Scopulariopsis and scopulariopsis-like species from indoor environments.

Scopulariopsis-like species are often reported from the indoor environment, as well as from clinical samples. The lack of type isolates and thorough phylogenetic studies in the Microascaceae hampered the correct identification of these isolates. Based on recent phylogenetic studies, which resulted in multiple name changes, the aim is to molecularly identify the Scopulariopsis and scopulariopsis-like species which occur in the indoor environment and give an overview of the current species in these genera and their habitats. Strains from the CBS culture collection were supplemented with almost 80 indoor strains of which the internal transcribed spacer 1 and 2 and intervening 5.8S nrDNA (ITS), beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1) gene regions were sequenced for phylogenetic inference. The multi-gene phylogenies recognise 33 Microascus species and 12 Scopulariopsis species and showed that the recently established genus Fuscoannellis, typified by Scopulariopsis carbonaria, should be synonymized with the genus Yunnania. Seven new Microascus species, four new Scopulariopsis species, and one new Yunnania species, are described, and a new name in Microascus and two new name combinations (one in Microascus, and one in Yunnania) are proposed. In the indoor environment 14 Microascus species and three Scopulariopsis species were found. Scopulariopsis brevicaulis (22 indoor isolates) and Microascus melanosporus (19 indoor isolates) are the most common indoor species, in number of isolates, followed by M. paisii (8 indoor isolates) and S. candida (7 indoor isolates). A genus phylogeny based on the ITS, tef1 and the large subunit 28S nrDNA (LSU) of the type or representative isolates of all here recognised species is provided depicting all species habitats. No correlation between phylogenetic relationship and habitat preference could be observed. Ten species which are found indoor are also found in relation with human-derived samples. A table showing recent name changes and a key to common species of Scopulariopsis and scopulariopsis-like genera found indoors is included.

Introduction

People spend up to 90 % of their time indoors (Höppe & Martinac 1998). Fungi present in these indoor environments can produce toxins or carry allergens which cause health hazards. Therefore it is important to know which fungal species are present indoors. Several reports are made on the presence of Microascaceae in the indoor environment. The species Scopulariopsis brevicaulis, S. candida, S. fusca (= S. asperula), S. brumptii (= Microascus paisii) and S. sphaerospora (= M. paisii) are often mentioned as indoor fungi (Samson ). However, in most of the indoor reports of scopulariopsis-like isolates, morphological examination has not been confirmed with molecular studies. Also the absence of thorough phylogenetic studies in these genera made it difficult to accurately identify the indoor Microascaceae. The first phylogenetic study of scopulariopsis-like species was based on the large subunit 28S nrDNA (LSU, Issakainen ). Here the potential relationship between asexual and sexually reproducing species was assessed, with a focus on clinically occurring species. The main microascoid clade, which contained all Microascus and Scopulariopsis species studied, was divided into seven clades. Further taxonomic study is suggested to redefine or split the genus Microascus. A study of clinical isolates in Poland confirmed that the LSU sequence alone is insufficient for species delimitation in Scopulariopsis (Jagielski ). A taxonomic study of cheese fungi used the beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1) gene regions next to LSU to identify their Scopulariopsis species (Ropars ). The internal transcribed spacer 1 and 2 and intervening 5.8S nrDNA (ITS) gave problems with amplification, and displayed a high variability, which made it not useful for phylogenetic study of their isolates. Translation elongation factor 1-alpha showed to be the most phylogenetically informative genomic region and was proposed for identifying Scopulariopsis species. A subsequent phylogenetic study on clinical Microascus and Scopulariopsis species made a combined phylogeny of the LSU and tef1 gene region (Sandoval-Denis ). They concluded that this combined analysis is useful for the identification of the most common clinically relevant Scopulariopsis species. However, further phylogenetic studies testing more genetic markers and reference strains are suggested, since nine phylogenetic clades in their combined phylogenies could not be properly named. This follow-up study with the aim to clarify the taxonomy and phylogeny of Microascus, Scopulariopsis and allied genera was published recently (Sandoval-Denis ). On a large set of clinical and environmental isolates, including ex-type strains of multiple species, a phylogenetic study was conducted based on the ITS, LSU, tef1 and tub2 gene regions, in combination with morphological and physiological analyses. In this polyphasic approach study the genera Microascus and Scopulariopsis are separated, the genus Pithoascus reinstated, and the new genus Pseudoscopulariopsis proposed. Seven new Microascus species and one new Scopulariopsis species are described, nine new name combinations are introduced, and several species are neotypified (Sandoval-Denis ). A second taxonomic study on a set of clinical and environmental scopulariopsis-like fungi followed soon (Jagielski ). Here another three new Microascus species, one new Scopulariopsis species and one new Pithoascus species are described, S. albo-flavescens is reinstated, M. trigonosporus var. terreus recombined in M. terreus, and the new genus Fuscoannellis proposed.

Although these two recent phylogenetic studies (Jagielski et al., 2016, Sandoval-Denis et al., 2016) make molecular identification of scopulariopsis-like isolates upon species level possible, the involved name changes can cause a lot of confusion. Commonly mentioned species from the indoor environment, like S. brumptii (now M. paisii), S. fusca (now S. asperula) and S. sphaerospora (now M. paisii), are renamed. The aim of this project is to molecularly identify the scopulariopsis-like taxa, which occur in the indoor environment. Simultaneously, a phylogenetic overview of these genera is constructed, and the species habitats are studied. All available Microascus and Scopulariopsis isolates from the Westerdijk Fungal Biodiversity Institute culture collection (CBS collection) and working collection of the Applied and Industrial Mycology department (DTO collection) are included in the study. Species phylogenetic inferences were conducted on sequence data of parts of the ITS, tub2 and tef1 gene regions, and a genus phylogenetic inference on the LSU, ITS and tef1 gene regions. Phylogenetic clades which contain indoor isolates are highlighted as indoor species. New species are described, and an overview of the current species and their habitats in the genera Microascus, Scopulariopsis and Yunnania is provided. Furthermore, a table showing recent name changes and a key to common species of Scopulariopsis and scopulariopsis-like genera found in the indoor environment is provided.

Materials and methods

Isolates

In total 248 isolates were included in this study, comprising of 152 Microascus isolates, 88 Scopulariopsis isolates, four Yunnania isolates, and four out-group isolates. The isolates were obtained from the culture collection of the Westerdijk Fungal Biodiversity Institute (former CBS-KNAW Fungal Biodiversity Centre), Utrecht, the Netherlands and the working collection of the Applied and Industrial Mycology department (DTO) housed at the Westerdijk Institute (Table 1). Isolates from the culture collection of the Westerdijk Institute (CBS collection) have a world-wide distribution and are isolated from a diverse range of substrates. Isolates from the working collection of DTO are mostly isolated from indoor environments or food, and include swab and air samples mainly from Europe, and house dust samples collected world-wide (Amend ). Freeze-dried strains from the CBS culture collection were revived in 2 mL malt/peptone (50 % / 50 %) and subsequently transferred to oatmeal agar (OA) (Samson ). Strains stored in the liquid nitrogen (CBS collection) or the DTO collection were transferred to OA directly from the −185 °C or −80 °C storage, respectively. They were cultured for 14 d at 25 °C in the dark. From eight isolates only their DNA sequences from GenBank were obtained (Table 1, isolates without a DTO number).

Table 1

Isolates used in this study and their GenBank accession numbers. Bold accession numbers were generated in other studies.

Name	Old name1	Strain numbers2	Host/Substrate	Country	GenBank accession number
					ITS	tub2	tef1	LSU
Cephalotrichum asperulum		CBS 582.71IT, DTO 104-B7, ATCC 26885	Soil	Argentina	KX923818		KX924043	KX924027
C. stemonitis		CBS 103.19NT, DTO 170-B3, MUCL 6960	Seed	Netherlands	KX923819		LN850953	LN850952
Microascus alveolaris	M. trigonosporus	CBS 268.49, DTO 342-D8	Avena sativa, grain	USA	KX923823	KX924257	KX924047
	M. trigonosporus	CBS 269.49, DTO 342-C2	Glycine soja, seed	USA	KX923824	KX924258	KX924048
	M. trigonosporus	CBS 270.49, DTO 345-F7	Hordeum vulgare, seed	USA	KX923825	KX924259	KX924049
	M. trigonosporus	CBS 271.49, DTO 342-D9	Hordeum vulgare, seed	USA	KX923826	KX924260	KX924050
	M. trigonosporus	CBS 272.49, DTO 342-C3	Avena sativa, leaf	USA	KX923827	KX924261	KX924051
	M. trigonosporus	CBS 150.64, DTO 342-E2	Allium cepa, seed	USA	KX923828	KX924262	KX924052
	M. trigonosporus	CBS 494.70, DTO 342-C8	Marine sediment	Norway	LN850757	LN850855	LN850903
		CBS 139501T, DTO 351-E2, FMR 12252, UTHSC 07-3491	Human, BAL fluid	USA	KX923829	KX924263	KX924053	KX924029
		DTO 223-A7	Indoor	Uruguay	KX923830	KX924264	KX924054
M. appendiculatus sp. nov.	M. senegalensis	CBS 594.78T, DTO 354-C3	Human, skin	Algeria	LN850781	LN850878	KX924055	LN850830
M. atrogriseus nom. nov.	Masonia grisea	CBS 295.52T, DTO 103-H6, IFO 6795, IMI 049908, MUCL 9003	Culture contaminant	UK	LM652433	KX924265	KX924056	KX924030
		CBS 897.68, DTO 356-C3, ATCC 16279, IFO 31245, MUCL 8993	Wheat field soil	Germany	LM652436	LM652649	LM652571
	S. chartarum	CBS 410.76, DTO 345-B1	Burnt soil	Netherlands	KX923831	KX924266	KX924057
		DTO 139-D7	Indoor	Germany	KX923832	KX924267	KX924058
		DTO 191-C2	Indoor horse arena	Netherlands	KX923833	KX924268	KX924059
M. brunneosporus		CBS 138276T, DTO 351-D8, UTHSC 06-4312, FMR 12343	Human, BAL fluid	USA	KX923834	KX924269	HG380420	HG380497
M. chartarus	Masonia chartarum	CBS 294.52T, IMI 049909, MUCL 9001	Wall paper	UK	LM652393	LM652607	HG380386	HG380463
M. chinensis		CBS 139628T, BMU 01837	Human, nail	China	LN850760	LN850858	LN850906	LN850809
M. cinereus	M. griseusT	CBS 365.65, DTO 104-A3, DTO 104-A4, ATCC 16204, HACC 1252, IMI 113680	Soil	India	LM652399	KX924270	KX924060
		CBS 664.71, DTO 104-B8	Human, lung	USA	KX923835	LN850860	KX924061
		CBS 324.72, DTO 342-G2	Clay	Namibia	KX923836	KX924271	KX924062
		CBS 138709NT, DTO 351-E1, UTHSC 10-2805, FMR 12217	Human, BAL fluid	USA	KX923837	KX924272	KX924063	KX924031
M. cirrosus		CBS 217.31T, DTO 103-G1	Prunus sp., leaf	Italy	KX923838	KX924273	KX924064	KX924032
		CBS 277.34, DTO 345-A7, MUCL 9050, MUCL 9055	Vitis vinifera, root	Italy	KX923839	KX924274	LM652556
	M. longirostris	CBS 267.49, DTO 170-B9, IFO 7029	Sciurus vulgaris, skin	Netherlands	KX923840	KX924275	KX924065
		CBS 240.58, DTO 342-E1, IMI 086913	Compost soil	Germany	KX923841	KX924276	KX924066
		CBS 301.61, DTO 345-A8, IMI 086914, NRRL 1689, MUCL 9054	Unknown	UK	KX923842	KX924277	KX924067
		CBS 302.61, DTO 345-D1	Unknown	Canada	KX923843	KX924278	KX924068
		CBS 424.62, DTO 345-A9	Polyvinylchloride	Netherlands	KX923844	KX924279	KX924069
		CBS 541.74, DTO 342-I8	Rodent dung	USA	KX923845	KX924280	KX924070
		CBS 157.92, DTO 342-G6, FRR 4174	Arachis hypogaea, nut	Indonesia	KX923846	KX924281	KX924071
		CBS 115860, DTO 345-D4, FMR 8575	Air	Spain	KX923847	KX924282	KX924072
		CBS 116405, DTO 345-D6	Antique tapestries	Poland	LN850763	LN850861	LN850909
		DTO 342-D4, RGR 84.0007	Helianthus annuus, oilseed	USA	KX923848	KX924283	KX924073
		DTO 342-D5, RGR 84.0033	Unknown	Unknown	KX923849	KX924284	KX924074
		DTO 342-D7, RGR 84.0051	Unknown	Unknown	KX923850	KX924285	KX924075
M. cleistocarpus sp. nov.		CBS 134638T, DTO 342-D2, CGMCC 3.15222	Discarded cloth	China	KX923851	KX924286	KX924076	KX924033
M. croci	S. croci	CBS 158.44T, DTO 103-H3, IMI 078261, MUCL 9002	Crocus sp.	Netherlands	KX923852	KX924287	KX924077	LM652508
	Masoniella tertiaT	CBS 296.61, DTO 103-I6, IMI 109550, MUCL 9005	Air	Brazil	KX923853	KX924288	KX924078
	S. chartarum	CBS 522.69, DTO 342-C7	Forest soil	Canada	KX923854	KX924289	KX924079
		DTO 220-I5	Indoor	Indonesia	KX923855	KX924290	KX924080
		DTO 252-D8	Indoor	Germany	KX923856	KX924291	KX924081
		DTO 305-B3	Indoor	Mexico	KX923857	KX924292	KX924082
		DTO 305-B5	Indoor	Mexico	KX923858	KX924293	KX924083
M. expansus		CBS 138127T, DTO 351-D6, UTHSC 06-4472, FMR 12266	Human, sputum	USA	KX923859	KX924294	KX924084	HG380492
M. fusisporus sp. nov.	M. paisii	CBS 896.68T, DTO 356-C2, ATCC 16278, IFO 31244, MUCL 8989	Wheat-field soil	Germany	LM652432	LM652645	HG380372	LN850825
M. gracilis	M. cinereus	CBS 126.14, DTO 347-C4, IMI 086916	Unknown	Unknown	KX923860	KX924295	KX924085
	M. cinereus	CBS 195.61, DTO 345-C8, IMI 075542, MUCL 9048	Soil	UK	LM652416	LM652629	HG380391
		CBS 300.61, DTO 345-C9, MUCL 9049	Zea mays, stored seed	USA	LM652417	KX924296	LM652563
		CBS 369.70T, DTO 104-B3, IFO 7561	Wheat flour	Japan	KX923861	KX924297	KX924086	HG380467
		CBS 794.91, DTO 345-D2	Rice flour	Australia	KX923862	KX924298	KX924087
		CBS 156.92, DTO 345-D3	Arachis hypogaea, nut	Thailand	KX923863	KX924299	KX924088
	M. cinereus	CBS 116059, DTO 345-D5	Polyethylene with starch	Poland	KX923864	LN850863	KX924089
		CBS 120886, DTO 342-D1	Prunus persica	South Africa	KX923865	KX924300	KX924090
		DTO 220-I4	Indoor	Indonesia	KX923866	KX924301	KX924091
	M. cinereus	DTO 342-D6, RGR 84.0038	Unknown	Unknown	KX923867	KX924302	KX924092
	M. cinereus	DTO 342-G8, RGR 84.0040	Unknown	Unknown	KX923868	KX924303	KX924093
M. hollandicus sp. nov.		CBS 141582T, DTO 191-C3	Indoor horse arena	Netherlands	KX923869	KX924304	KX924094	KX924034
M. hyalinus		CBS 766.70T, DTO 170-F2	Cow dung	USA	KX923870	KX924305	LM652564	LM652513
		CBS 134639, DTO 342-I9	Goat dung	China	KX923871	KX924306	KX924095
M. intricatus		CBS 138128T, DTO 351-D7, UTHSC 07-156, FMR 12264	Human BAL fluid	USA	KX923872	KX924307	HG380419	HG380496
		DTO 223-A6	Indoor	Micronesia	KX923873	KX924308	KX924096
M. longicollis		CBS 752.97, DTO 338-G8	Anacardium occidentale, nut	Brazil	KX923874	KX924309	KX924097	KX924035
M. longirostris		CBS 196.61NT, IMI 086908, MUCL 9058, NRRL 1717	Wasp's nest	USA	LM652421	LM652634	LM652566	LM652515
		CBS 415.64, IFO 7554	Soil	Japan	LM652422	LM652635	LM652567
M. macrosporus		CBS 662.71, DTO 170-F6, NRRL A-8018	Soil	USA	LM652423	LM652636	LM652568	LM652517
	M. cirrosus	CBS 540.74, DTO 347-D1	Soil	USA	KX923875	KX924310	KX924098
M. melanosporus comb. nov.	S. melanospora	CBS 272.60T, DTO 103-I1, IFO 6441, IMI 078257, LCP 59.1590, MUCL 9040, NHL 6045	Oryza sativa, milled	USA	KX923876	KX924311	LM652572	KX924036
		CBS 854.68, DTO 220-H7	Compost soil	Germany	KX923877	KX924312	KX924099
	S. fusca	CBS 102829, DTO 342-E7	Cheese warehouse	Netherlands	KX923878	KX924313	KX924100
		CBS 116060, DTO 136-G8	Antique tapestries	Poland	LN850775	LN850872	KX924101
		DTO 043-A1	Unknown	Unknown	KX923879	KX924314	KX924102
		DTO 043-A2	Unknown	Unknown	KX923880	KX924315	KX924103
		DTO 049-E4	Archive	Netherlands	KX923881	KX924316	KX924104
		DTO 049-E5	Archive	Netherlands	KX923882	KX924317	KX924105
		DTO 049-F2	Office	Netherlands	KX923883	KX924318	KX924106
		DTO 053-H2	Between concrete Floor and carpet	Netherlands	KX923884	KX924319	KX924107
		DTO 067-G7	Bakery	Netherlands	KX923885	KX924320	KX924108
		DTO 138-B6	Indoor	Germany	KX923886	KX924321	KX924109
		DTO 220-H9	Indoor	South Africa	KX923887	KX924322	KX924110
		DTO 220-I1	Indoor	South Africa	KX923888	KX924323	KX924111
		DTO 220-I2	Indoor	South Africa	KX923889	KX924324	KX924112
		DTO 220-I3	Indoor	South Africa	KX923890	KX924325	KX924113
		DTO 223-A9	Indoor	Germany	KX923891	KX924326	KX924114
		DTO 240-A9	Archive	Netherlands	KX923892	KX924327	KX924115
		DTO 240-B1	Archive	Netherlands	KX923893	KX924328	KX924116
		DTO 240-B3	Archive	Netherlands	KX923894	KX924329	KX924117
		DTO 240-B4	Archive	Netherlands	KX923895	KX924330	KX924118
		DTO 252-D7	Indoor	Germany	KX923896	KX924331	KX924119
		DTO 255-A5	Airsampling	Germany	KX923897	KX924332	KX924120
		DTO 255-A6	Airsampling	Germany	KX923898	KX924333	KX924121
		DTO 255-A7	Airsampling	Germany	KX923899	KX924334	KX924122
		DTO 255-B1	Plaster	Germany	KX923900	KX924335	KX924123
		DTO 255-B3	Polystyrene	Germany	KX923901	KX924336	KX924124
		DTO 255-B5	Oriented strand board	Germany	KX923902	KX924337	KX924125
		DTO 255-B6	Wood	Germany	KX923903	KX924338	KX924126
		DTO 255-C3	Unknown	Germany	KX923904	KX924339	KX924127
M. micronesiensis sp. nov.		CBS 141523T, DTO 220-I9	Indoor	Micronesia	KX923905	KX924340	KX924128	KX924037
		DTO 223-A5	Indoor	Micronesia	KX923906	KX924341	KX924129
M. murinus		CBS 621.70, DTO 347-C8	Composted municipal waste	Germany	KX923907	LN850868	KX924130
		CBS 830.70T, DTO 104-B5, DTO 170-F5, IMI 161540	Composted municipal waste	Germany	KX923908	KX924342	KX924131	HG380481
		CBS 864.71, DTO 347-C9	Municipal waste	Germany	KX923909	LN850867	KX924132
M. onychoides		CBS 139629T, BMU 03911	Human, nail	China	LN850774	LN850871	LN850920	LN850823
M. paisii	Torula paisii	CBS 213.27T, DTO 103-F9, IMI 036480, MUCL 7915, VKM F-424	Human	Italy	LM652434	KX924343	KX924133	LM652518
	S. sphaerosporaT	CBS 402.34, DTO 103-G8, MUCL 9045	Unknown	Austria	LM652437	KX924344	LM652651
	S. brumptiiIT (?)	CBS 333.35, DTO 220-H5	Small-pox vaccine	France	KX923910	KX924345	KX924134
		CBS 345.58, DTO 220-H6	Human, skin and hair	Germany	LN850777	LN850874	LN850923
		DTO 073-F1	Moist wall of archive	Netherlands	KX923911	KX924346	KX924135
		DTO 109-G6	Indoor	Denmark	KX923912	KX924347	KX924136
		DTO 220-I6	Indoor	New Zealand	KX923913	KX924348	KX924137
		DTO 220-I7	Indoor	New Zealand	KX923914	KX924349	KX924138
		DTO 252-D9	Indoor	Germany	KX923915	KX924350	KX924139
		DTO 255-A8	Airsampling	Germany	KX923916	KX924351	KX924140
		DTO 255-A9	Airsampling	Germany	KX923917	KX924352	KX924141
		DTO 255-B2	Plaster	Germany	KX923918	KX924353	KX924142
		DTO 255-B4	Oriented strand board	Germany	KX923919	KX924354	KX924143
		DTO 255-B7	Plaster	Germany	KX923920	KX924355	KX924144
		DTO 255-B8	Polystyrene	Germany	KX923921	KX924356	KX924145
		DTO 255-B9	Airsampling	Germany	KX923922	KX924357	KX924146
M. pseudolongirostris	M. cirrosus	CBS 462.97T, DTO 351-D5	Human, nail	Netherlands	LN850782	LN850879	KX924147	LN850831
M. pseudopaisii sp. nov.		CBS 141581T, DTO 116-A3	Air, basement	Netherlands	KX923923	KX924358	KX924148	KX924038
		DTO 116-A4	Air, basement	Netherlands	KX923924	KX924359	KX924149
M. pyramidus		CBS 212.65T, DTO 104-A1, DTO 104-A2, ATCC 36763, IMI 109887	Desert soil	USA	KX923925	KX924360	KX924150	HG380435
		CBS 668.71, DTO 342-E4	Pocket mouse, hair	USA	KX923926	LN850876	LN850925
	M. trigonosporus	CBS 663.71, DTO 342-G1	Soil	USA	KX923927	KX924361	KX924151
M. restrictus		CBS 138277T, DTO 347-B4, UTHSC 09-2704, FMR 12227	Human, left hallux	USA	KX923928	KX924362	KX924152	HG380494
M. senegalensis		CBS 277.74T, DTO 351-E4	Mangrove soil	Senegal	KX923929	KX924363	KX924153	LM652523
		CBS 760.84, DTO 347-D2	Helianthus annuus, seed	USA	KX923930	KX924364	KX924154
		CBS 761.84, DTO 342-G5	Helianthus annuus, seed	USA	KX923931	KX924365	KX924155
		CBS 775.84, DTO 342-C9	Helianthus annuus, seed	Germany	KX923932	KX924366	KX924156
		DTO 342-F1, RGR 84.0112	Unknown	Unknown	KX923933	KX924367	KX924157
		DTO 342-G9, RGR 84.0113	Unknown	Unknown	KX923934	KX924368	KX924158
		DTO 342-H1, RGR 84.0158	Unknown	Unknown	KX923935	KX924369	KX924159
		DTO 342-H2, RGR 84.0159	Unknown	Unknown	KX923936	KX924370	KX924160
		DTO 342-H4, RGR 85.0058	unknown	Unknown	KX923937	KX924371	KX924161
M. terreus		CBS 601.67T, DTO 104-A8, ATCC 22360, NRRL A-18283, VKM F-1144	Soil	Ukraine	LN850783	LN850880	LN850928	LN850832
	M. trigonosporus	CBS 665.71, DTO 342-E3	Soil	USA	KX923938	KX924372	KX924162
	M. trigonosporus	CBS 807.73, DTO 342-E5	Saline desert soil	Kuwait	KX923939	KX924373	KX924163
		CBS 138275, UTHSC 07-1823, FMR 12342	Human, sputum	USA	LM652384	LM652600	HG380412
	M. trigonosporus	DTO 342-D3, RGR 84.0004, ATCC 62716	Helianthus annuus, confectionary seed	USA	KX923940	KX924374	KX924164
	M. trigonosporus	DTO 343-A1, RGR 84.0003	Helianthus annuus, seed	USA	KX923941	KX924375	KX924165
M. trautmannii sp. nov.		CBS 141583T, DTO 255-C1	oriented strand board	Germany	KX923942	KX924376	KX924166	KX924039
M. trigonosporus		CBS 218.31T, DTO 103-G2, HACC 178, IMI 113702	Unknown	Puerto Rico	KX923943	KX924377	HG380359	HG380436
		CBS 198.61, DTO 342-C5, IMI 086911, NRRL 1570	Unknown	Unknown	KX923944	KX924378	KX924167
		CBS 199.61, DTO 342-C6, IFO 7027, IMI 086912, MUCL 9061, NHL 2265	Oryza sativa, milled	Burma	LM652444	KX924379	HG380361
		CBS 366.65, DTO 342-F9, ATCC 16203, HACC 178	Unknown	India	KX923945	KX924380	KX924168
		CBS 158.92, DTO 342-E6, FRR 4046	Arachis hypogaea, nut	Thailand	KX923946	KX924381	KX924169
		DTO 220-I8	Indoor	Micronesia	KX923947	KX924382	KX924170
		DTO 223-A1	Indoor	Micronesia	KX923948	KX924383	KX924171
		DTO 223-A2	Indoor	Micronesia	KX923949	KX924384	KX924172
M. verrucosus		CBS 138278T, DTO 351-D9, UTHSC 10-2601, FMR 12219	Human, BAL fluid	USA	KX923950	LM652658	HG380416	HG380493
	S. sphaerospora	CBS 210.61, DTO 347-B3, IMI 086939	Unknown	Unknown	KX923951	KX924385	KX924173
Pithoascus stoveri		CBS 176.71T, DTO 104-B6, ATCC 11173	Beta vulgaris, root seedling	USA	KX923952	KX924386	KX924174	LM652532
Pseudoscopulariopsis schumacheri		CBS 435.86NT, DTO 170-H5	Soil	Spain	KX923953	KX924387	KX924175	LM652534
Scopulariopsis africana sp. nov.	M. manginii	CBS 118736T, DTO 336-D1	Mud, salt pan	South Africa	KX923954	KX924388	KX924176	KX924040
S. albida sp. nov.	S. flava	CBS 119.43T, DTO 334-G7	Soil	Netherlands	LN850800	LN850897	LM652592	LN850849
	S. acremonium	CBS 415.51, DTO 334-H2	Unknown	Germany	KX923955	KX924389	KX924177
S. alboflavescens	S. koningii	CBS 152.22, DTO 347-C5, IMI 086928, MUCL 9044	Unknown	France	LN850785	LN850882	KX924178
		CBS 399.34T, DTO 103-G6, UAMH 934	Human, skin	Austria	KX923956	JQ434537	KX924179	LM652539
	S. koningii	CBS 208.61, DTO 170-C6, IMI 086926, FMR 3654	Elephant	Unknown	LN850786	LN850883	LN850931
		DTO 104-C7	Unknown	Unknown	KX923957	KX924390	KX924180
S. asperula	S. arnoldiiT	CBS 204.27, DTO 334-F5, MUCL 9009, UAMH 923	Unknown	France	KX923958	KX924391	KX924181
	S. fuscaT	CBS 401.34, DTO 103-G7, IFO 8181, IMI 086934, MUCL 9032, UAMH 930	Rabbit carcass	Austria	LM652463	KX924392	KX924182
		CBS 105.35, DTO 334-F8, IMI 086925	Unknown	Unknown	KX923959	KX924393	KX924183
	Torula bestaeT	CBS 289.38, DTO 103-H1, IMI 086927, MUCL 9012, UAMH 924	Human	Italy	KX923960	KX924394	KX924184	LM652538
	S. fusca	CBS 351.49, DTO 342-C4, MUCL 9033	Sciurus vulgaris, dead	Unknown	KX923961	KX924395	KX924185
		CBS 390.52, DTO 334-H4, IMI 086924, LCP 224, MUCL 9043	Calliphora vomitoria, proboscis	France	KX923962	KX924396	KX924186
	S. fusca	CBS 334.53, DTO 334-H5	Human, nail	Netherlands	LN850788	LN850885	KX924187
		CBS 298.67, DTO 334-I4	Triticum aestivum	Turkey	LN850789	LN850886	LN850934
		CBS 853.68, DTO 334-I5	Compost soil	Germany	KX923963	JQ434558	KX924188
		CBS 872.68, DTO 334-I6, ATCC 16281	Wheat field soil	Germany	KX923964	KX924397	KX924189
		CBS 668.74, DTO 335-A5	Soil	Egypt	KX923965	KX924398	KX924190
		CBS 373.76, DTO 170-G6	Unknown	Netherlands	KX923966	KX924399	KX924191
		CBS 114063, DTO 038-B3, DTO 335-B3	Wood sample	Germany	KX923967	KX924400	KX924192
		CBS 117767, DTO 038-B6	Wood sample	Germany	KX923968	LN850884	KX924193
	S. fusca	CBS 138116, DTO 335-D1	Alkaline soil	Russia	KX923969	KX924401	KX924194
S. brevicaulis		CBS 120.20, DTO 334-F4, MUCL 9021	Unknown	Unknown	KX923970	KX924402	KX924195
		CBS 273.30, DTO 334-F6, VKM F-175	Unknown	Unknown	KX923971	KX924403	KX924196
	S. flava	CBS 334.35, DTO 334-G1	Arge berberidis, pupa	Czech Republic	LN850790	LN850887	LN850935
	S. flava	CBS 335.35, DTO 334-G2, IMI 086922, MUCL 9035	Pteronus pini, pupa	Netherlands	LM652477	KX924404	KX924197
		CBS 340.39, DTO 336-C4	Bone	South Africa	KX923972	KX924405	KX924198
		CBS 341.39, DTO 334-G4	Unknown	Unknown	KX923973	KX924406	KX924199
		CBS 147.41, DTO 334-G5	Human, nail	Netherlands	KX923974	KX924407	KX924200
		CBS 467.48, DTO 103-H4, ATCC 7903, IMI 040026, IMI 061534, NRRL 1096	Unknown	Unknown	KX923975	KX924408	KX924201
		CBS 398.54, DTO 170-C3, IMI 086919	Human, toe nail	UK	KX923976	KX924409	KX924202
		CBS 112377, DTO 011-H5, DTO 038-A8	Indoor	Germany	KX923977	KX924410	KX924203
		CBS 115540, DTO 335-B4	Air biofilter	Mexico	KX923978	KX924411	KX924204
		CBS 116112, DTO 335-B5	Tattoo-paint	Czech Republic	KX923979	KX924412	KX924205
		CBS 117277, DTO 001-F7, DTO 012-E7	Hat-rack in museum	Netherlands	KX923980	KX924413	KX924206
		CBS 118469, DTO 338-H1	Tattoo-paint	UK	KX923981	KX924414	KX924207
		CBS 118470, DTO 336-C8	Tattoo-paint	UK	KX923982	KX924415	KX924208
		CBS 118471, DTO 335-B6	Tattoo-paint	UK	KX923983	KX924416	KX924209
		CBS 118472, DTO 335-B7	Tattoo-paint	UK	KX923984	KX924417	KX924210
		CBS 118473, DTO 338-H2	Tattoo-paint	UK	KX923985	KX924418	KX924211
		CBS 118474, DTO 336-C9	Tattoo-paint	UK	KX923986	KX924419	KX924212
		CBS 118993, DTO 335-B8	Unknown	France	KX923987	KX924420	KX924213
		CBS 119549, DTO 335-B9	Human, skin biopsy	USA	KX923988	KX924421	KX924214
		CBS 119550, DTO 335-C1	Human, blood culture	USA	KX923989	KX924422	KX924215
	M. brevicaulisT	CBS 127812, DTO 138-E6, DTO 138-E7, UAMH 7770, MUCL 40726	Indoor air	Canada	LM652465	KX924423	HG380363	HG380440
		CBS 127825, DTO 138-E8, DTO 138-E9, UAMH 7880	Indoor air	Canada	KX923990	KX924424	KX924216
		CBS 137631, DTO 335-C8	Alkaline soil	Russia	KX923991	KX924425	KX924217
		CBS 137632, DTO 335-C9	Alkaline soil	Russia	KX923992	KX924426	KX924218
		DTO 012-C7	Indoor air	Germany	KX923993	KX924427	KX924219
		DTO 012-D9	Wall paper	Unknown	KX923994	KX924428	KX924220
		DTO 012-F6	Plaster	Germany	KX923995	KX924429	KX924221
		DTO 106-B6	Indoor, giraffes stay	Netherlands	KX923996	KX924430	KX924222
		DTO 109-H4	Indoor	Denmark	KX923997	KX924431	KX924223
		DTO 145-C7	Indoor	Germany	KX923998	KX924432	KX924224
		DTO 168-A4	Indoor air, poultry house	Poland	KX923999	KX924433	KX924225
		DTO 168-A5	Indoor air, poultry house	Poland	KX924000	KX924434	KX924226
		DTO 168-A6	Indoor air, poultry house	Poland	KX924001	KX924435	KX924227
		DTO 195-A1	Indoor, swab sample bakery	Netherlands	KX924002	KX924436	KX924228
		DTO 197-F3	Indoor air sample	Netherlands	KX924003	KX924437	KX924229
		DTO 240-A8	Archive	Netherlands	KX924004	KX924438	KX924230
		DTO 305-A2	Indoor	South Africa	KX924005	KX924439	KX924231
		DTO 305-A3	Indoor	USA	KX924006	KX924440	KX924232
		DTO 305-A4	Indoor	USA	KX924007	KX924441	KX924233
		DTO 305-A5	Indoor	USA	KX924008	KX924442	KX924234
		DTO 305-A8	Indoor	USA	KX924009	KX924443	KX924235
S. candida	Nephrospora manginiiT	CBS 170.27, DTO 347-C6, IMI 086931, UAMH 9135	Unknown	France	LM652488	LM652694	LM652594
	M. manginii	CBS 205.27, DTO 347-C7, MUCL 9026	Unknown	France	LM652483	KX924444	KX924236
	S. acremonium	CBS 305.31, DTO 336-C3	Unknown	USA	KX924010	KX924445	KX924237
	M. manginii	CBS 353.36, DTO 334-G3	Indoor air, hospital	unknown	KX924011	KX924446	KX924238
	S. acremonium	CBS 389.52, DTO 334-H3	Unknown	Italy	KX924012	KX924447	KX924239
	M. manginii	CBS 254.69, DTO 335-A1	Greenhouse soil	Netherlands	KX924013	KX924448	KX924240
	M. manginii	CBS 132.78, DTO 342-G3	Human, dentine	France	KX924014	LN850898	KX924241
		DTO 032-A6	Indoor air, house	Netherlands	KX924015	KX924449	KX924242
		DTO 138-B7	Indoor air	Germany	KX924016	KX924450	KX924243
		DTO 139-D8	Indoor	Germany	KX924017	KX924451	KX924244
		DTO 139-E7	Indoor	Germany	KX924018	KX924452	KX924245
		DTO 139-E8	Indoor	Germany	KX924019	KX924453	KX924246
		UAMH 9004ET, MUCL 40743	Indoor air	Canada	LM652484	LM652690	HG380381	HG380458
S. caseicola sp. nov.	S. acremonium	CBS 480.62T, DTO 342-F8	Cheese-coating	Netherlands	KX924020	KX924454	KX924247	KX924041
S. cordiae	M. manginii	CBS 816.73, DTO 335-A4	Soil	Australia	KX924021	KX924455	KX924248
		CBS 138129T, DTO 335-D2, UTHSC 09-866, FMR 12338	Human, finger	USA	KX924022	KX924456	KX924249	HG380499
S. flava		CBS 207.61NT, DTO 170-C5, IMI 086921, MUCL 9031	Cheese	UK	KX924023	KX924457	HG380387	HG380464
	S. brevicaulis	CBS 108960, DTO 335-B2	Cheese	Denmark	LN850804	LN850901	LN850949
S. macurae	M. manginii	CBS 506.66T, DTO 334-I3, ATCC 16685	Chicken litter	Canada	LN850805	LN850902	KX924250	LN850854
S. sexualis sp. nov.	M. manginii	CBS 250.64T, DTO 338-G3, IFO 7555, NHL 2278, UAMH 1923	Oryza sativa, milled	Burma	KX924024	KX924458	KX924251	KX924042
	M. manginii	CBS 667.71, DTO 335-A3, NRRL A-8022	Bat dung	USA	KX924025	KX924459	KX924252
	M. manginii	CBS 332.78, DTO 342-G4	Brassica oleracea, seed	India	KX924026	KX924460	KX924253
S. soppii		UAMH 9169T	Populus tremuloides, wood	Canada	LM652495	LM652698	LM652595	LM652552
Yunnania carbonaria	S. carbonaria	CBS 205.61T, DTO 103-I3, IFO 8116, IMI 086941, MUCL 9027, NRRL 1860	Soil	Panama	KX923820	KX924254	KX924044	HG380462
	S. brumptii	CBS 121662, DTO 220-H8	Dead hardwood branch	USA	KX923821	KX924255	KX924045
Y. penicillata		CBS 130296T, DTO 139-F4	Molded pork sample	China	JN831361	KY659807	KY659808	KY659809
Y. smithii sp. nov.	S. carbonaria	CBS 855.68T, DTO 354-C2	Garden soil	Germany	KX923822	KX924256	KX924046	KX924028

The T indicates the ex-type isolate of the synonymised species.

ATCC: American Type Culture Collection, Manassas, VA, USA; BMU: Beijing Medical University (Peking University), Beijing China; CBS: Culture Collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CGMCC: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; DTO: Working Collection of the Applied and Industrial Mycology Group of the Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; FMR: Facultat de Medicina i Ciències de la Salut, Reus, Spain; FRR: Division of Food Research, Food Research Laboratory, CSIRO, North Ryde, Australia; HACC: Research Laboratory, Hindustan Antibiotics Ltd., Pimpri, Pune, India; IFO: Institute for Fermentation Culture Collection, Osaka, Japan; IMI: Culture Collection of CABI Europe-UK, Egham, UK; LCP: Laboratory of Cryptogamy, National Museum of Natural History, Paris, France; MUCL: (Agro)Industrial Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Louvain-la Neuve, Belgium; NHL: National Institute of Hygienic Sciences, Tokyo, Japan; NRRL: ARS Culture Collection, U.S. Department of Agriculture, Peoria, IL, USA; RGR: Personal Collection of Rodney G. Roberts; UAMH: University of Toronto, UAMH Centre for Global Microfungal Biodiversity, Toronto, Canada; UTHSC: Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center, San Antonio, TX, USA; VKM: All-Russian Collection of Microorganisms, Moscow, Russia. Ex-epitype, -isotype, -type, and -neotype isolates are indicated with ET, IT, T and NT, respectively, and printed in bold.

DNA isolation, PCR and sequencing

DNA extraction was performed using the Ultraclean® Microbial DNA Isolation Kit (MoBio laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. The LSU, ITS, tub2 and tef1 gene regions were amplified and sequenced with respectively the primers LR0R (Rehner & Samuels 1994)/LR5 (Vilgalys & Hester 1990), V9G (De Hoog & Gerrits van den Ende 1998)/LS266 (Masclaux ), Bt2a/Bt2b (Glass & Donaldson 1995) and EF1-983F/EF1-2218R (Rehner & Buckley 2005). The PCRs were performed in an Applied Biosystems® 2720 Thermal Cycler (Thermo Fisher Scientific, Bleiswijk, the Netherlands) in a total volume of 12.5 μl. The PCR mixture consisted of 1 μl genomic DNA, 1 × NH4 reaction buffer (Bioline, Luckenwalde, Germany), 0.2 μM of each primer, 5 % dimethyl sulfoxide (DMSO), 20 μM (tub2) or 40 μM (LSU/ITS/tef1) of each dNTP, 1 mM (ITS) or 1.6 mM (tef1) or 2 mM (LSU/tub2) MgCl2, and 0.25 U Taq DNA polymerase (Bioline). The PCR conditions for LSU, ITS and tub2 consisted of an initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 47 °C (LSU) or 55 °C (ITS) or 59 °C (tub2) and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. For tef1 a touchdown PCR protocol of 9 cycles of 30 s at 94 °C, 30 s at 66 °C (−1 °C every cycle) and 90 s at 72 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 56 °C and 90 s at 72 °C and a final elongation step of 7 min at 72 °C was used. The PCR products were sequenced in both directions using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and analysed with an ABI Prism 3730xl DNA Analyser (Thermo Fisher Scientific) according to the manufacturer's instructions. Consensus sequences were computed from forward and reverse sequences using the Bionumerics v. 4.61 software package (Applied Maths, St-Marthens-Latem, Belgium). Several sequences obtained in this study had one or multiple nucleotide differences and length differences with already published sequenced of the same isolates. All new sequences and sequences which were longer in length or had nucleotide differences with already published sequenced were submitted to GenBank (Table 1).

Phylogenetic analyses

Multiple sequence alignments of the separate LSU, ITS, tub2 and tef1 sequences were generated with MAFFT v. 7.271 (http://mafft.cbrc.jp/alignment/server/index.html) using the L-INS-i method. With Findmodel (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html) the best nucleotide substitution models were determined. On both the single gene-sequence alignments and the combined gene-sequence alignment Bayesian analyses were performed with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist, 2001, Ronquist and Huelsenbeck, 2003). The Markov Chain Monte Carlo (MCMC) analysis used four chains and started from a random tree topology. The sample frequency was set at 1 000 and the temperature value of the heated chain was set at 0.1. The run stopped when the average standard deviation of split frequencies reached below 0.01. Burn-in was set to 25 % after which the likelihood values were stationary. Tracer v. 1.5.0 (Rambaut & Drummond 2009) was used to confirm the convergence of chains. Maximum-likelihood analyses including 500 bootstrap replicates using RAxML v. 7.2.6 (Stamatakis & Alachiotis 2010) were additionally run on both the single gene-sequence alignments and the combined gene-sequence alignment. The resulting trees were printed with TreeView v. 1.6.6 (Page 1996) and, together with the alignments, deposited into TreeBASE (http://www.treebase.org).

In order to get optimal sequence alignments, the dataset was divided in three different phylogenies. Based on the ITS, tub2 and tef1 sequences a separate species phylogeny for Microascus (157 isolates) and for Scopulariopsis (89 isolates) was constructed. Following these species phylogenies a genus phylogeny including one isolate per recognised species (when present the ex-type isolate) was constructed based on the LSU, ITS and tef1 sequences (52 isolates). The tub2 sequences were omitted in the genus phylogeny because of alignment difficulties. Based on former phylogenetic studies (Jagielski et al., 2016, Sandoval-Denis et al., 2016) Pithoascus stoveri (CBS 176.71) was used as out-group in the Microascus phylogeny, Pseudoscopulariopsis schumacheri (CBS 435.86) in the Scopulariopsis phylogeny, and Cephalotrichum stemonitis (CBS 103.19) in the genus phylogeny.

Habitat study

All studied Microascus, Scopulariopsis and Yunnania isolates are assigned to one of nine different habitats (animal, dung, food, human, indoor, insect, plant, soil, others) based on their origin of isolation. Subsequently, these habitats are plotted behind the species name, to which the isolate belongs, in the genus phylogeny tree. The habitats from isolates studied by others (Ropars et al., 2012, Jagielski et al., 2016, Sandoval-Denis et al., 2016) are also included. Finally, a table showing which Microascus, Scopulariopsis and Yunnania species are present in which habitat is constructed.

Morphology

Cultures were incubated on oatmeal agar (OA), malt extract agar (MEA) and dichloran 18 % glycerol agar (DG18) plates (recipes Samson ) at 25 °C in the dark. After 14 d the colony diameters were measured and the colony characters noted. Colony colours were rated according to Rayner (1970). Measurements and descriptions of microscopic structures were made from cultures grown on synthetic nutrient agar (SNA, Samson ) at 25 °C in the dark for 14 d or longer to ensure ascomata development. Slide preparations of the asexual morph structures were made with the sellotape technique (Schubert ) or mounted in 85 % lactic acid, like the sexual morph structures. Photographs of characteristic structures were made with a Zeiss Axio Imager A2 microscope equipped with a Nikon DS-Ri2 high-definition colour camera head using differential interference contrast (DIC) optics and the Nikon software NIS-elements D v. 4.50. Furthermore, growth at 36 °C and 40 °C in the dark on OA was tested.

Results

Microascus phylogeny

For the phylogeny 157 isolates were selected to represent the genus Microascus (Table 1) including the outgroup-isolate Pithoascus stoveri (CBS 176.71). The aligned sequences of the ITS (474 characters), tub2 (529 characters), and tef1 (898 characters) gene regions had a total length of 1 901 characters, with respectively 147, 253, and 221 unique site patterns. The GTR model with a gamma-distributed rate variation was suggested as model for the ITS and tef1 alignments and the HKY model with a gamma-distributed rate variation for the tub2 alignment. After discarding the burn-in phase trees, the multi-gene Bayesian analysis resulted in 4 172 trees from two runs from which the majority rule consensus tree and posterior probabilities were calculated. The multi-gene phylogeny divided the isolates in 33 Microascus species (clades, Fig. 1), and three Yunnania species (clades, Fig. 1). As a result of this study, seven new Microascus species (M. appendiculatus, M. cleistocarpus, M. fusisporus, M. hollandicus, M. micronesiensis, M. pseudopaisii, and M. trautmannii), one new Yunnania species (Y. smithii), one new name (M. atrogriseus) and two new name combinations (M. melanosporus, and Y. carbonaria) are proposed. The recently established genus Fuscoannellis is synonymised under Yunnania. All descriptions are provided below in the taxonomy section. For the genus Microascus, only the tef1 phylogeny can distinguish all identified species. This is in congruence with Ropars and Jagielski . With tub2, the species M. restrictus and M. verrucosus cannot be separated, they can molecularly only clearly be distinguished based on their tef1 sequence (ITS 2 nt difference, tub2 1 nt difference, tef1 20 nt difference). The ITS single gene phylogeny is least distinctive. Besides M. restrictus and M. verrucosus, M. alveolaris and M. terreus, M. intricatus and M. onychoides, M. paisii and M. melanosporus and the three Yunnania species cannot be separated based on their ITS sequences. Furthermore, the four isolates of M. cinereus are split into two clades based on their ITS sequence alone (data not shown, all single gene phylogenies are submitted to TreeBase).

Fig. 1

Maximum likelihood tree based on the ITS, tub2 and tef1 sequences of 157 isolates representing the genera Microascus and Yunnania. The RAxML bootstrap support values ≥75 % (BS) and Bayesian posterior probabilities ≥0.95 (PP) are given at the nodes. Thickened lines indicate a BS of 100 % and a PP of 1.0. Ex-type strain numbers are in bold face and indicated with T (or NT when ex-neotype). Species names between parentheses represent synonymised species names. The tree was rooted to Pithoascus stoveri (CBS 176.71).

Scopulariopsis phylogeny

For the phylogeny 89 isolates were selected to represent the genus Scopulariopsis (Table 1) including the outgroup-isolate Pseudoscopulariopsis schumacheri (CBS 435.86). The aligned sequences of the ITS (441 characters), tub2 (502 characters), and tef1 (887 characters) gene regions had a total length of 1 830 characters, with respectively 58, 143, and 109 unique site patterns. The TrN model with a gamma-distributed rate variation was suggested as model for the ITS alignment, the GTR model with a gamma-distributed rate variation as model for the tef1 alignment and the HKY model with a gamma-distributed rate variation for the tub2 alignment. After discarding the burn-in phase trees, the multi-gene Bayesian analysis resulted in 2 214 trees from both runs from which the majority rule consensus tree and posterior probabilities were calculated. The multi-gene phylogeny divided the isolates in 12 species (clades, Fig. 2) of which four are proposed as new; S. africana, S. albida, S. caseicola and S. sexualis. Their descriptions are provided below in the taxonomy section. Both with only the tub2 or tef1 sequence all 12 species can be identified, although the S. candida isolates do not form a monophyletic clade. Based on ITS alone, only five species can be identified, S. alboflavescens, S. brevicaulis, S. flava, S. macurae and S. soppii (data not shown, all single gene phylogenies are submitted to TreeBase).

Fig. 2

Maximum likelihood tree based on the ITS, tub2 and tef1 sequences of 89 isolates representing the genus Scopulariopsis. The RAxML bootstrap support values ≥75 % (BS) and Bayesian posterior probabilities ≥0.95 (PP) are given at the nodes. Thickened lines indicate a BS of 100 % and a PP of 1.0. Ex-type strain numbers are in bold face and indicated with T (or NT or ET when ex-neotype or ex-epitype respectively). Species names between parentheses represent synonymised species names. The tree was rooted to Pseudoscopulariopsis schumacheri (CBS 435.86).

Genus phylogeny with habitat study

For the genus phylogeny (Fig. 3) 52 isolates were selected to represent all above recognised species in the genera Microascus, Scopulariopsis and Yunnania together with Pithoascus stoveri (CBS 176.71), Pseudoscopulariopsis schumacheri (CBS 435.86), Cephalotrichum asperulum (CBS 582.71) and the out-group isolate Cephalotrichum stemonitis (CBS 103.19). The phylogeny was constructed based on the LSU, ITS and tef1 sequences. The aligned sequences of the LSU (533 characters), ITS (474 characters), and tef1 (814 characters) gene regions had a total length of 1 821 characters, with respectively 93, 175, and 209 unique site patterns. The GTR model with a gamma-distributed rate variation was suggested as model for the ITS and tef1 alignments and the TrN model with a gamma-distributed rate variation for the LSU alignment. After discarding the burn-in phase trees, the multi-gene Bayesian analysis resulted in 1 180 trees from two runs from which the majority rule consensus tree and posterior probabilities were calculated. The habitats are plotted behind the species names in the genus tree (Fig. 3) and placed in an overview table depicting the species per habitat (Table 2). No specific clustering of habitat preference related to phylogenetic relationships can be found, the different habitats are scattered over the phylogenetic tree (Fig. 3).

Fig. 3

Maximum likelihood tree based on the LSU, ITS and tef1 sequences of 52 isolates. The RAxML bootstrap support values ≥75 % (BS) and Bayesian posterior probabilities ≥0.95 (PP) are given at the nodes. Thickened lines indicate a BS of 100 % and a PP of 1.0. When no collection number is mentioned behind the species name, the type-isolate is used. The tree was rooted to Cephalotrichum stemonitis (CBS 103.19). The habitats where the species are found are plotted behind the species names:  = indoor;  = animal;  = dung;  = food;  = human;  = insect;  = plant;  = soil;  = others.

Table 2

Current species within Microascus, Scopulariopsis and Yunnania per habitat.

Habitat	Species
Indoor	M. alveolaris, M. atrogriseus, M. chartarus, M. cirrosus, M. croci, M. gracilis, M. hollandicus, M. intricatus, M. melanosporus, M. micronesiensis, M. paisii, M. pseudopaisii, M. trautmannii, M. trigonosporus, S. asperula, S. brevicaulis, S. candida
Animal	M. cirrosus, M. pyramidus, S. alboflavescens, S. asperula
Dung	M. cirrosus, M. hyalinus, S. asperula, S. sexualis, S. macurae
Food	M. gracilis, M. longicollis, M. trigonosporus, S. asperula, S. brevicaulis, S. candida, S. caseicola, S. flava, Y. penicillata
Human	M. alveolaris, M. appendiculatus, M. brunneosporus, M. chinensis, M. cinereus, M. cirrosus, M. croci, M. expansus, M. gracilis, M. intricatus, M. longicollis, M. melanosporus, M. onychoides, M. paisii, M. restrictus, M. terreus, M. verrucosus, M. pseudolongirostris, S. alboflavescens, S. asperula, S. brevicaulis, S. candida, S. cordiae
Insect	M. longirostris, S. asperula, S. brevicaulis
Plant	M. alveolaris, M. cirrosus, M. croci, M. gracilis, M. melanosporus, M. paisii, M. senegalensis, M. terreus, M. trigonosporus, S. asperula, S. sexualis, S. soppii, Y. carbonaria
Soil	M. alveolaris, M. atrogriseus, M. cinereus, M. cirrosus, M. croci, M. fusisporus, M. gracilis, M. intricatus, M. longirostris, M. macrosporus, M. melanosporus, M. murinus, M. pyramidus, M. senegalensis, M. terreus, S. africana, S. albida, S. asperula, S. brevicaulis, S. candida, S. cordiae, Y. carbonaria, Y. smithii
Others	M. paisii, M. cleistocarpus, S. brevicaulis

Seventeen species are found in the indoor environment, 14 Microascus species and three Scopulariopsis species (Table 2). Most of them are only occasionally found in the indoor environment. Scopulariopsis brevicaulis (22 indoor isolates) and M. melanosporus (19 indoor isolates) are the most common indoor species, in number of isolates, followed by M. paisii (8 indoor isolates), S. candida (7 indoor isolates), and M. croci (5 indoor isolates). Ten species which are found indoor are also found in relation with humans (Fig. 3), but mostly only from skin or nail infections, and more rarely in other tissues like pulmonary tissue (e.g. M. cirrosus) or blood culture (e.g. S. brevicaulis). This needs to be taken into account when trying to indicate the risk for human health.

Scopulariopsis asperula can be found in all included habitats, followed by S. brevicaulis and M. cirrosus which both are found in six different habitats and M. gracilis found in five different habitats (Fig. 3). These species are all also found indoor, which is not surprisingly considering their non-selective habitats. Five species, M. chartarus, M. hollandicus, M. micronesiensis, M. pseudopaisii and M. trautmannii, are only found in the indoor environment. Of these five species, three are single isolate species and the other two only include two isolates (M. micronesiensis and M. pseudopaisii). The two isolates of M. pseudopaisii are isolated from the same place, and could be seen as duplicates. Microascus micronesiensis has been found in two different houses in Micronesia in different cities on separate occasions, and has therefore the most potential in being a true indoor species.

Taxonomy

Based on the multi-gene species phylogenies (Fig. 1, Fig. 2) 33 Microascus species, 12 Scopulariopsis species, and three Yunnania species are recognised. In total 12 new species (four Scopulariopsis, seven Microascus and one Yunnania species), and a new name and two new name combination are proposed, which descriptions are provided below. Additionally, all recent name changes are summarised in an overview table (Table 3).

Table 3

Overview of recent name changes in Scopulariopsis and scopulariopsis-like species.

Old name	Current name	Old name	Current name
Kernia hyalinus	Microascus hyalinus	Scopulariopsis brevicaulis var. glabra	Scopulariopsis candida
Masonia chartarum	Microascus chartarus	S. brumptii	Microascus paisii
M. grisea	Microascus atrogriseus	S. carbonaria	Yunnania carbonaria
Masoniella chartarum	Microascus chartarus	S. casei	Scopulariopsis flava
M. croci	Microascus croci	S. chartarum	Microascus chartarus
M. grisea	Microascus atrogriseus	S. croci	Microascus croci
M. tertia	Microascus croci	S. fusca	Scopulariopsis asperula
Microascus brevicaulis	Scopulariopsis brevicaulis	S. gracilis	Microascus gracilis
M. exsertus	Pithoascus exsertus	S. grylli	Scopulariopsis flava
M. griseus	Microascus cinereus	S. hibernica	Pseudoscopulariopsis hibernica
M. intermedius	Pithoascus intermedius	S. hominis	Scopulariopsis brevicaulis
M. manginii	Scopulariopsis candida	S. insectivora	Scopulariopsis brevicaulis
M. nidicola	Pithoascus nidicola	S. ivorensis	Scopulariopsis asperula
M. niger	Scopulariopsis asperula	S. koningii	Scopulariopsis brevicaulis
M. schumacheri	Pseudoscopulariopsis schumacheri	S. murina	Microascus murinus
M. soppii	Scopulariopsis soppii	S. paisii	Microascus paisii
M. stoveri	Pithoascus stoveri	S. penicilloides	Scopulariopsis brevicaulis
M. stysanophorus	Pseudoscopulariopsis schumacheri	S. roseola	Scopulariopsis asperula
M. trigonosporus var. terreus	Microascus terreus	S. rufulus	Scopulariopsis brevicaulis
Nephrospora manginii	Scopulariopsis candida	S. sphaerospora	Microascus paisii
Pithoascus schumacheri	Pseudoscopulariopsis schumacheri	S. stercoraria	Scopulariopsis brevicaulis
P. stysanophorus	Pesudoscopulariopsis schumacheri	S. trigonospora	Microascus trigonosporus
Scopulariopsis arnoldii	Scopulariopsis asperula	S. versicolor	Microascus paisii
S. atra	Pithoascus ater	Torula asperula	Scopulariopsis asperula
S. aurea	Scopulariopsis flava	T. bestae	Scopulariopsis asperula
S. bestae	Scopulariopsis asperula	T. paisii	Microascus paisii
S. brevicaulis var. alba	Scopulariopsis flava

Woudenb. & Samson sp. nov. MycoBank MB818278. Fig. 4.

Fig. 4

Microascus appendiculatus sp. nov. CBS 594.78. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D. Ascoma. E. Ascomatal wall. F. Asci and ascospores. G. Ascospores. H, J–K. Conidiophores, annellides and conidia. I. Conidia. Scale bars = 10 μm.

Etymology: name refers to its conidia with a basal appendage.

Ascomata abundant, immersed, ostiolate, globose to subglobose with a short (up to 45 μm long) cylindrical ostiolar neck, (134–)158–208(–218) μm diam., black, glabrous; peridium with a textura angularis. Asci irregularly ellipsoidal, (19.5–)21–24.5(–25) × (10–)12.5–17.5(–20) μm. Ascospores fusiform, (5.5–)6.5–7.5(–8) × (3.5–)4–4.5(–5) μm, honey, pale luteous in mass, smooth, with a single inconspicuous germ pore. Conidiophores arising from substrate mycelium, indistinctive or simple, rarely branched, bearing terminally a single annellide. Annellides lageniform to ampulliform, (6–)7.5–11(–13.5) μm long, (2–)2.5–3(–3.5) μm broad at the widest part, tapering abruptly to a cylindrical annellate zone 0.5–1(–1.5) μm wide, hyaline to subhyaline, smooth-walled. Conidia subglobose with small basal appendage, (5–)5.5–7 × (3.5–)4–5(–5.5) μm, subhyaline, older conidia covered with hazel mucilaginous coating, smooth, thick-walled, arranged in short chains.

Culture characteristics: Colonies on OA attaining a diameter of 27–28 mm after 14 d at 25 °C, flat, white to cream-coloured with smoke grey zones and olivaceous grey ascomata, margin undulated. On MEA attaining a diameter of 17–19 mm, convex, white to cream-coloured, radially striated with dentate margin. On DG18 attaining a diameter of 19 mm, low convex, white to cream-coloured with partly a grey olivaceous ring close to the edge, margin undulate. On OA able to grow at 36 and 40 °C.

Specimen examined: Algeria, from human skin, collection date and collector unknown, (holotype CBS H-22744, culture ex-type CBS 594.78).

Notes: The ex-type strain of M. appendiculatus (CBS 594.78) was recently published as M. senegalensis (Jagielski ). However, the sequences of their two included M. senegalensis isolates deposited on GenBank (which are confirmed by resequencing the isolate) only have 94 % identity based on ITS, 99 % on LSU, 90 % on tub2 and 96 % on tef1. Although they seem to cluster together in their phylogenetic tree, our phylogenies (all single gene phylogenies, and the combined phylogeny Fig. 2) places CBS 594.78 as a separate species, which is described here as M. appendiculatus. Also morphologically it is distinct from M. senegalensis with the subglobose conidia with small basal appendage and the hazel mucilaginous coating around the older conidia.

Woudenb. & Samson nom. nov. MycoBank MB818284. Fig. 5.

Fig. 5

Microascus atrogriseus nom. nov. A–C. Fourteen day old colonies of DTO 139-D7 on OA (A), MEA (B) and DG18 (C). D–F. Conidiophores, annellides and conidia CBS 295.52. G. Conidiophores, annellides and conidia DTO 139-D7. H. Conidia DTO 191-C2. Scale bars = 10 μm.

Basionym: Masonia grisea G. Sm., Trans. Brit. Mycol. Soc. 35: 149. 1952, non Microascus griseus P.N. Mathur & Thirum,. 1963.

≡ Masoniella grisea (G. Sm.) G. Sm., Trans. Brit. Mycol. Soc. 35: 237. 1952.

Etymology: name refers to the original description of the basionym where “atro-griseis coloniis” are described.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or indistinctive, bearing one or multiple annellides. Annellides ampulliform, (4.5–)5.5–8.5(–10) μm long, 2–3 μm broad at the widest part, tapering abruptly to a cylindrical annellate zone 1–1.5(–2) μm wide, hyaline, smooth-walled. Conidia broadly ellipsoidal to short clavate with truncate base, (3–)3.5–4(–4.5) × (2.5–)3(–3.5) μm, hyaline when young turning hazel when ageing, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 17–20 mm after 14 d at 25 °C, flat, white to cream-coloured with (pale) olivaceous grey to iron grey centre, margin crenated. On MEA attaining a diameter of 13–14 mm, convex, white to very pale olivaceous grey, radially striated edge with crenated margin. On DG18 attaining a diameter of 10–20 mm, low convex, white to pale olivaceous grey, margin entire. On OA no growth at 36 and 40 °C.

Specimens examined: England, London, isolated as culture contaminant, 1946, G. smith, (culture ex-type CBS 295.52). Germany, indoor environment, before Aug. 2010, collector unknown, DTO 139-D7. Netherlands, from a swab sample of an indoor horse arena, Mar. 2012, Houba, DTO 191-C2.

Notes: Microascus atrogriseus is morphologically indistinguishable from M. paisii. Sequence data is necessary to distinguish it from M. paisii. All three genes used in this manuscript can separate the two species (ITS 5 nt difference, tub2 20 nt difference, and tef1 9 nt difference between the type isolates of both species).

Woudenb., X. Wei Wang & Samson sp. nov. MycoBank MB803264. Fig. 6.

Fig. 6

Microascus cleistocarpus sp. nov. CBS 134638. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D, G. Ascomata. E. Ascomatal wall. F. Asci and ascospores. H–J. Conidiophores, annellides and conidia. K. Conidia. Scale bars = 10 μm.

Etymology: named after the non-ostiolate ascomata.

Ascomata abundant, immersed, non-ostiolate, globose to subglobose, (50–)52–71(–83) μm diam., dark brown, glabrous; peridium with a textura angularis. Asci ovoid to subglobose, (11–)11.5–14(–15) × (7–)7.5–9.5(–10.5) μm. Ascospores broad fusiform to ellipsoidal, (6.5–)7–8 × 4–5 μm, buff to honey, smooth, with a single inconspicuous germ pore. Conidiophores arising from substrate mycelium, indistinctive, simple or occasionally branched, bearing terminally a single annellide. Annellides lageniform to ampulliform, (8–)9–14.5(–17.5) μm long, (2–)2.5–3 μm broad at the widest part, tapering gradually to a cylindrical annellate zone 1–1.5(–2) μm wide, hyaline to subhyaline, smooth-walled. Conidia obovoid with truncate base, (4.5–)5–6(–6.5) × 3.5–4.5 μm, hyaline, turning to hazel when ageing, smooth or finely roughened, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 21–24 mm after 14 d at 25 °C, flat, white to cream-coloured with greenish olivaceous to olivaceous buff and pale olivaceous grey zones, radially striated with dentate margin. On MEA attaining a diameter of 18–19 mm, crateriform, pale olivaceous grey to olivaceous grey and greyish sepia, radially striated and folded in the centre, margin crenated. On DG18 attaining a diameter of 13–14 mm, crateriform, buff to rosy buff, radially striated and folded in the centre, margin undulate. On OA still growth at 36 °C, no growth at 40 °C.

Specimen examined: China, Inner Mongolia, Ulanqab city, Huade county, from discarded cloth, 24 Jul. 2011, Y-Y Huo, (holotype HMAS 2444424, culture ex-type CBS 134638 = CGMCC 3.15222).

Notes: The newly described M. cleistocarpus is closely related to M. hyalinus (Fig. 1). Microascus cleistocarpus and M. hyalinus are the only two species in Microascus producing cleistothecial ascocarps. However, M. hyalinus produces hyaline conidia and M. cleistocarpus has hazel conidia. Based on sequence data M. cleistocarpus can be distinguished from M. hyalinus on all three genes (ITS 3 nt difference, tub2 5 nt difference, tef1 10 nt difference).

Woudenb. & Samson sp. nov. MycoBank MB818280. Fig. 7.

Fig. 7

Microascus fusisporus sp. nov. CBS 896.68. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–G. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Etymology: name refers to the fusiform conidia.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or branched, occasionally indistinctive, bearing one to multiple annellides. Annellides ampulliform, (7–)9–12(–14) μm long, 2–3(–3.5) μm broad at the widest part, tapering gradually to a cylindrical annellate zone, sometimes thickened, 1–1.5(–2) μm wide, hyaline, smooth-walled. Conidia obovoid to broad clavate or fusiform, with truncate base, (5–)5.5–6.5 (–7) × 2.5–3.5 μm, hyaline or subhyaline, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 15 mm after 14 d at 25 °C, flat, white at the margin with grey olivaceous to olivaceous centre, margin crenated. On MEA attaining a diameter of 10–12 mm, convex, olivaceous grey with white to cream-coloured sectors and margin, margin crenated. On DG18 attaining a diameter of 15–18 mm, crateriform, cinnamon with buff tufts of mycelium at the outer ring, olivaceous grey with white velvet mycelium at the centre, margin dentate. On OA no growth at 36 and 40 °C.

Specimen examined: Germany, Schleswig-Holstein, Kiel-Kitzeberg, from wheat-field soil, collection date unknown, K.H. Domsch & W. Gams, (holotype CBS H-22743, culture ex-type CBS 896.68 = ATCC 16278 = IFO 31244 = MUCL 8989).

Notes: Morphologically M. fusisporus resembles M. trautmannii, but can be distinguish based on its shorter annellides (9–12 μm long in M. fusisporus against 16–22 μm long in M. trautmannii) and the ability to grow on OA at 36 °C of M. trautmannii. Both species can easily be distinguished from the other M. paisii-like species based on their obovoid to broad clavate or fusiform conidia with truncate base.

Woudenb. & Samson sp. nov. MycoBank MB818279. Fig. 8.

Fig. 8

Microascus hollandicus sp. nov. CBS 141582. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–F. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Etymology: name refers to the country of isolation, the Netherlands.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or indistinctive, bearing one or multiple annellides. Annellides ampulliform, (3.5–)4–6(–8) μm long, (2.0–)2.5–3(–3.5) μm broad at the widest part, tapering abruptly to a cylindrical annellate zone 1–1.5 μm wide, hyaline, smooth-walled. Conidia broadly ellipsoidal to short clavate with truncate base, (3.5–)4–4.5(–5) × (2.5–)3–3.5(–4) μm, hyaline when young turning honey when ageing, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 17–18 mm after 14 d at 25 °C, flat to slightly raised, white to cream-coloured with olivaceous grey to olivaeous buff centre, margin dentate. On MEA attaining a diameter of 12–13 mm, raised, white to very pale olivaceous grey, radially striated with crenated margin. On DG18 attaining a diameter of 10–11 mm, raised, olivaceous grey, woolly with long white mycelium hairs growing out, margin entire. On OA no growth at 36 and 40 °C.

Specimen examined: Netherlands, from a swab sample of an indoor horse arena, Mar. 2012, Houba, (holotype CBS H-22716, culture ex-type CBS 141582).

Notes: Microascus hollandicus morphologically resembles M. pseudopaisii. Sequence data is necessary to distinguish both species. All three genes used in this manuscript can separate the two species (ITS 8 nt difference, tub2 21 nt difference, and tef1 10 nt difference between the type isolates of both species). Microascus hollandicus and M. pseudopaisii can be differentiated from the other M. paisii-like species by their shorter annellides (4–6 μm long).

(Udagawa) Woudenb. & Samson comb. nov. MycoBank MB817657. Fig. 9.

Fig. 9

Microascus melanosporus comb. nov. DTO 255-B1. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–F. Conidiophores, annellides and conidia. G. Conidia. Scale bars = 10 μm.

Basionym: Scopulariopsis melanospora Udagawa, J. agric. Sci. (Tokyo) 5: 18. 1959.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or indistinctive, bearing one to multiple annellides. Annellides ampulliform, (5.5–)7.5–11(–13) μm long, (2–)2.5–3.5(–4) μm broad at the widest part, tapering abruptly to a cylindrical annellate zone 1–1.5(–2) μm wide, hyaline, smooth-walled. Conidia broadly ellipsoidal to short clavate with truncate base, 4–4.5(–5) × (2.5–)3–3.5 μm, hyaline when young turning hazel when ageing, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 21–25 mm after 14 d at 25 °C, low convex, white to cream-coloured with olivaceous grey to iron grey zones, margin crenated. On MEA attaining a diameter of 19–23 mm, convex or crateriform, pale olivaceous grey to olivaceous grey, margin undulate. On DG18 attaining a diameter of 20–24 mm, crateriform, white to cream-coloured with grey olivaceous to greyish sepia and vinaceous grey zones, margin undulate. On OA some isolates grow at 36 °C, no growth at 40 °C.

Specimens examined: Germany, from indoor air sample, 2013, C. Trautmann, DTO 255-A5; from indoor air sample, 2013, C. Trautmann, DTO 255-A7; from plaster, 2013, C. Trautmann, DTO 255-B1. South Africa, Somerset West, from house dust, 12 Feb. 2009, Karin Jacobs, DTO 220-H9. USA, from milled Oryza sativa, 1955, S. Udagawa, (culture ex-type CBS 272.60 = MUCL 9040 = IMI 078257).

Notes: Microascus melanosporus is morphologically and phylogenetically closely related to M. paisii. Morphologically it can be differentiated from the other M. paisii-like species by its faster growth on OA and MEA at 25 °C (21–25 mm versus 16–20 mm and 19–23 versus 15–18 mm in diam. respectively). Based on sequence data both the tub2 and tef1 can separate M. melanosporus from the other M. paisii-like species. The ITS sequence is identical to M. paisii.

Woudenb., Seifert & Samson sp. nov. MycoBank MB818281. Fig. 10.

Fig. 10

Microascus micronesiensis sp. nov. CBS 141523. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–G. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Etymology: name refers to the country of isolation, Micronesia.

Sexual morph not observed. Conidiophores arising from substrate mycelium, indistinctive, simple or branched, bearing terminally one or occasionally two annellides. Annellides ampulliform, (6–)7–11(–14) μm long, 2–2.5(–3) μm broad at the widest part, tapering gradually to a cylindrical annellate zone (0.5–)1–1.5 μm wide, hyaline, smooth-walled. Conidia broadly obovoid with truncate base, 3–4(–4.5) × (2–)2.5–3(–3.5) μm, hyaline or subhyaline, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 15–17 mm after 14 d at 25 °C, flat, white to cream-coloured with pale grey olivaceous to grey olivaceous rings, margin undulated. On MEA attaining a diameter of 16–17 mm, low convex, white to cream-coloured, margin undulate. On DG18 attaining a diameter of 10–11 mm, low convex, white to cream-coloured, margin undulate. On OA reduced growth at 36, no growth at 40 °C.

Specimens examined: Micronesia, Kosrae, Kosrae Island, Malem, from house dust, 15 Mar. 2009, Wayne Law, (holotype CBS H-22739 culture ex-type CBS 141523); Kosrae, Kosrae Island, Tofol, from house dust, 2009, Wayne Law, DTO 223-A5.

Notes: Phylogenetically M. micronesiensis is closely related to the two recently described sexual species M. brunneosporus (Sandoval-Denis ) and M. chinensis (Jagielski ). Morphologically M. micronesiensis can be distinguished from M. brunneosporus and M. chinensis by the lack of producing sexual structures in culture and its much slower growth on OA at 25 °C (15–17 mm for M. micronesiensis versus 21–25 and 25–28 mm for M. brunneosporus and M. chinensis respectively after 14 d). Microascus micronesiensis has been found in house-dust samples from two different houses in Micronesia in different cities on separate occasions.

(Pollacci) Sandoval-Denis, Gené & Guarro, Persoonia 36:21. 2016. Fig. 11.

Fig. 11

Microascus paisii DTO 255-B2. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–G. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Basionym: Torula paisii Pollacci (as ‘pais’), Atti Ist. Bot. Univ. Pavia, ser. 2, 18:130. 1921.

≡ Phaeoscopulariopsis paisii (Pollacci) M. Ota, Jap. J. Dermatol. Urol. 28:5. 1928. nom. inval.

≡ Scopulariopsis paisii (Pollacci) Nann., Repert. Sist. dei Miceti dell'Uomo e degli Anim.: 259. 1934.

= Scopulariopsis sphaerospora Zach, Oesterr. Bot. Z. 83: 180. 1934.

= Scopulariopsis brumptii Salv.-Duval, Thèse Fac. Pharm. Paris. 23: 58. 1935.

= Scopulariopsis versicolor Salv.-Duval, Thèse Fac. Pharm. Paris 23: 63.1935.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or indistinctive, bearing one or multiple annellides. Annellides ampulliform, (5.5–)6.5–9.5(–12) μm long, (2–)2.5–3(–3.5) μm broad at the widest part, tapering abruptly to a cylindrical annellate zone 1–1.5(–2) μm wide, hyaline, smooth-walled. Conidia broadly ellipsoidal to short clavate with truncate base, 3.5–4(–4.5) × 3–3.5(–4) μm, hyaline when young turning hazel when ageing, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 17–20 mm after 14 d at 25 °C, low convex, white to cream-coloured with olivaceous grey to iron grey centre, margin crenated. On MEA attaining a diameter of 15–18 mm, crateriform, olivaceous grey to iron grey, radially striated, margin crenated. On DG18 attaining a diameter of 19–22 mm, crateriform, vinaceous buff with purplish grey zones at the margin and greyish sepia centre, margin entire. On OA some isolates grow at 36 °C, no growth at 40 °C.

Specimens examined: Austria, from unknown substrate, 1934, F. Zach, (S. sphaerospora culture ex-type CBS 402.34 = MUCL 9045). France, from small-pox vaccine, 1935, M. Langeron, (probably S. brumptii culture ex-type CBS 333.35). Germany, from plaster, 2013, C. Trautmann, DTO 255-B2; from oriented strand board, 2013, C. Trautmann, DTO 255-B8. Italy, from human, 1927, G. Pollacci (T. paisii culture ex-type CBS 213.27 = MUCL 7915).

Notes: The new name combination Microascus paisii for Torula paisii was recently proposed, together with the synonymy of several well-known species underneath it (Sandoval-Denis ). In this manuscript two synonymies are reinstated, namely Masonia grisea as Microascus atrogriseus and Scopulariopsis melanospora as Microascus melanosporus, and four new species are described, M. fusisporus, M. hollandicus, M. pseudopaisii and M. trautmannii. Morphologically M. fusisporus and M. trautmannii can be distinguished from M. paisii by their shape of conidia (see notes of the respective species), and M. pseudopaisii and M. hollandicus by their shorter annellides (see notes of the respective species). Microascus melanosporus can be distinguished by its faster growth rate on OA and MEA at 25 °C (see notes of M. melanosporus). Microascus atrogriseus is morphological identical to M. paisii, molecular data is necessary to distinguish the species (see notes of M. atrogriseus). CBS 333.35 isolated from small-pox vaccine in France is recognised here as the probable ex-type isolate of S. brumpti. It was deposited to the CBS in 1935 by Prof. Dr. Langeron who worked in the Université de Paris, Faculté de Médecine. The original description of S. brumptii by Salvanet-Duval was published in Thése Faculté de Pharmacie at the Université de Paris, on research on the small-pox vaccine (Salvanet-Duval 1935). All three studied ex-type isolates (CBS 213.27, CBS 402.34 and CBS 333.35) showed reduced growth and are therefore excluded from the culture descriptions.

Woudenb. & Samson sp. nov. MycoBank MB818282. Fig. 12.

Fig. 12

Microascus pseudopaisii sp. nov. CBS 141581. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–H. Conidiophores, annellides and conidia. Scale bars = 10 μm.

Etymology: name refers to the morphological and phylogenetic close relationship to M. paisii.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or branched, occasionally indistinctive, bearing one to multiple annellides. Annellides lageniform to ampulliform, (3.5–)4.5–6(–6.5) μm long, 2–3(–3.5) μm broad at the widest part, tapering abruptly to a cylindrical annellate zone 1–1.5 μm wide, hyaline, smooth-walled. Conidia broadly ellipsoidal to short clavate with truncate base, (3–)3.5–4.5(–5) × 2.5–3(–3.5) μm, hyaline when young turning honey when ageing, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 17–20 mm after 14 d at 25 °C, slightly raised, white to cream-coloured with olivaceous grey centre, margin crenated. On MEA attaining a diameter of 14–15 mm, crateriform, olivaceous grey with white to cream-coloured edge, radially striated with crenated margin. On DG18 attaining a diameter of 10–12 mm, crateriform, pale olivaceous grey, woolly with long white mycelium hairs growing out, margin entire. On OA no growth at 36 and 40 °C.

Specimens examined: Netherlands, Nederwetten, from an air sample of the basement of a house, 16 Dec. 2009, J. Houbraken, (holotype CBS H-22715, culture ex-type CBS 141581); additional strain from the same source, DTO 116-A4.

Notes: Microascus pseudopaisii morphologically resembles M. hollandicus. Sequence data is necessary to distinguish both species (see notes M. hollandicus). Microascus hollandicus and M. pseudopaisii can be differentiated from the other M. paisii-like species by their shorter annellides (4–6 μm long).

Woudenb. & Samson sp. nov. MycoBank MB818283. Fig. 13.

Fig. 13

Microascus trautmannii sp. nov. CBS 141583. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–G. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Etymology: named after Dr. Christoph Trautmann, who collected numerous Microascus isolates from the indoor environment.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or indistinctive, bearing terminally one or multiple annellides. Annellides slender ampulliform, (13.5–)16–22(–25) μm long, (1.5–)2–2.5(–3) μm broad at the widest part, with a sometimes thickened cylindrical annellate zone (1–)1.5–2(–2.5) μm wide, hyaline, smooth-walled. Conidia obovoid to broad clavate or fusiform, with truncate base, (5–)5.5–6.5(–7) × (2–)2.5–3 μm, hyaline or subhyaline, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 17 mm after 14 d at 25 °C, flat to slightly raised, white to cream-coloured with olivaceous grey centre, margin dentate. On MEA attaining a diameter of 13–16 mm, raised, buff to (pale) olivaceous grey with zones of white woolly mycelium, edge radially striated with crenated margin. On DG18 attaining a diameter of 10–11 mm, crateriform, pale olivaceous grey to pale greenish grey, margin entire. On OA reduced growth at 36 °C, no growth at 40 °C.

Specimen examined: Germany, from oriented strand board, C. Trautmann, 2013 (holotype CBS H-22717, culture ex-type CBS 141583).

Notes: Morphologically M. trautmannii resembles M. fusisporus, but they can be distinguished based on the size of their annellides and the growth at 36 °C on OA (see notes M. fusisporus). Both species can easily be distinguished from the other M. paisii-like species based on their obovoid to broad clavate or fusiform conidia with truncate base.

Woudenb. & Samson sp. nov. MycoBank MB818274. Fig. 14.

Fig. 14

Scopulariopsis africana sp. nov. CBS 118736. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–F. Conidiophores, annellides and conidia. G–H. Conidia. Scale bars = 10 μm.

Etymology: name refers to the country of isolation, South Africa.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple to indistinctive, occasionally branched. Annellides cylindrical to slight ampulliform, (5–)8–15(–21.5) μm long, (2–)3–4(–4.5) μm broad at the widest part, tapering gradually to a cylindrical annellate zone 2– 3(–3.5) μm wide, hyaline, smooth-walled. Conidia subglobose to broadly ovoid with truncate base, (5.5–)6–7(–8) × (4–)4.5–5.5(–6) μm, hyaline, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 35 mm after 14 d at 25 °C, flat, white to cream-coloured with olivaceous zones, margin crenated. On MEA attaining a diameter of 12–14 mm, crateriform, white to cream-coloured, folded, margin crenated. On DG18 attaining a diameter of 25–28 mm, low convex, white to cream-coloured, margin undulate to erose to fimbriate. On OA no growth at 36 and 40 °C.

Specimen examined: South Africa, Free State, Lemoenskloof, from mud sample from salt pan, before Sep. 2004, M.E. Setati, (holotype CBS H-22741, culture ex-type CBS 118736).

Notes: Morphologically S. africana resembles S. albida, S. candida and S. alboflavescens, although S. africana shows olivaceous zones on OA (S. albida and S. candida are characterised by white colonies, S. alboflavescens by white-cream to pale yellowish colonies). Molecularly, S. africana can be distinguished from other Scopulariopsis species based on its tub2 and tef1 sequence.

Woudenb. & Samson sp. nov. MycoBank MB818275. Fig. 15.

Fig. 15

Scopulariopsis albida sp. nov. CBS 119.43. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–G. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Etymology: name refers to the white colonies.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple to indistinctive. Annellides cylindrical to slight ampulliform, (6)8.5–19.5(–29.5) μm long, (2.5–)3–5(5.5) μm broad at the widest part, annellate zone (2–)2.5–3.5(–4) μm wide, single, hyaline, smooth-walled. Conidia globose to subglobose with truncate base, (5–)6.5–8(–8.5) × (6–)6.5–7.5(–8) μm, hyaline, smooth, thick-walled, arranged in chains.

Culture characteristics: Colonies on OA attaining a diameter of 55–60 mm after 14 d at 25 °C, flat, white to cream-coloured, margin entire. On MEA attaining a diameter of 20–23 mm, crateriform, white to cream-coloured, folded, margin undulate. On DG18 attaining a diameter of 21–25 mm, low convex, white to cream-coloured, folded, margin undulate to erose to fimbriate. On OA no growth at 36 and 40 °C.

Specimens examined: Netherlands, from soil, collection date and collector unknown (holotype CBS H-22740, culture ex-type CBS 119.43). Germany, substrate and collection date unknown, P. Höhle, CBS 415.51.

Notes: Morphologically S. albida resembles S. candida. Although S. candida does not form a monophyletic clade, S. albida can molecularly be distinguished from S. candida based on its tub2 and tef1 sequence. Also S. africana and S. alboflavescens morphologically resemble S. albida. Here the olivaceous zones on OA of S. africana isolates and the cream-white to pale yellowish colonies and subhyaline conidia of S. alboflavescens isolates can be used to distinguish the species.

Woudenb. & Samson sp. nov. MycoBank MB818276. Fig. 16.

Fig. 16

Scopulariopsis caseicola sp. nov. CBS 480.62. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–G. Conidiophores, annellides and conidia. H. Conidia. Scale bars = 10 μm.

Etymology: name refers to the substrate of isolation, cheese.

Sexual morph not observed. Conidiophores arising from substrate mycelium, simple or branched, bearing terminally a single annellide (at each branch). Annellides cylindrical, (10.5–)22.5–47.5(–67.5) × (2.5–)3–4(–5) μm, tapering gradually to a cylindrical annellate zone 2–3.5(–4) μm wide, subhyaline becoming darker with age, smooth-walled. Conidia broad ovoid with truncate base, (4.5–)6–7(–8) × (4–)5–6(–7) μm, buff to honey, smooth, thick-walled, arranged in long chains.

Culture characteristics: Colonies on OA attaining a diameter of 30 mm after 14 d at 25 °C, flat, white to opaque, margin entire. On MEA attaining a diameter of 22–23 mm, low convex, white to cream-coloured, margin undulate. On DG18 attaining a diameter of 7 mm, flat, pale olivaceous grey to smoke grey, margin fimbriate. On OA no growth at 36 and 40 °C.

Specimen examined: Netherlands, from cheese-coating, collection date unknown, M.B. Schol-Schwarz, (holotype CBS H-22738, culture ex-type CBS 480.62).

Note: Sporulation was only observed on OA after 2 months cultivation, re-isolation of a fresh culture might influence the morphological description. Morton & Smith (1963) discussed the synonyms of S. flava, a species frequently found on cheese. Among the synonyms they also discussed S. casei Loubière of which no type material is known to exist.

Woudenb. & Samson sp. nov. MycoBank MB818277. Fig. 17.

Fig. 17

Scopulariopsis sexualis sp. nov. CBS 250.64. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D. Ascomata. E. Ascomatal wall. F. Asci. G. Ascospores. Scale bars = 10 μm.

Etymology: name refers to the presence of only sexual structures and lack of asexual structures.

Ascomata abundant, superficial or immersed, ostiolate, globose with a short cylindrical ostiolar neck (up to 20 μm) or ovoid, 108–145(171) μm diam., dark brown to black, glabrous; peridium with a textura angularis. Asci irregularly ellipsoidal, (9.5–)10.5–12(–13) × 9–11.5(–13) μm. Ascospores reniform to broadly lunate, (4.5–)5–5.5(–6.5) × 3.5–4.5(–5) μm, buff to honey, luteous to orange in mass, smooth, with a single inconspicuous germ pore. Asexual morph not observed.

Culture characteristics: Colonies on OA attaining a diameter of 60–67 mm after 14 d at 25 °C, flat, dull green with white edge, margin entire. On MEA attaining a diameter of 47–57 mm, crateriform, white to cream-coloured with olivaceous grey to iron grey centre, radially striated with entire margin. On DG18 attaining a diameter of 38–40 mm, flat, white to cream-coloured, margin undulate to crenated. On OA still growth at 36 °C, no growth at 40 °C.

Specimens examined: Burma, from milled rice, 1954, S. Udagawa, (holotype CBS H-14445, culture ex-type CBS 250.64 = IFO 7555 = UAMH 1923 = NHL 2278). India, Delhi, from seed of Brassica oleracea (Brassicaceae), collection date unknown, K.G. Mukerji, CBS 332.78. USA, Arizona, Tucson, from bat dung, collection date unknown, G.F. Orr, CBS 667.71 = NRRL A-8022.

Note: Morphologically S. sexualis resembles the sexual morph of S. cordiae. Scopulariopsis sexualis can be differentiated from S. cordiae by its much faster growth on OA (60–70 mm for S. sexualis, 35–36 mm for S. cordiae at 25 °C after 14 d) and shorter cylindrical ostiolar neck (S. sexualis up to 20 μm, S. cordiae up to 390 μm).

H.Z. Kong, Mycotaxon 69: 320. 1998.

= Fuscoannellis Sandoval-Denis, Jagielski, Jin Yu & Gené, Fungal Biol. 120: 593. 2016.

(F.J. Morton & G. Sm.) Woudenb., Houbraken & Samson comb. nov. MycoBank MB820189.

Basionym: Scopulariopsis carbonaria F.J. Morton & G. Sm., Mycol. Pap. 86: 59. 1963.

≡ Fuscoannellis carbonaria (F.J. Morton & G. Sm.) Sandoval-Denis, Jagielski, Jin Yu & Gené, Fungal Biol. 120: 593. 2016.

Specimens examined: Panama, from soil, collection date unknown, R. Cogill, (culture ex-type CBS 205.61 = NRRL 1860 = IFO 8116 = MUCL 9027 = IMI 086941); USA, Hawaii, on dead hardwood branch, 3 Nov. 2002, D.T. Wicklow, CBS 121662.

Woudenb. & Samson sp. nov. MycoBank MB818273. Fig. 18.

Fig. 18

Yunnania smithii sp. nov. CBS 855.68. A–C. Fourteen day old colonies on OA (A), MEA (B) and DG18 (C). D–F. Conidiophores, annellides and conidia. G–H. Conidia. Scale bars = 10 μm.

Etymology: named after late George Smith (1895–1967), a British mycologist who extensively studied the closely related fungal genera Microascus and Scopulariopsis, and collected the type isolate.

Sexual morph not observed. Conidiophores arising from substrate mycelium, frequently branched, bearing terminally a group of annellides (at each branch). Annellides ampulliform, (4–)5–7 μm long, 2–2.5(–3) μm broad at the widest part, with a short annellate zone, (1–)1.5–2 μm wide, hyaline to subhyaline, smooth-walled. Conidia ovoid to ellipsoidal with truncate base, 4–5(–5.5) × 2–2.5(–3) μm, vinaceous buff to hazel, smooth, thick-walled, arranged in (long) chains.

Culture characteristics: Colonies on OA attaining a diameter of 33–35 mm after 14 d at 25 °C, flat, olivaceous to olivaceous grey, margin crenated. On MEA attaining a diameter of 26–30 mm, crateriform, greyish blue to (pale) olivaceous grey and slate blue, radially striated with dentate margin. On DG18 attaining a diameter of 29–30 mm, crateriform, pale olivaceous grey to olivaceous grey, radially striated, margin entire. On OA no growth at 36 and 40 °C.

Specimen examined: Germany, Kiel-Kitzeberg, from garden soil, 1963, G. Smith, (holotype CBS H-22742, culture ex-type CBS 855.68).

Notes: Yunnania smithii morphologically resembles the other two species in the genus Yunnania, Y. carbonaria and Y. penicillata. It can be distinguished from Y. penicillata by its faster growth on OA in 7 d (Y. smithii 33–35 mm, Y. penicillata 20 mm). The colour of the conidiophores and annellides can be used to distinguish it from Y. carbonaria (hyaline in Y. smithii, pale brown to brown in Y. carbonaria). Molecularly Y. smithii can best be distinguished based on its tef1 sequence (19 nt difference Y. carbonaria, 19 nt difference Y. penicillata), followed by its tub2 sequence (12 nt difference Y. carbonaria, 14 nt difference Y. penicillata). The LSU and ITS sequences are not suited for identification, the LSU sequences are all 100 % identical and the ITS sequence of Y. smithii is identical to Y. penicillata and has only 1 nt difference with the type isolate of Y. carbonaria.

Discussion

This manuscript presents a molecular phylogenetic study of species in the genera Microascus and Scopulariopsis known from culture, with the intention to identify the common indoor species. Since fungi present in indoor environments can produce toxins or carry allergens which cause health hazards, it is important to know which fungal species are present indoors. Scopulariopsis and scopulariopsis-like species are mainly found in soil, but also frequently isolated from food and building materials like drywall paper and wood (Samson ). Little is known about the health effects of these fungi, although several species seem to be able to cause human onychomycosis and superficial tissue infections (e.g. Tosti et al., 1996, Wu et al., 2009). Rare cases of more severe diseases are reported, but only in immunocompromised patients (e.g. Baddley et al., 2000, Miossec et al., 2011). The ability of Scopulariopsis species to deteriorate building materials (Gutarowska, 2014, Lavin et al., 2016), and to accumulate various elements and turning these into toxic volatiles (Cheng and Focht, 1979, Boriová et al., 2014) also makes them an important group to study in the indoor environment.

As stated in the results section, 17 species are mentioned in this manuscript to occur in the indoor environment (Table 2), but most of them are only occasionally found indoors. Besides the number of isolates found indoors, the substrate of isolation should also be taken into consideration when labelling species as indoor species. The isolates assigned to the indoor habitat include swab samples and house dust or air samples. Since swab sample are mostly taken from sites suspicious of fungal growth, they can (often) be related to actual indoor growth. A dust or air sample only implies the presence of the fungus. Since the concentration of fungal spores in the indoor air is to certain extent dependent on the outside spore concentration, it is recommended to also sample the outside air for comparison (Samson ). This information is not known for our isolates, and also information on the abundance of the species in the air or dust sample is unknown. Ten of the species which are mentioned here to occur in the indoor environment are also found in relation with humans (Fig. 3). However, isolates assigned in this study to the human habitat are mainly isolated from human-derived specimens, which is merely an indication of a possible pathogenic role. For the majority of the species known from human specimens there is no proven relation with disease (Sandoval-Denis ). Especially for isolates obtained from superficial sites and the upper respiratory tracts it should be taken into account that these can be environmental contaminants. Another point of attention is the indoor environment in which the species are found. How much time people spend in the different indoor environments (archives, homes, offices, stables, animal pens, etc.) varies, although all of them are treated here as indoor habitat. As example we will take M. alveolaris, only 1 out of 9 studied isolates is isolated from the indoor environment. This indoor isolate came from a house-dust sample, but no additional information is known on the abundance of the fungus in the dust sample. Additional information is needed to label M. alveolaris as true indoor species, although this study shows it can be found in homes. One other M. alveolaris isolate was human-derived, although an earlier study linked multiple human-derived isolates to this species (Sandoval-Denis ). Most of them are bronchoalveolar lavage isolates, as the name of the fungus already applies. Although for most of these isolates their relation with disease is not known, the finding of multiple isolates from the respiratory tract of human patients, and the ability of the species to grow at 40 °C are good indications of the potential pathogenicity of the species.

The most commonly found indoor species, both in swab and air/dust samples are M. melanosporus, M. paisii, S. brevicaulis and S. candida. All four are also placed in relation with the human habitat. Scopulariopsis brevicaulis and S. candida are known to be involved in onychomycosis, and S. brevicaulis is also recognised as important human opportunistic pathogens, as well as S. brumptii (now M. paisii) (De Hoog ). For M. melanosporus, which was previously regarded a synonym of S. brumptii (Morton & Smith 1963, as S. melanospora), the pathogenic abilities are unknown. However, one can expect that it can also act as opportunistic pathogen, as it close relatives, especially with the ability of some isolates to grow at 36 °C.

Based on the multi-gene phylogeny (Fig. 2) and congruent single gene trees (Fig. 19) the newly combined M. paisii (Sandoval-Denis ) is split in this study into seven species of which four (M. fusisporus, M. hollandicus, M. pseudopaisii, and M. trautmannii) are newly described, one was given a new name (M. atrogriseus) and one a new name combination (M. melanosporus). Scopulariopsis brumptii is still regarded as synonym of M. paisii (Table 3). Based on their tub2 and tef1 sequences all seven M. paisii-like species can be molecularly identified (Fig. 19). Microascus melanosporus seems to be the most prevalent species found indoor, and M. paisii is recognised as second most common indoor Microascus species. For the studied isolates M. melanosporus can morphologically be distinguished from M. paisii based on the growth rate and colony colour after 2 wk incubation on OA at 25 °C (Fig. 20). Microascus melanosporus grows slightly faster than M. paisii (21–25 mm versus 16–20 mm in diam. respectively), and M. melanosporus has slightly lighter grey colonies than M. paisii (Fig. 20A, B). Microascus fusisporus and M. trautmannii can be distinguished from the other M. paisii-like species based on their obovoid to broad clavate or fusiform conidia versus the broadly ellipsoidal to short clavate conidia from the other M. paisii-like isolates. Microascus hollandicus and M. pseudopaisii can be distinguished by their shorter annellides (4–6 μm long versus 6–11 μm long on average for the other M. paisii-like isolates). To distinguish M. atrogriseus from M. paisii and M. hollandicus from M. pseudopaisii molecular data is needed. The two most common indoor Scopulariopsis species, S. brevicaulis and S. candida, are both molecularly as morphologically easy to distinguish. Morphologically the growth rate and colony colour after 2 wk incubation on OA at 25 °C, and the conidia morphology can be used to distinguish the species (Fig. 20). Scopulariopsis brevicaulis grows faster than S. candida (75 mm and 38–48 mm in diam. respectively), and S. brevicaulis has buff to rosy buff colonies versus white colonies in S. candida (Fig. 20C, D). Another distinction are the roughened conidia of S. brevicaulis, versus the smooth conidia of S. candida (Fig. 20G, H). A growth test at 36 °C can also be used to distinguish the species since S. brevicaulis is able to grow at 36 °C and S. candida is not.

Fig. 19

Maximum likelihood trees based on respectively the ITS, tub2 or tef1 sequences of 56 isolates, representing the Microascus paisii clade and the out-group isolate M. hyalinus (CBS 766.70). The RAxML bootstrap support values ≥75 % (BS) and Bayesian posterior probabilities ≥0.95 (PP) are given at the nodes. Thickened lines indicate a BS of 100 % and a PP of 1.0. Species names between parentheses indicate the ex-type isolates of those species names. The red and orange printed isolates represent M. melanosporus, the blue printed isolates represent M. paisii, the green printed isolates represent M. atrogriseus and the pink printed isolates represent M. pseudopaisii.

Fig. 20

Most common indoor Microascus and Scopulariopsis species. A, E.M. melanosporus DTO 255-B1. B, F.M. paisii DTO 255-B2. C, G.S. brevicaulis CBS 118474. D, H.S. candida DTO 138-B7. A–D. Fourteen day old colonies on OA. E–H. Conidiophores, annellides and conidia. Scale bars = 10 μm.

Below we will discuss some phylogenetic unclarities which we encountered during this molecular phylogenetic study of Scopulariopsis and scopulariopsis-like species.

The phylogenetic position of M. longirostris and M. pseudolongirostris is doubtful. In our Microascus phylogeny, based on the ITS, tub2 and tef1 sequences, M. longirostris and M. pseudolongirostris cluster closest to the genus Yunnania rather than Microascus but without phylogenetic support (Fig. 2). In the genus tree, based on the LSU, ITS and tef1 sequences, M. longirostris and M. pseudolongirostris cluster closest to Microascus, although again without phylogenetic support (Fig. 3). We choose to keep them in the genus Microascus following earlier publications (Jagielski et al., 2016, Sandoval-Denis et al., 2016) although they will need further study. The genus Yunnania is supported as separate genus in the genera tree, which is congruent with the study of Jagielski . They already stated that the S. carbonaria isolates did not belong to Scopulariopsis, and proposed the new genus Fuscoannellis. However, since the genus Yunnania was described earlier (Kong 1998), this genus name has priority according to the rules of the ICN and Fuscoannellis will become a synonym of Yunnania. The CBS collection contained four isolates named Scopulariopsis carbonaria, including the type isolate CBS 205.61. However, the type isolate only clustered together with CBS 121662 (Fig. 2), which was originally stored as S. brumptii in the CBS collection (as was already noticed by Jagielski ). Two other ‘S. carbonaria’ isolates, CBS 687.68 and 253.69, cluster together in the genus Kernia (data not shown). CBS 855.68 does cluster with the type isolate of S. carbonaria based on its ITS sequence (only 1 nt difference), but based on its tub2 (12 nt difference) and tef1 (19 nt difference) sequence, in combination with morphological study, we describe it here as a new species Y. smithii. These “S. carbonaria” isolates form a good example of the problems with morphological identification in these genera. The isolate DTO 223-A6 clusters close to the type isolates of the recent described species M. intricatus and M. onychoides. These two species have identical ITS sequences, but differ 9 nt in their tub2 sequences, and 10 nt in their tef1 sequences. Isolate DTO 223-A6 clusters closest to M. onychoides, although it is not 100 % molecularly identical. Also based on morphology we could not clearly place DTO 223-A6 in one of the two species, since the measurements of the spores did not exactly match one of them. We choose however to name DTO 223-A6 M. onychoides for now, but collection of more isolates will be necessary to establish the species boundaries of M. intricatus and M. onychoides. The phylogenetic species M. terreus contains two supported clades (Fig. 2). Because the isolates in these two clades are morphologically identical, and are isolated from the same substrates, we choose to keep them as one species. Also in the species M. croci there is some sequence variation. Isolates CBS 158.44 and DTO 220-I5 deviate from the other M. croci isolates in their ITS sequence only (3 nt in a short stretch). DTO 305-B5 deviates on all three loci from the other isolates (ITS 3 nt, tub2 6 nt and tef 8 nt difference). We choose to keep it as a M. croci for now, since there is no support in the tree for the split. With the collection of more isolates, M. croci might split into two or maybe three Microascus species. The new species M. longicollis was published while preparing this manuscript (Crous , Fungal Planet description sheet 444). The morphological description matches the morphology of isolate CBS 752.97, stored as S. gracilis in the CBS collection. Sequence comparison of CBS 752.97 with the ex-type isolate of M. longicollis gave 99 % identity matches between all three sequenced loci. We therefore named CBS 752.97 M. longicollis. Multiple isolates stored as M. trigonosporus are now identified as M. alveolaris (Table 1). Also many stored as M. manginii now cluster in different species clades than the type isolate of M. manginii which falls within the S. candida clade. Four of them actually cluster within Scopulariopsis, corresponding to two new species (S. sexualis and S. africana). These are more examples of the problems with identification based on morphology in these genera. Microascus campaniformis CBS 138126 has been omitted from the study awaiting a new culture deposit. The isolate deposited to the CBS collection turned out to be a M. melanosporus isolate. The published sequences from the ex-type isolate were also not included in this study, since the tef1 sequence (GenBank HG380418) seems to belong to the M. melanosporus clade. The deposited ITS (GenBank LM652391), and tub2 (GenBank LM652606) sequences are unique sequences suggesting it is indeed a new species in Microascus. The ex-type isolate need to be recovered and re-sequenced to place it into the phylogenetic tree. Scopulariopsis candida has a lot of molecular variation and is non-monophyletic in the species phylogeny (Fig. 1). This is congruent with previous publications (Jagielski et al., 2016, Sandoval-Denis et al., 2016). No solution for this problem could be provided in this study, since the single gene trees were not congruent. Further study including more isolates will be necessary to solve this (for now) non-monophyletic species.

Conclusions

In the genus Microascus 33 phylogenetic species can be distinguished based on (parts) of the ITS, tub2 and tef1 gene regions. From these 33 species, seven are described here as new species, and one new name and new combination are proposed. Thirteen Microascus species are found in the indoor environment of which M. melanosporus is most commonly found, followed by M. paisii. In the genus Scopulariopsis 12 phylogenetic species can be distinguished based on (parts) of the ITS, tub2 and tef1 gene regions. From these 12 species, four are described here as new species. Three Scopulariopsis species are found in the indoor environment of which S. brevicaulis and S. candida are most common. No correlation was found between phylogenetic relationships and habitat preference in the genera Microascus and Scopulariopsis. The genus Fuscoannellis is placed in synonymy with Yunnania. The genus Yunnania, which is not known for indoor environments, now includes three species of which one is described here as new and one has a new name combination proposed.

Key to the most common Microascus, Scopulariopsis and Cephalotrichum species from the indoor environment

1a.	Synnemata absent …………………………………………………………… 2
1b.	Synnemata present …………………………………… Cephalotrichum, 8
2a.	Annellides lageniform or ampulliform …………………….. Microascus, 3
2b.	Annellides cylindrical …………………………………... Scopulariopsis, 7
3a.	Colony diam. on OA after 14 d at 25 °C > 20 mm ………………………………………. M. melanosporus
3b.	Colony diam. on OA after 14 d at 25 °C < 20 mm …………………………………………………………… 4
4a.	Conidia obovoid to broad clavate or fusiform ……………………………. 5
4b.	Conidia broadly ellipsoidal to short clavate ……………………………… 6
5a.	Annellides 9–12 μm long …………………………………… M. fusisporus
5b.	Annellides 16–22 μm long ………………………………… M. trautmannii
6a.	Annellides 4–6 μm long …………... M. hollandicus or M. pseudopaisii
6b.	Annellides 6–11 μm long …………………… M. atrogriseus or M. paisii
7a.	Roughened conidia, able to grow at 36 °C on OA ……………………………………………… S. brevicaulis
7b.	Smooth conidia, no growth at 36 °C on OA ………………………………………………….. S. candida
8a.	Coiled setae present on the upper part of the synnemata ………………………………………………... C. gorgonifer
8b.	Setae absent …………………………………………………………………. 9
9a.	Conidia distinctively rough ……………………………... C. verrucisporum
9b.	Conidia smooth ……………………………………………………………... 10
10a.	Conidia 3.5–5 × 2–3 μm ………………………………... C. microsporum
10b.	Conidia larger …... C. pseudopurpureofuscum or C. purpureofuscum