Botany 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? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 27 Botany 1 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 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 27 2 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 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 27 Botany 3 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 & https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 27 4 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 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 27 Botany 5 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 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 27 6 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