Botryotrichum Domesticum Sp. Nov., a New Hyphomycete from an Indoor Environment

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Botryotrichum domesticum sp. nov., a new hyphomycete from an indoor environment

Journal: Botany

Manuscript ID cjb-2018-0196.R2

Manuscript Type: Article

Date Submitted by the 14-Jan-2019 Author:

Complete List of Authors: Schultes, Neil; The Connecticut Agricultural Experiment Station, Department of Plant Pathology and Ecology Strzalkowski, Noelle; The Connecticut Agricultural Experiment Station, Department of Plant Pathology and Ecology Li, De-Wei;Draft The Connecticut Agricultural Experiment Station, Valley Laboratory

Keyword: asexual fungi, Chaetomium, Desertella, homonym, multi-loci

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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Botryotrichum domesticum sp. nov., a new hyphomycete from an indoor environment

Neil P. Schultes1*, Noelle Strzalkowski1 and De-Wei Li2, 3*

1 The Connecticut Agricultural Experiment Station, Department of Plant Pathology and Ecology,

123 Huntington Street, New Haven, CT 06511, USA. email: [email protected];

[email protected]

2 The Connecticut Agricultural Experiment Station, Valley Laboratory, 153 Cook Hill Road, Windsor, CT 06095, USA. email: [email protected]

3 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University,

Nanjing, Jiangsu 210037, China

*Corresponding authors

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Abstract

Here we report on a fungus that is new to science and was isolated from a swab sample collected in a Massachusetts (USA) residence. Morphological characters of the fungus were studied and

DNA sequences generated from ITS, LSU, rpb2, and tub2 ribosomal loci were used to establish a proper phylogenetic relationship with allied genera. The fungus was named Botryotrichum domesticum. The newly named species has thick-walled conidia globose to subglobose, 17.7 ±

2.6 × 17.3 ± 2.5 μm, developing on both aerial and immersed hyphae, with an absence of setae.

Key Words: asexual fungi, Chaetomium, Desertella, homonym, multi-loci, phylogeny, sexual state. Draft

Introduction

Indoor environments are distinctive man-made habitats for fungi. Despite the fact that new taxa from indoor environments are increasingly described, fungal diversity in indoor environments remains insufficiently studied. Microfungi that are often found in indoor environments are ordinarily known as indoor molds. These organisms have attracted enormous public attention in the last two decades due to concerns about potential health effects. Indoor environments are unique man-made habitats that include diverse substrates which can be colonized by fungi under permissive conditions (Flannigan and Miller 1994; Li and Yang 2004). Common examples include molds that develop on wet or damp wood, clothing, fabric, paper products, foodstuffs, due to leaks or flooding (Adan et al. 2011; Yang et al. 2016). Even indoor dust, composed of

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varied substrates, can nourish growth of some xerophilic fungi (Li and Yang 2004). Despite the

fact that several new taxa from indoor environments have been described in the past, fungal

diversity, ecological function, and evolution in indoor environments have not been sufficiently

studied.

Botryotrichum Sacc. & Marchal was erected and typified with Botryotrichum piluliferum

Sacc. & Marchal as an asexual species (Marchal 1885). Daniels (1961) found the sexual stage of

B. piluliferum and a mono-phialidic synanamorph (Acremonium-like); the sexual stage was

named Chaetomium piluliferum J. Daniels. In addition to the type species Botryotrichum

piluliferum, 11 epithets and two varieties are published to date. Botryotrichum murorum and Botryotrichum spirotrichum (≡ EmilmuelleriaDraft spirotricha (R.K. Benj.) Arx) are known only as a sexual stage and Botryotrichum lachnella Sacc. (= Peziotrichum lachnella) has been reassigned

to Nectriaceae and removed from Botryotrichum (Petch 1927; Wang et al. 2016a).

Botryotrichum nematophagus M.A. Santos et al. collected from nematode eggs in Brazil is nom.

inval., [Art. 40.3 (Melbourne Code)] (McNeill et al. 2012; Index Fungorum 2018). A later name

Emilmuelleria Arx is considered a synonym of Botryotrichum (MycoBank 2018).

Several species of Chaetomium and Farrowia (sexual genera) develop an asexual state

with Botryotrichum-like aleurioconidia (Daniels 1961; Hawksworth 1975; Seifert et al. 2011).

Chaetomium Kunze was established and typified with Chaetomium globosum Kunze based on its

sole sexual state (Kunze and Schmidt 1817). Wang et al. (2016a) studied Chaetomium sense lato

and chaetomium-like genera collected from indoor environments using multiple-locus

phylogenetic analysis (ITS, LSU, rpb2, and tub2) and segregated them into 14 genera, among

which five genera: Amesia X. Wei Wang, Samson & Crous, Arcopilus X. Wei Wang, Samson &

Crous, Collariella X. Wei Wang, Samson & Crous, Dichotomopilus X. Wei Wang, Samson &

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Crous, Ovatospora X. Wei Wang, Samson & Crous were newly erected. Their phylogenetic analyses showed a monophyletic lineage containing B. piluliferum, B. atrogriseum, B. peruvianum, Chaetomium murorum (≡ Botryotrichum murorum) and Emilmuelleria spirotricha

(≡ Botryotrichum spirotrichum), the type species of the monotypic genus Emilmuelleria (the extype of generic type used in this study). Wang et al. (2016a) kept the genus Botryotrichum based on its generic type B. piluliferum, but included the species of both asexual and sexual species and moved Chaetomium murorum and the genus Emilmuelleria to Botryotrichum. Wang et al. (2016a) narrowed Chaetomium to the monophyletic lineage as Chaetomium sensu stricto, which included the generic type (neotype CBS 160.62) of Chaetomium globosum (Wang et al.

2016b). Draft A fungus collected from a residence belongs to the genus Botryotrichum, yet displays unique morphological characters. We describe our novel indoor isolate as a new species of

Botryotrichum, supported by both morphological and multilocus (ITS, LSU, rpb2, and tub2) molecular data.

Materials and Methods

The fungus was present in a swab sample collected from the blade of a ceiling fan in a residence in Boston, MA on March 13, 2012. The swab was vortexed in 10 mL distilled water for 30s and suspension was serial diluted 10 to 10,000 times and plated on malt extract medium (MEA) (20 g malt, 20 g agar, and 1 L distilled water). The unknown fungus was purified and subsequently grown on MEA and V8 at 25°C for 7-30 days depending on mycelial growth. MEA plates were prepared from the purified isolate and incubated at 25°C on MEA for a month to observe the colony growth, and for preparing extype and molecular work. Each plate was inoculated at three

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equidistant points in a triangle. To monitor sporulation, aerial hyphae and colony development,

MEA, and V8 media were used. The fungus was mounted in 85% lactic acid for microscopic

observation. All observations and measurements employed a compound microscope (Zeiss

Imager.M2) with differential interference contrast (DIC), and photomicrographs were taken with

an Axiocam 506 color camera (Carl Zeiss AG, Oberkochen, Germany). Measurements of the

fungal structures were made under 40–100 × objective lenses and statistically analyzed for

means and standard deviations with 95% confidence interval of means.

The type specimen has been deposited in The Connecticut Agricultural Experiment

Station (NHES) in the USA. An extype culture has been deposited in The UAMH Centre for Global Microfungal Biodiversity at UniversityDraft of Toronto (UAMH), Canada.

DNA extraction, amplification, and sequencing

The isolate was grown on MEA at 25°C for a month. Genomic DNA was extracted from

colonies grown in Petri plates according to the procedure in ZR Fungal/Bacterial DNA

MicroPrep Kit (Zymo Research, Irvine, CA, USA). ITS, LSU rpb2, and tub2 were sequenced. A

combination of oligonucleotides V9G or ITS5 with LR1 were used to amplify a fragment

corresponding to the partial small subunit rDNA and internal transcribed spacer 1 and 2 region

(ITS) by polymerase chain reaction (PCR) (Vilgalys and Hester 1990; White et al. 1990; Van

den Ende and De Hoog 1999). Oligonucleotides LROR and LR7 were used to amplify a portion

of the large subunit rDNA region (LSU) by PCR (Vilgalys and Hester 1990; White et al. 1990;

Hopple Jr and Vilgalys 1999). The RPB2 PCR product was generated using oligonucleotides

RPB2AM-1gf and RPB2AM-7R (Miller and Huhndorf 2005). Oligonucleotides TUB2T1 and

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TUB2T2 were used to amplify partial Tubulin2 locus sequences by PCR (O'Donnell and

Cigelnik 1997). The parameters for the PCR amplification protocol were 94 ºC 3 minutes; 94 ºC

30 seconds; 45 ºC 30 seconds; 72 ºC 2 minutes, repeat 35×, 72ºC 7 minutes.

The resulting PCR products were purified using QIA quick PCR Purification columns

(Qiagen, Valencia, CA, USA) and the DNA concentrations were determined on a NanoDrop Lite

Spectrophotometer (ThermoScientific, Waltham, MA, USA). The ITS PCR products were sequenced using oligonucleotides ITS1, ITS4, ITS2, ITS3 and ITS5 (Vilgalys and Hester 1990;

White et al. 1990). The LSU PCR products were sequenced using primers LROR, LR7, LR5,

LR3R, LR3B and LR16 (Vilgalys and Hester 1990; White et al. 1990). The Tubulin PCR product was sequenced with primer T1 & 2 (O'Donnell and Cigelnik 1997) and the RPB2 PCR product, was sequenced with RPB2AM-1bfDraft and RPB2AM-7R (Miller and Huhndorf 2005). All

DNA sequencing was performed at the W. M. Keck Biotechnology Resource Laboratory, Yale

School of Medicine (New Haven, CT, USA). DNA sequence information was deposited to

GenBank and accession numbers of the taxa and isolates, which were newly sequenced in the study are listed in Table 1.

All four genes/region of fungal sequences were employed as a query for BLASTn analysis in databases at GenBank (http://blast.ncbi.nlm.nih.gov). The BLAST search was carried out to choose identified allied taxa and fungi from uncultured/unidentified samples, environmental samples, and samples with questionable identifications were excluded (Wheeler et al. 2003).

Fungi, whose identities were linked to voucher cultures and which had close matches with the query sequence from BLAST analysis, were chosen for phylogenetic analysis. GenBank was searched for the allied taxa and the corresponding ITS, LSU, rbp2 and tub2 sequences obtained.

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Sixteen fungi with 30 sequences in total were selected for phylogenetic analyses (Table 1). The

sequences of the unknown fungus generated from the present study were deposited to GenBank

(Table 1).

Alignment and phylogenetic analysis

ITS, LSU, rpb2, and tub2 sequence data were aligned independently using Muscle (Edgar 2004)

and followed by manual correction. Sequences of aligned ITS, LSU, rpb2, and tub2 were

trimmed and concatenated with FABOX sequence alignment jointer (http://users-

birc.au.dk/biopv/php/fabox/alignment_joiner.php#).

Phylogenetic analyses were conducted using the maximum likelihood procedures with

MEGA7 (Tamura 1992; Kumar et al. 2016).Draft The dataset had 30 nucleotide sequences with 2206

positions, and all sites were treated equally for the analysis. All sites were equally weighted.

Gaps were treated as missing data. A bootstrap was calculated with 1000 replicates. Chaetomium

globosum CBS 160.62 (neotype) was designated as an outgroup.

Bayesian inference (BI) was analyzed for the combined dataset of sequences of ITS, LSU,

rpb2, and tub2 using MrBayes3.2.6 (Ronquist et al. 2012). Four Markov chains were used for

one run from random starting trees for two million generations. Trees sampling frequency was

1000 generations. The first one-tenth generations were discarded as burn-in. Majority rule

consensus tree of all remaining trees was computed. Branches that received Bayesian posterior

probabilities 0.95 (BPP) were set as significantly supported. Phylogenetic trees were constructed

using TreeGraph2 (Stöver and Müller 2010).

Results

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The results of phylogenetic analyses with ITS, LSU, RPB2, TUB 2 conducted using the

maximum likelihood procedures with MEGA7 and Bayesian inference were identical. Thus, the

phylogenetic trees from two analyses were merged into one tree (Figure 1).

Phylogenetic analyses demonstrated that Botryotrichum domesticum UAMH11929 is

sister to B. piluliferum (generic type), in the same clade as B. atrogriseum, B. peruvianum, B.

murorum and B. spirotrichum, and subtended by eight species of Subramaniula (Figure 1). The

clade of genus Botryotrichum was well separated from genus Chaetomium and phylogenetically

distant from the generic type of Chaetomium, C. globosum (CBS 160.62 neotype) (Figure 1).

Taxonomy Draft

Botryotrichum domesticum D.W. Li and N.P. Schultes sp. nov. Fig. 2.

MycoBank # MB 828185

Diagnosis: B. domesticum is differentiated from B. piluliferum and other asexual species of

Botryotrichum by its absence of setae, growing very slowly on MEA, having tuberculate aerial hyphae and from the species with sole sexual state by lack of sexual state.

Asexual state— Colonies growing very slowly on MEA at 25 °C, attaining 5.5 mm in diameter in a month, velvety, hyphae immersed, septate, 2−4 μm wide, colorless, smooth, no aerial hyphae; attaining 16 mm in a week on V8 at 25 °C, gray to dark gray, fluffy, developing

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immersed and aerial hyphae, immersed hyphae colorless, smooth, thin-walled, aerial hyphae,

dark brown, tuberculate or rarely smooth when young, rigid, thick-walled. Blister-like structure

on tuberculate aerial hyphae may burst and leaves covering remnant. Setae absent.

Conidiophores micronematous on both immersed and aerial hyphae, mostly reduced to

conidiogenous cells; when multiple celled, unbranched or branched, colorless to light brown.

Conidiogenous cells terminal, intercalary or rarely integrated on the hyphae, solitary,

monoblastic, barrel, cylindrical, or T shaped, hyaline, smooth. Conidial secession rhexolytic.

Conidia mostly solitary, subhyaline to pale brown, 1-celled, rarely 1-septate, globose to

subglobose, thick-walled, (13.5–)15.1−20.3(–25.5) × (12.5–)14.8−19.8 (–23.8) μm, (mean ± SD:

17.7 ± 2.6 × 17.3 ± 2.5 μm, n=30), conidial cell wall 1.1−2 μm thick (mean ± SD: 1.6 ± 0.4 μm,

n=30), smooth or some verruculose, withDraft a conspicuous truncate hilum or frill. Sexual state not

observed.

Holotype: USA, Massachusetts, Boston, 1 Charles St., a swab sample from a fan of residence,

March 13, 2012, NHES L1707, extype UAMH 11929.

ETYMOLOGY: Latin, domesticum, referring to indoors where the holotype was collected.

Comments: Young stage of aerial hyphae resembles setae, but develop indeterminately and are

fertile, on which conidiophores and conidia are developed. Botryotrichum domesticum is

morphologically similar and phylogenetically sister to B. piluliferum and phylogenetic analyses

using multiple locus sequences placed it in Botryotrichum. However, there are several

morphological characters to differentiate the two species. B. domesticum lacks setae, grows very

slowly, and develops only immersed hyphae on MEA. Botryotrichum domesticum is

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differentiated from other asexual members of Botryotrichum by lack of setae and from two sole

sexual species (B. spirotrichum and B. murorum) by absence of a sexual state. Botryotrichum

peruvianum develops both sporodochia and setae and conidia are spherical, 10–20 μm in diam

(Matsushima 1975; 12–16 μm in Wang et al. 2016a). Botryotrichum atrogriseum develops dark

gray colonies, colorless sterile hyphae and light brown fertile hyphae, colorless

conidiophores >150 μm long, conidia spherical, pale brown, verrucose, 10–25 μm in diameter,

and lacks setae (Beyma 1929). Botryotrichum gorakhpurense has setae, its conidia are spherical

to subspherical, 15–20 μm in diameter, and lacks a phialidic synanamorph (Rakesh et al. 1994).

Botryotrichum indicum Subrahm. does not have setae, but its conidia are oblong or ellipsoid,

colorless, 9.7–17 × 9.7–11 μm, in slimy heads and it has phialidic synanamorphic state

(Subrahmanyam 1979). Botryotrichum keratinophilumDraft has no setae or phialidic synanamorph, is

keratinophilic and its colorless conidia are 8.4–25 μm (Kushwaha and Agrawal 1976).

Botryotrichum lachnella also has setae and its smaller conidia are described as spherical, smooth

to granulose, subhyaline, 12 μm in diameter (Saccardo 1893). Botryotrichum pampeanum also

has setae and its conidia are spherical, rough, pale olivaceous, 16-18 μm in diameter (Spegazzini

1926). Botryotrichum villosum has setae and elliptical or ovate, smooth, colorless conidia 12-15

× 7-9 μm (Spegazzini 1899).

Discussion

Botryotrichum was erected as an asexual genus (Marchal 1885; Seifert et al. 2011). According to

Ellis (1971) the generic concept of this genus is setae unbranched, flexuous, pale to mid brown or greyish brown, often verrucose or encrusted; conidiophores straight or flexuous, often

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branched, smooth, colourless; conidiogenous cells of bimorphic, (1) monoblastic, cylindrical: (2)

monophialidic, subulate; conidia solitary, colourless, 1-celled, smooth, very thick-walled,

spherical; phialoconidia catenate or aggregated in slimy heads, colourless, 1-celled, smooth,

obovoid or clavate. Later it was expanded by including: setae are present or absent, and conidia

have rhexolytic secession (Seifert et al. 2011).

Wang et al. (2016a) retained Botryotrichum, in which species of both asexual and sexual

state were included, but the generic concept of Botryotrichum was not emended accordingly.

Such treatment of the genus would make identification of the members of Botryotrichum rather

difficult, if not impossible, based on morphology for the species, which develop only sexual state. It is necessary to redefine the genericDraft concept of Botryotrichum from the study by Wang et al. (2016a) to avoid confusion.

Botryotrichum-like asexual state was reported in several genera in Chaetomiaceae: C.

bostrychodes Zopf ( Collariella bostrychodes (Zopf) X. Wei Wang & Samson), Chaetomium

cuyabenoensis Decock & Hennebert ( Humicola cuyabenoensis (Decock & Hennebert) X. Wei

Wang & Houbraken), C. distortum L. Ames ( Humicola distorta (L.M. Ames) X. Wei Wang &

Houbraken), C. gangligerum Ames, C. homophilatum Omvik ( Humicola homopilata (Omvik)

X. Wei Wang & Houbraken), C. megasporum Sorgel ex Seth, Chaetomium seminudum L. Ames

( Humicola seminuda (L.M. Ames) X. Wei Wang & Houbraken) C. piluliferoides Udagawa &

Y. Horie, C. pulchellum L. Ames (= C. spirale Zopf), C. semispirale Udagawa & Cain (

Humicola semispiralis (Udagawa & Cain) X. Wei Wang & Houbraken), C. sulphureum Sorgel

ex Seth, C. torulosum Bainier (Udagawa 1960; Calviello 1971; Malloch and Cain 1973;

Hawksworth 1975; Cannon 1986; Von Arx et al. 1986; Decock and Hennebert 1997; Wang et al.

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2016a; Wang et al. 2018 [2019]). The conidia of these taxa are all smaller than 12 μm diameter and some of these taxa have been moved to Humicola (Wang et al. 2018 [2019]). B. domesticum is differentiated from the asexual state of these taxa by its much larger conidia and the presence of tuberculate aerial hyphae. Thielavia cephalothecoides Malloch & Benny (≡ Chaetomidium cephalothecoides (Malloch & Benny) Arx) developed a Botryotrichum or Staphylotrichum-like asexual state (Malloch and Benny 1973), which is different from B. domesticum by its thin- walled conidia and lack of tuberculate aerial hyphae.

Chaetomium s.l. is linked to several additional asexual genera: Acremonium-like (monophialidic state) (Zopf 1881; Moreau and Moreau 1954; Udagawa 1960; Domsch and Gams 1970; Cannon 1986; Von Arx et al. 1986), ScopulariopsisDraft-like (Corlett 1966; Von Arx et al. 1986), Sporothrix-like (Cannon 1986), and Chrysosporium-like (Wang and Zheng 2005).

Previous studies showed that synanamorphic Proteophiala morph of Harzia and characters of asexual states in Erysiphales not only are an important morphological characters, but also have phylogenetic significance in redefining generic concepts for Harzia and

Erysiphales, respectively (Braun and Takamatsu 2000; Schultes et al. 2017). However, the phylogenetic significance of asexual states, especially Botryotrichum in Chaetomium and allied genera remain unclear and warrant further studies in the future.

On MEA B. domesticum is rather similar to Desertella in morphology. Desertella

Mouch. was established by Mouchacca (1979 [1978]) and typified using Desertella globulifera

Mouch. isolated from ferruginous desert soil in Egypt (Mouchacca 1979 [1978]). At present only two species have been described to this genus, the type and D. fumimontarum Raja & Shearer

(Raja et al. 2007). The genus is defined by its ochre cultures, hyaline to yellow, 1-celled,

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subglobose, solitary, thick-walled, conidia, monoblastic conidiogenous cells and reduced,

hyaline conidiophores. Although conidiogenesis is identical, the conidia of Desertella are much

larger than those of B. domesticum. At present the phylogenetic placement of Desertella remains

unknown, since the extype of Desertella globulifera - the type species of Desertella - is not

available for phylogenetic study.

Some warts on the tuberculate hyphae of B. domesticum collapsed releasing their liquid

contents and left their covering on the aerial hyphae. This phenomenon has not been observed in

other species of Botryotrichum. The functions of the warts and their contents are unknown and

warrant future studies.

Botryotrichum domesticum, isolatedDraft from an indoor environment, was determined to be a new species by both morphological characters and multi-locus phylogenetic analysis. Since B.

domesticum does not have setae, which is a major morphological character shared by other

asexual species in Botryotrichum, in future the morphological delineation of species in

Botryotrichum should be based on features of both asexual and sexual states.

Acknowledgements

The authors are grateful to Ariunaa Jalsrai for providing the isolate. We would like express our

gratitude to Dr. Donald H. Pfister and Ms. Judith A. Warnement, Harvard University for

obtaining several papers for us and Dr. Carole Cheah for providing access to some literature.

This study is supported by USDA Hatch grants (CONH00813).

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2 Table 1. Taxa, their sequences and GenBank accession numbers used in the phylogenetic analysis.

Taxon Strain# Type Substrate Country ITS LSU rpb2 tub2 Reference

Botryotrichum The Wang et al. atrogriseum CBS 130.28 T Dung of rabbit Netherlands KX976589 KX976714 KX976813 KX976931 2016a

Wang et al. CBS 604.69 Corn field soil Canada KX976590 KX976715 KX976814 KX976932 2016a A swab Botryotrichum sample from a Draft domesticum UAMH 11929 T residential fan USA MH899168 MH899169 MH899171 MH899172 This study

Botryotrichum Wang et al. murorum CBS 163.52 USA KX976591 KX976716 KX976815 KX976933 2016a

Liquor cerebrospinalis of Homo Wang et al. CBS 173.68 sapiens Netherlands KX976592 KX976717 KX976816 KX976934 2016a

DTO 324-G9; Wang et al. DTO 324-H9 Air China KX976593 KX976718 KX976817 KX976935 2016a

DTO 333-E6 (= IBT Wang et al. 42175) Ceiling tile Denmark KX976594 KX976719 KX976818 KX976936 2016a

CBS Botryotrichum 460.90/CBS Dung of peruvianum 112386 herbivore Spain KX976595 KX976720 KX976819 KX976937 Air CBS 421.93 Cuba KX976596 KX976721 KX976820 KX976938 Wang et al.

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2016a

Botryotrichum The Wang et al. piluliferum CBS 654.79 Pastry Netherlands KX976597 KX976722 KX976821 KX976939 2016a

Wang et al. CBS 105.14 Unknown KX976598 KX976723 KX976822 KX976940 2016a

The Wang et al. DTO 194-F7 Plaster wall Netherlands KX976599 KX976724 KX976823 KX976941 2016a

DTO 254-B8; The Wang et al. DTO 254-B9 Wall in villa Netherlands KX976600 KX976725 KX976824 KX976942 2016a

Botryotrichum Wang et al. spirotrichum CBS 211.55 T Dung of deer USA KX976601 KX976726 KX976825 KX976943 2016a Botryotrichum Dung of Draft Wang et al. spirotrichum CBS 828.71 T donkey Algeria KX976602 KX976727 KX976826 KX976944 2016a

Chaetomium Wang et al. globosum CBS 160.62 neoT Compost Germany KT214565 KT214596 KT214666 KT214742 2016b

Humicola Wang et al. fuscoatra CBS 118.14 T Soil Norway KX976675 KX976769 KX976882 KX977017 2016a

Subramaniula Peritonitis of Wang et al. anamorphosa CBS 137114 T Homo sapiens Kuwait KP862598 KP970641 KP900667 KP900704 2016a

Subramaniula Keratitis of Wang et al. asteroides CBS 123294 T Homo sapiens USA HQ906667 JX280731 KP900666 KP900703 2016a Corneal ulcer of Homo Wang et al. CBS 128466 sapiens USA JX280843 JX280732 KP900656 KP900695 2016a

Subramaniula Wang et al. cristata CBS 156.52 T Dung of rabbit USA KX976690 KX976788 KX976903 KX977038 2016a

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4 DTO 324-H8; Wang et al. DTO 324-H7 Air China KX976691 KX976789 KX976904 KX977039 2016a 5 Subramaniula Wang et al. cuniculorum CBS 800.83 Soil Spain KX976692 KX976790 KX976905 KX977040 2016a 6 Subramaniula Wang et al. flavipila CBS 446.66 T Dead leaves Bulgaria KP862600 KP970647 KP900669 KP900706 2016a 7 Wang et al. CBS 227.82 Dung Spain KP862599 KP970646 KP900668 KP900705 2016a 8 Subramaniula Dung of Wang et al. fusispora CBS 199.84 marmot Canada KP862601 KP970645 KP900653 KP900707 2016a 9 Subramaniula Tinea pedis of Wang et al. obscura CBS 132916 T Homo sapiens KuwaitDraft KP862595 KP970653 KP900662 KP900700 2016a 10 Subramaniula Wang et al. thielavioides CBS 122.78 T Dung of nilgai India KP862597 KP970654 KP900670 KP900708 2016a 11

14 Note: The new taxon and newly generated sequences are in bold. T, type; neoT, neotype; Abbreviations of isolates and

15 culture collections: ATCC = American Type Culture Collection, Manassas, USA; CBS = The Westerdijk Fungal

16 Biodiversity Institute (formerly Centraalbureau voor Schimmel cultures), Utrecht, the Netherlands, Utrecht, Netherlands;

17 IMI = CABI Bioscience, UK; UAMH, The UAMH Centre for Global Microfungal Biodiversity at University of Toronto.

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Draft

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23 Figure legends

24 Fig. 1. Maximum Likelihood analysis of Botryotrichum domesticum (ex-type) and allied taxa

25 based on concatenated ITS, LSU, RPB2, TUB 2 sequence data. Chaetomium globosum CBS

26 160.62 neoT is included as outgroup. The bootstrap test was conducted with 1000 replicates.

27 Bootstrap values >80% (before the slash) and Bayesian posterior probabilities (>0.95) (after the

28 slash) were indicated at the nodes. The scale bar indicates the number of expected changes per

29 site. T indicated the ex-types used in the analysis.

31 Fig. 2. Botryotrichum domesticum (NHES L1707, extype UAMH 11929). a. Colonies growing

32 on MEA for a week. b. Colonies growingDraft on V8 for a week. c. Aerial hyphae and conidia, d.

33 Conidia and conidiogenous cell on aerial hypha. e-f. Conidia and conidiogenous cells on

34 immersed hyphae. g. Conidia. h. tuberculate aerial hypha.

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Botryotrichum piluliferum CBS 654.79 Botryotrichum piluliferum DTO 194-F7 99/1 Botryotrichum piluliferum DTO 254-B9 98/1 Botryotrichum piluliferum CBS 105.14 98/1 Botryotrichum domesticum sp. nov.

Botryotrichum atrogriseum CBS 130.28T 100/1 100/1 Botryotrichum atrogriseum CBS 604.69 Botryotrichum peruvianum CBS 421.93 97/1 100/1 Botryotrichum peruvianum CBS 460.90 Botryotrichum murorum DTO 324-G9 Botryotrichum murorum CBS 163.52 100/1 100/1 Botryotrichum murorum CBS 173.68 69/- Botryotrichum murorum DTO 333-E6 Botryotrichum spirotrichum CBS 211.55T 100/1 Botryotrichum spirotrichum CBS 828.71 81/- Subramaniula flavipila CBS 227.82 100/1 89/0.98 Subramaniula flavipila CBS 446.66T 100/1 Subramaniula anamorphosa CBS 137114T Subramaniula fusispora CBS 199.84T Subramaniula cristata CBS 156.52T Draft 98/1 100/1 100/1 Subramaniula cristata DTO 324-H7 Subramaniula cuniculorum CBS 800.83 Subramaniula asteroides CBS 123294T 97/1 100/1 Subramaniula asteroides CBS 128466 100/1 Subramaniula obscura CBS 132916T 71/- Subramaniula thielavioides CBS 122.78T Humicola fuscoatra CBS 118.14T Chaetomium globosum CBS 160.62 neoT

0.02

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Draft

Fig. 2. Botryotrichum domesticum (NHES L1707, extype UAMH 11929). a. Colonies growing on MEA for a week. b. Colonies growing on V8 for a week. c. Aerial hyphae and conidia, d. Conidia and conidiogenous cell on aerial hypha. e-f. Conidia and conidiogenous cells on immersed hyphae. g. Conidia. h. tuberculate aerial hypha.

152x243mm (300 x 300 DPI)

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