungiZalaria
Humphries et al.ARTICLE
300 I M A F U N G U S
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 first proposed by Von Arx & Müller (1975), and introducing Aureobasidiaceae for Aureobasidium and closely related genera. The polythetic morphological definitions 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 typified genera were keyed out separately. Sexually typified 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 clarified with the single name classification system, but presently remains difficult 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 identification 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 typified genera in Dothideales, and although no sexual morph is definitively known for the most frequently reported species A. pullulans, an ascospore-derived strain identified 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 typified 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 difficult to distinguish morphologically when grown in culture. For example, neither Hermanides-Nijhof (1977) nor De Hoog & Yurlova (1994) could find morphological differences among asexual morphs in cultures of the sexually typified 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) defined
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 difficult 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 modified 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 dustARTIC
LE
301V O L U M E 8 · N O . 2
Tabl
e 1.
Info
rmat
ion
on h
ouse
dus
t iso
late
s in
clud
ed in
this
stu
dy.
Spec
ies
Stra
in n
umbe
rIs
olat
ion
med
ium
Orig
inC
olle
ctor
aD
ate
colle
cted
Isol
ator
b
Aur
eoba
sidi
um m
elan
ogen
umS
LOA
N 1
260
= A
A07
MX
-884
ME
AM
exic
o, N
ayar
it, S
ayul
itaA
. Am
end
31 J
an. 2
009
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um m
elan
ogen
umS
LOA
N 1
606
= B
H02
AU
-110
ME
AA
ustra
lia, T
asm
ania
, Hob
art
B. H
orto
n10
Feb
. 200
9E
. Whi
tfiel
d &
K. M
wan
ge
Aur
eoba
sidi
um m
elan
ogen
umS
LOA
N 5
623
= TA
10N
Z-21
4aM
EA
New
Zea
land
, Wel
lingt
on,
Wel
lingt
onT.
Atk
inso
n3
May
200
9E
. Whi
tfiel
d &
K. M
wan
ge
Aur
eoba
sidi
um m
elan
ogen
umK
AS
584
0M
Y50
GC
anad
a, O
ntar
io, S
titts
ville
K.A
. Sei
fert
20 D
ec. 2
014
C.M
. Vis
agie
Aur
eoba
sidi
um m
elan
ogen
umK
AS
791
7M
Y10
12U
SA
, Haw
aii,
Kai
lua
A. A
men
d11
May
201
5C
.M. V
isag
ie
Aur
eoba
sidi
um m
elan
ogen
umK
AS
795
6D
G18
US
A, H
awai
i, K
ailu
aA
. Am
end
11 M
ay 2
015
C.M
. Vis
agie
Aur
eoba
sidi
um m
elan
ogen
umS
LOA
N 7
256
= A
A04
US
-587
20%
S-M
EA
US
AA
. Am
end
unkn
own
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
726
1 =
AA
02U
S-3
0620
%S
-ME
AU
SA
, Cal
iforn
ia, B
erke
ley
A. A
men
d31
Mar
. 200
5E
. Whi
tfiel
d &
K. M
wan
ge
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
203
= 7
0500
35.7
9-65
n.a.
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
12 M
ar. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
207
= 7
0500
35.7
9-71
n.a.
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
12 M
ar. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
48
= 70
5003
5.79
-127
20%
S-M
EA
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
12 M
ar. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
62
= 70
5003
5.79
-148
20%
S-M
EA
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
12 M
ar. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
349
= 7
3300
09.3
3-45
20%
S-M
EA
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
21 A
ug. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
359
= 7
3300
09.3
3-60
20%
S-M
EA
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
21 A
ug. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
724
9 =
7330
009.
34-9
25n.
a.C
anad
a, S
aska
tche
wan
, Reg
ina
Hea
lth C
anad
a21
Aug
. 200
7E
. Whi
tfiel
d &
K. M
wan
ge
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
165
2 =
BH
02A
U-1
54a
20%
S-M
EA
Aus
tralia
, Tas
man
ia, H
obar
tB
. Hor
ton
10 F
eb. 2
009
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
165
3 =
BH
02A
U-1
54b
20%
S-M
EA
Aus
tralia
, Tas
man
ia, H
obar
tB
. Hor
ton
10 F
eb. 2
009
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
SLO
AN
321
4 =
KJ0
9SA
-65
ME
AS
outh
Afri
ca, W
este
rn C
ape,
K
uils
rivie
rK
. Jac
obs
24 J
ul. 2
009
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um p
ullu
lans
KA
S 5
951
DG
18C
anad
a, B
ritis
h C
olum
bia,
Vic
toria
B. K
endr
ick
27 J
an. 2
015
C.M
. Vis
agie
Aur
eoba
sidi
um s
peci
esS
LOA
N 4
1 =
7050
035.
79-1
19M
EA
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
12 M
ar. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Aur
eoba
sidi
um s
ubgl
acia
leS
LOA
N 7
263
= A
A02
US
-332
20%
S-M
EA
US
A, C
alifo
rnia
, Ber
kele
yA
. Am
end
31 M
ar. 2
005
E. W
hitfi
eld
& K
. Mw
ange
Hor
taea
wer
neck
iiK
AS
794
2M
Y50
GU
SA
, Haw
aii,
Kai
lua
A. A
men
d11
May
201
5C
.M. V
isag
ie
Hor
taea
wer
neck
iiK
AS
794
7M
Y50
GU
SA
, Haw
aii,
Kai
lua
A. A
men
d11
May
201
5C
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ie
Hor
taea
wer
neck
iiK
AS
794
9M
Y10
12U
SA
, Haw
aii,
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lua
A. A
men
d11
May
201
5C
.M. V
isag
ie
Hor
taea
wer
neck
iiK
AS
795
3M
Y10
12U
SA
, Haw
aii,
Kai
lua
A. A
men
d11
May
201
5C
.M. V
isag
ie
Rhi
zosp
haer
a pi
niD
AO
MC
251
499
= K
AS
630
9M
Y50
GC
anad
a, N
ew B
rusn
wic
k, L
ittle
Le
prea
uA
. Wal
ker
29 J
an. 2
015
C.M
. Vis
agie
Syd
owia
pol
yspo
raD
AO
MC
251
470
= K
AS
591
8D
G18
Can
ada,
Brit
ish
Col
umbi
a, V
icto
riaB
. Ken
dric
k27
Jan
. 201
5C
.M. V
isag
ie
Syd
owia
pol
yspo
raD
AO
MC
251
471
= K
AS
591
9D
G18
Can
ada,
Brit
ish
Col
umbi
a, V
icto
riaB
. Ken
dric
k27
Jan
. 201
5C
.M. V
isag
ie
Syd
owia
pol
yspo
raD
AO
MC
251
469
= K
AS
599
9D
G18
Can
ada,
New
Bru
snw
ick,
Litt
le
Lepr
eau
A. W
alke
r29
Jan
. 201
5C
.M. V
isag
ie
Zala
ria a
lba
DA
OM
C 2
5084
7 =
SLO
AN
52
= 70
5003
5-79
-132
20%
S-M
EA
Can
ada,
Sas
katc
hew
an, R
egin
aH
ealth
Can
ada
12 M
ar. 2
007
E. W
hitfi
eld
& K
. Mw
ange
Humphries et al.ARTICLE
302 I M A F U N G U S
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 fluid. An Olympus BX50 compound microscope attached with an InfinityX camera powered by Infinity Analyze v. 6.5.1 software (Lumenera, 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/ fRPB2-7cR (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 (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.
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. Ta
ble
1. (C
ontin
ued)
.
Spec
ies
Stra
in n
umbe
rIs
olat
ion
med
ium
Orig
inC
olle
ctor
aD
ate
colle
cted
Isol
ator
b
Zala
ria a
lba
DA
OM
C 2
5084
8 =
SLO
AN
352
=
7330
009.
33-5
ME
AC
anad
a, S
aska
tche
wan
, Reg
ina
Hea
lth C
anad
a21
Aug
. 200
7E
. Whi
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A new family, genus, and species from house dustARTIC
LE
303V O L U M E 8 · N O . 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/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 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 identified 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 Ta
ble
2. B
LAS
T re
sults
sho
win
g or
igin
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nes
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aria
.
Gen
eG
enB
ank
nr
Orig
inal
iden
tifica
tion
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rent
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tifica
tion
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lone
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, Hen
an, N
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ity, B
aotia
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Nat
ural
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erve
Are
a;
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ture
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, Mis
sour
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ansa
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ity; H
ouse
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our e
t al.
(201
4)
ITS
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ood
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lula
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nds;
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sp.
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f sp2
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land
, Pra
chua
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n, P
ran
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t in
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ural
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ound
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pes
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Italy,
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t Bla
nc, M
iage
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cier
; sup
ragl
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imen
tsTu
rche
tti e
t al.
(201
3)
Humphries et al.ARTICLE
304 I M A F U N G U S
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
Myr
iang
iale
s
Myriangiaceae
Elsinoaceae
insertae sedis
Zalariaceae
Dothideaceae
Dot
hide
ales
Aureobasideaceae
NatipusillalesMicrothyrium 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)
JahnulalesAsterinales
Trypetheliales | Lichenotheliaceae
Zalaria obscura DAOMC250852 (KX579102)
Hysteriales | Strigulales
Dothidea hippophaes CBS188.58 (DQ678048)
Dothiora cannabinae CBS737.71 (DQ470984)
Phaeosclera dematioides CBS157.81 (GU301858)
Mytilinidiales | GloniaceaeMicropeltis 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
*
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86
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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 dustARTIC
LE
305V O L U M E 8 · N O . 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
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0.99/-
*/99
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*/93
*/91
0.99/90
0.97/-
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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)
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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.
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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)
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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)
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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)
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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.
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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 confirmed sequence-based identifications 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 identified 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 identified 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 classified in Dothideales and Myriangiales based on a short unique 28S rDNA sequence flanked by two conserved regions. The section defining 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 fimbriate. 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 identification markers: 28S rDNA: KX579099, RPB2: KX579105, 18S rDNA: KX579111, BenA: KX579117.
Type: Canada: Saskatchewan: Regina, isol. ex house dust, 12 Mar. 2007, E. Whitfield & 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
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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.
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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 (x̄ = 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 (x̄ = 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 significantly 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. Whitfield & 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 identification markers: 28S rDNA: KX579100, RPB2: KX579106, 18S rDNA: KX579112, BenA: KX579118
Type: Canada: Saskatchewan: Regina, isol. ex house dust, 21 Aug. 2007, E. Whitfield & 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 (x̄ = 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 (x̄ = 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. Whitfield & K. Mwange (DAOMC 250850). – USA: California: Berkeley, isol. ex house dust, 31 Mar. 2005, A. Amend, E. Whitfield & 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.
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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.
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Because of the black yeast asexual morphology, Zalaria is difficult to distinguish from Aureobasidium and Hormonema. Conidiogenesis has been used to differentiate the latter two genera, but does not consistently provide accurate identification 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 confidently 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 identified 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 identified 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 specifically 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 Whitfield and Kalima Mwange who made all isolations from house dust samples. We also thank everybody who collected house dust used during this project.
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