Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus

Mycol. Res. 108 (8): 864–872 (August 2004). f The British Mycological Society 864 DOI: 10.1017/S0953756204000607 Printed in the United Kingdom. Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus# Lisa A. CASTLEBURY1*, Amy Y. ROSSMAN1, Gi-Ho SUNG2, Aimee S. HYTEN1 and Joseph W. SPATAFORA2 1 Systematic Botany and Mycology Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705, USA. Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA. E-mail : castlebury@nt.ars-grin.gov 2 Received 21 February 2004; accepted 5 May 2004. Stachybotrys chartarum is an asexually reproducing fungus commonly isolated from soil and litter that is also known to occur in indoor environments and is implicated as the cause of serious illness and even death in humans. Despite its economic importance, higher level phylogenetic relationships of Stachybotrys have not been determined nor has a sexual state for S. chartarum been reported. DNA sequences from four nuclear and one mitochondrial gene were analyzed to determine the ordinal and familial placement of Stachybotrys within the Euascomycota. These data reveal that species of Stachybotrys including S. chartarum, S. albipes, for which the sexual state Melanopsamma pomiformis is reported, species of Myrothecium, and two other tropical hypocrealean species form a previously unknown monophyletic lineage within the Hypocreales. These results suggest that Stachybotrys and Myrothecium are closely related and share characteristics with other hypocrealean fungi. In addition, S. chartarum may have a sexual state in nature that consists of small, black, fleshy perithecia similar to Melanopsamma. INTRODUCTION Stachybotrys chartarum is an asexually reproducing fungus that is commonly isolated from soil, leaf litter, and dung from throughout the world (Domsch, Gams & Anderson 1980, Koster et al. 2003). In recent years this species has achieved notoriety as an airborne house-inhabiting fungus that produces mycotoxins and can cause serious illness and even death in humans (Vesper et al. 2000). Considerable effort is given to monitoring the presence of this fungus particularly in situations where building interiors, especially substrates rich in cellulose, have become wet, thus supporting the growth of this and other mycotoxin-producing molds (Andersen, Nielsen & Jarvis 2002, Shelton et al. 2003, Spurgeon 2003). A sexual state for S. chartarum has never been reported. As an asexually reproducing fungus, S. chartarum is, at least theoretically, a clonal organism. An increasing body of evidence suggests that, despite the lack of a sexual state, a number of fungi known to reproduce only asexually appear to be undergoing * Corresponding author. # Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. the equivalent of sexual reproduction resulting in genetically diverse populations (Taylor, Jacobson & Fisher 1999, Taylor et al. 2000, Bidochka & Koning 2001). In addition, many asexually reproducing fungi are derived from within groups that include sexually reproducing species (O’Donnell, Cigelnik & Nirenberg 1998, Chaverri et al. 2003). Only one species of Stachybotrys, S. albipes, has been linked to a sexual state reported as Melanopsamma pomiformis (Booth 1957), although this research has not been repeated. M. pomiformis is a small black perithecial ascomycete less than 300 mm diam that occurs on decorticated rotten wood, especially hardwoods (Booth 1957, Samuels & Barr 1997). This species is widely distributed throughout the north temperate regions of the world (Samuels & Barr 1997, Farr et al. 2004) including a report of the asexual state from black rush, Juncus roermerius, in salt marshes (Fell & Hunter 1979). The genus Melanopsamma has been placed in an obscure family, Niessliaceae, in the Hypocreales (Samuels & Barr 1997, Rossman et al. 1999). Intrageneric relationships within Stachybotrys and the synonymous genus Memnoniella have been investigated (Haugland, Vesper & Harmon 2001) and species level studies of S. chartarum have resulted in the description of a new species and the presence of L. A. Castlebury and others two chemotypes in S. chartarum (Andersen et al. 2002, 2003, Cruse et al. 2002). Despite its economic importance, higher level phylogenetic relationships of the genus Stachybotrys have not been investigated. To determine if S. chartarum is derived from within the Hypocreales, we sequenced both the nuclear encoded small subunit ribosomal DNA (nrSSU) and the nuclear encoded large subunit ribosomal DNA (nrLSU) for isolates of Stachybotrys, including S. chartarum, and analyzed them with major orders of the Sordariomycetes. To determine family placement within the Hypocreales, we sequenced regions of RNA polymerase II largest subunit (RPB1), translation elongation factor 1-a (EF1-a) and mitochondrial ATP synthase 6 (ATP6) and analyzed them in combination with the ribosomal RNA genes for representatives of the known families of the Hypocreales. 865 sequencer. The PCR primers were used as sequencing primers for all genes. The following internal primers were used for sequencing nrSSU, in addition to the PCR primers listed previously : NS2, NS3, NS4, NS5, NS6, NS7 (White et al. 1990). The following internal primers were used for sequencing nrLSU in addition to the previously mentioned PCR primers : LR3R and LR5 (Vilgalys & Hester 1990, Rehner & Samuels 1995). A few genes were unable to be sequenced for certain isolates (Table 1) and were treated as missing data in the analyses. For RPB1, the primer CRPB1A rather than CRPB1 was used for the following four isolates : Myrothecium leucotrichum CBS 114052, Ochronectria calami CBS 125.87, Peethambara spirostriata CBS 110115, and Stilbocrea macrostoma GJS 73-26. Sequences were deposited in GenBank and listed in Table 1. Phylogenetic analyses MATERIAL AND METHODS Nucleic acid extraction and PCR amplification Culture or specimen numbers for species used in this study are listed in Table 1. Mycelia for DNA extractions were grown on PDA plates, scraped from the plates with a sterile scalpel, placed in 1.7 ml tubes and extracted using the PureGene DNA Extraction Kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. Individual genes were amplified in 50 ml reactions on a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, CA) and or I-Cycler (Bio-Rad, Hercules, CA) using the following previously published primers : nrLSU, LR0R and L7 (Vilgalys & Hester 1990, Rehner & Samuels 1994); nrSSU, NS1 and NS8 (White et al. 1990) ; EF1-a, EF1-983f and EF1-2218r (Rehner 2001, Currie et al. 2003) ; and RPB1Cr (Hall 2003). The following primers were designed by G.-H. S. for use with hypocrealean taxa : RPB1 forward primers, CRPB1 (CCWGGYTTYATCAAGAARGT) and CRPB1A (CAYCCWGGYTTYATCAAGAA) and ATP6 forward and reverse primers, ATP6-C1A (AGAWCAATTYGAARTRAGAG) and ATP6-C2A (ACAAAYACTTGWGCTTGKATWAAIGC). Failsafe PCR master mix B (Epicentre, Madison, WI) was used according to the manufacturer’s instructions for amplifications that were problematic. Standard cycling parameters with a 55 xC annealing temperature were used for nr-SSU, nr-LSU, and EF1a. For the RPB1 and ATP6 genes the thermal cycler program was as follows : (1) 1 min at 94 x, 35 s at 37 x, 1 x every 4 s to 72 x, 1 min at 72 x run for four cycles ; (2) 35 s at 94 x, 55 s at 45 x, 1 x every 4 s to 72 x, 1 min at 72 x, 29 cycles ; and (3) final extension 10 min at 72 x. The PCR products were purified using ExoSAP-IT (USB, Cleveland, OH) according to the manufacturer’s instructions. Amplified products were sequenced with the BigDye version 3.1 dye terminator kit (Applied Biosystems) on an ABI 3100 automated DNA Maximum parsimony (MP) analyses were conducted using PAUP 4.0b10 (Swofford 2002) and Bayesian analyses were conducted with MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001) for each alignment. MP analyses were conducted heuristically with 1000 random taxon addition replicates with TBR-branch swapping and MULTREES on. 1000 bootstrapping pseudoreplicates with ten random taxon addition replicates per pseudoreplicate were performed. The Bayesian analyses employed the GTR+C+I model and included three separate runs each consisting of 500 000 Markov Chain Monte Carlo (MCMC) generations, each with a burn-in of 100 000 generations. Posterior probabilities from the three runs were pooled and indicated on Figs 1–2. Alignment 1 was derived from Kohlmeyer, Spatafora & Volkmann-Kohlmeyer (2000), with the addition of sequences from Melanospora and Sphaerodes (Zhang & Blackwell 2002) and the Diaporthales (Castlebury et al. 2001) as well as newly sequenced species from this study. The alignments have been deposited in TreeBASE. Two datasets were analyzed as follows : (1) an alignment of nrSSU and nrLSU sequences for the major orders of the Sordariomycetes, including representative taxa within Stachybotrys, Myrothecium and known hypocrealean families, with the Pezizomycetes as outgroup taxa to determine if S. chartarum belongs within the Hypocreales ; and (2) an alignment of the nrSSU, nrLSU, EF1-a, ATP6, and RPB1 for an expanded set of taxa from Stachybotrys, Myrothecium and families within the Hypocreales, using Glomerella cingulata and Verticillium dahliae (Phyllachorales) as outgroup taxa, in order to determine the closest relatives of S. chartarum and related species. RESULTS Alignment 1 consisted of nrSSU (1024 bp) and nrLSU (596 bp) sequences for 67 taxa. Of the total of 1620 Multigene phylogeny of Stachybotrys chartarum 866 Table 1. List of taxa sequenced in this study. Taxon Sourcea Habitat or host LSU Aphysiostroma stercorarium Spain, on cow dung AF543792 AF543769 AF543782 AY489566 AY489633 Balansia henningsiana Bionectria ochroleuca B. pityrodes Claviceps purpurea ATCC 62321 ex-type GAM 16112 CBS 114056 ATCC 208842 GAM 12885 AY489715 AY489176 AY489728 AF543789 Cordyceps capitata OSC 71233 C. gunnii OSC 76404 C. heteropoda OSC 106404 C. ophioglossoides OSC 106405 Cosmospora coccinea CBS 114050 Didymostilbe echinofibrosa AR 2824 Epichloe typhina ATCC 56429 Glomerella cingulata CBS 114054 Georgia, on Panicum sp. Venezuela, on bark Mauritius, on bark Georgia, on Dactylis glomerata France, on Elaphomyces sp. Australia, on Lepidoptera Australia, on Hemiptera North Carolina, on Elaphomyces sp. Germany, on Inonotus nodulosus Mexico, isol. from litter New Zealand, on Festuca rubra Florida, isol. from Fragaria Guyana, on bark Alabama, on bark North Carolina, on bark New York, on decorticated conifer wood North Carolina, on Trametes versicolor Louisiana, isol. from soil England, Ulmus sp. (single ascospore isolate by C. Booth) Georgia, on Andropogon virginicus Ukraine, isol. from soil UK, on Russula nigricans New Jersey, isol. from decaying grass leaf Germany, isol. from soil Washington, DC, isol. from baled cotton New York, on Betula Germany, on Fuligo septica Germany, on bark of Picea abies UK, on decaying needle of Pinus sylvestris Indonesia, on palm Cameroon, on liana Ecuador, on brooms of Crinipellis perniciosa Spain, on leaves of Buxus sempervirens New Zealand, on polypore Haematonectria haematococca CBS 114067 Hydropisphaera peziza CBS 102038 Hypocrea rufa CBS 114374 H. lutea ATCC 208838 Hypomyces polyporinus ATCC 76479 Leuconectria clusiae/ Gliocephalotrichum bulbilium Melanopsamma pomiformis/ Stachybotrys albipes ATCC 22228 ex type ATCC 18873 Myriogenospora atramentosa AEG 96-32 Myrothecium cinctum ATCC 22270 M. inundatum IMI 158855 M. leucotrichum AR 3506 M. roridum ATCC 16297 M. verrucaria ATCC 9095 Nectria cinnabarina Nectriopsis violacea CBS 114055 CBS 424.64 Niesslia exilis CBS 357.70 N. exilis CBS 560.74 Ochronectria calami Ophionectria trichospora Peethambara spirostriata CBS 125.87 CBS 109876 CBS 110115 Pseudonectria rousseliana CBS 114049 Sphaerostilbella berkeleyana CBS 102308 SSU AY489683 AY489684 AY489696 AF543765 EF1 AY489610 AY489611 AY489623 AF543778 ATP6 AY489576 – AY489589 – RPB1 AY489643 – AY489658 AY489648 AY489721 AY489689 AY489615 AY489581 AY489649 AF339522 AF339572 AY489616 AY489582 AY489650 AY489722 AY489690 AY489617 – AY489651 AY489723 AY489691 AY489618 AY489583 AY489652 AY489734 AY489702 AY489629 AY489596 AY489667 AY489706 AY489674 AY489601 AY489567 AY489634 U17396 U32405 AF543777 AY489584 AY489653 AF543786 AF543762 AF543773 AY489590 AY489659 AY489729 AY489697 AY489624 – AY489660 AY489730 AY489698 AY489625 AY489591 AY489661 AY489726 AY489694 AY489621 AY489587 AY489656 AF543791 AF543768 AF543781 AY489592 AY489662 AF543793 AF543771 AF543784 AY489593 AY489663 AY489732 AY489700 AY489627 AY489595 AY489664 AY489709 AY489677 AY489604 AY489570 AY489637 AY489733 AY489701 AY489628 – AY489665 AY489710 AY489678 AY489605 AY489571 AY489638 AY489731 AY489699 AY489626 AY489594 – AY489707 AY489675 AY489602 AY489568 AY489635 AY489708 AY489676 AY489603 AY489569 AY489636 AY489713 AY489681 AY489608 AY489574 AY489641 U00748 U32412 AF543785 – AY489666 AY489719 AY489687 – AY489579 AY489646 AY489718 AY489686 AY489613 AY489578 AY489645 AY489720 AY489688 AY489614 AY489580 AY489647 AY489717 AY489685 AY489612 AY489577 AY489644 AF543790 AF543766 AF543779 – AY489669 AY489724 AY489692 AY489619 AY489585 AY489654 U17416 AF543767 AF543780 AY489598 AY489670 U00756 AF543770 AF543783 – – L. A. Castlebury and others 867 Table 1. (Cont.) Taxon Sourcea Stachybotrys chartarum ATCC 9182 S. chartarumb S. echinata (=Memnoniella echinata)c S. subsimplex (=M. subsimplex) Stephanonectria keithii Stilbocrea macrostoma Verticillium dahliae Viridispora diparietispora Habitat or host LSU Washington DC, isol. from paper ATCC 66238 Namibia, isol. from (=UAMH 6417, desert sand =CBS 25089) UAMH 6594 Canada, Alberta, isol. from indoor air ATCC 32888 Florida, isol. from water hyacinth CBS 114057 France, on bark of Eleagnus CBS 114375 New Zealand, on Geniostoma ligustifolia ATCC 16535 Canada, Quebec, isol. from tomato ATCC MYA 627 New York, on Crataegus crus-galli SSU EF1 ATP6 RPB1 AY489714 AY489682 AY489609 AY489575 AY489642 AY489712 AY489680 AY489607 AY489573 AY489640 AY489736 AY489704 AY489631 AY489599 AY489672 AY489711 AY489679 AY489606 AY489572 AY489639 AY489727 AY489695 AY489622 AY489588 AY489657 AY489725 AY489693 AY489620 AY489586 AY489655 AY489737 AY489705 AY489632 AY489600 AY489673 AY489735 AY489703 AY489630 AY489597 AY489668 a ATCC, American Type Culture Collection, Manassas, VA; AR, Amy Y. Rossman personal collection; BPI, US National Fungus Collections, Beltsville, MD; CBS, Centraalbureau voor Schimmelcultures, Utrecht; GAM, Julian H. Miller Mycological Herbarium, Athens, GA; IMI, CAB International, Egham; NY, New York Botanical Garden, Bronx, NY ; OSC, Oregon State University Herbarium, Corvallis, OR; UAMH, University of Alberta Microfungus Collection and Herbarium, Edmonton, AB. b Mistakenly initially identified as S. kampalensis ; see Haugland & Heckman (1998). c Epitype isolate fide Haugland et al. (2001). characters, 549 are parsimony informative. All characters were used in the analyses. MP phylogenetic analysis of alignment 1 resulted in 48 equally parsimonious trees, differing slightly in the arrangement of the terminal taxa within the major lineages (length=2948, CI=0.384, RI=0.636, RC=0.244, HI=0.616). Bayesian and MP phylogenetic analyses of alignment 1 both identified the same groups corresponding to the major orders of the Sordariomycetes. Fig. 1 shows one of three Bayesian trees generated from alignment 1, with posterior probabilities above and MP bootstrap supports below the branches for the major orders. Within the major orders, only minor branching differences were noted in the trees resulting from the two analyses. Isolates of Stachybotrys were placed within the Hypocreales with 70 % MP bootstrap support and 100 % posterior probability in the Bayesian analyses. In these analyses, S. chartarum was most closely allied with species of Myrothecium, another asexually reproducing genus. Melanospora and Sphaerodes, however, were not placed within the Hypocreales in any of the analyses contrary to previous hypotheses (Rehner & Samuels 1995, Zhang & Blackwell 2002) and were excluded from the hypocrealean dataset (alignment 2). Alignment 2 consisted of nrSSU (1022 bp), nrLSU (914 bp), EF1-a (1014 bp), RPB1 (829 bp), and ATP6 (586 bp) sequences for 41 hypocrealean taxa and two phyllachoralean outgroup taxa for a total of 4365 total characters. Due to difficulties in aligning an introncontaining region in the RPB1 gene, 295 characters were excluded from that region in the analysis resulting in 4070 included characters of which 1119 are parsimony informative. MP phylogenetic analysis of alignment 2 resulted in two equally parsimonious trees (length=7172, CI=0.31, RI=0.463, RC=0.143, HI=0.690). Fig. 2 shows one of three Bayesian trees generated for alignment 2, with posterior probabilities above, and MP bootstrap supports below, the family level branches. Supports for branches within recognized families are not shown and branches leading to family level groups that are present in the strict MP consensus tree are shown with thickened lines. In both analyses Stachybotrys, Myrothecium, Peethambara spirostriata, and Didymostilbe echinofibrosa form a monophyletic group with 100% MP bootstrap support and 100% Bayesian posterior probabilities. DISCUSSION The Hypocreales represent an order of ascomycetous fungi that are most successful and well-known in their asexually reproducing states, such as Acremonium, Clonostachys, Cylindrocladium, Fusarium, and Trichoderma (Rossman 2000). Because of their ability to degrade a wide range of substrates, hypocrealean fungi function as virulent plant pathogens, producers of powerful antibiotics, sources of potent mycotoxins, and effective agents for the biocontrol of invasive fungi, plants, or insects. Within the Hypocreales, the major families have been defined based on morphological characteristics and recognized as the Bionectriaceae, Clavicipitaceae, Hypocreaceae, Nectriaceae, and Niessliaceae (Rossman et al. 1999). When tested using DNA sequence data, these families continue to define the major known lineages of the Hypocreales (Okada, Takematsu & Takamura 1997, Rossman et al. 2001, Zhang & Blackwell 2002). Multigene phylogeny of Stachybotrys chartarum 100 100 100 56 868 Myrothecium roridum Myrothecium cinctum Myrothecium verrucaria Myrothecium inundatum Didymostilbe echinofibrosa 100 Peethambara spirostriata 77 Stachybotrys chartarum Stachybotrys chartarum Stachybotrys echinata Niesslia exilis Niesslia exilis Bionectria ochroleuca Bionectria pityrodes 100 Stephanonectria keithii Nectriopsis violacea Stilbocrea macrostoma Hypocreales Ochronectria calami 64 Hypocrea schweinitzii Hypocrea rufa Aphysiostroma stercorarium 100 100 Sphaerostilbella aureonitens 70 100 Hypomyces polyporinus Leuconectria clusiae Viridispora diparietispora Pseudonectria rousseliana 79 Haematonectria haematococca Nectria cinnabarina Cordyceps ophioglossoides Paecilomyces tenuipes 94 79 Claviceps paspali 93 Balansia sclerotica Halosphaeriopsis mediosetigera 100 Halosarphei a fibrosa Halosphaeriales Halosphaeri a appendiculata 100 95 Aniptodera chesapeakensis Petriella setifera 100 100 87 100 100 Microascus trigonosporus 98 95 Microascales Ceratocystis virescens 100 Ceratocystis fimbriata 100 67 100 Glomerella cingulata Phyllachorales Colletotrichu m glo eosporioides 100 96 Melanospora za miae 100 Melanospora tiffani Melanosporales Sphaerodes compressa 100 Sphaerodes fimicola Lindra marinera Lindra thallassiae 100 Lulworthiales Lulworthia grandispora 99 Lulworthia medusa Chaetomium globosum 100 Cercophora septentrionalis Sordariales Neurospora crassa 100 Ophiostoma piliferum 100 Ophiostomatales Fragosphaeria purpurea 100 Pleuroceras pleurostyla Gnomonia setacea 100 Diaporthales Melanconis marginalis 100 Diaporthe phaseolorum Xylaria hypoxylon 100 Xylaria curta Xylariales Diatrype disciformis 98 Daldinia concentrica Morchella esculenta Gyromitra esculenta Helvella lacunosa Tuber gibbosum Peziza badia 0.01 substitutions/site Fig. 1. Bayesian tree resulting from analysis of 1625 bp from nrSSU and nrLSU for the major orders of the Sordariomycetes. The numbers above the branches indicate pooled posterior probabilities obtained from three independent Bayesian analyses, each consisting of 500 000 Markov Chain Monte Carlo generations (GTR+C+I model), with a burn-in of 100 000 generations. Numbers below the branches indicate MP bootstrap support proportions from 1000 pseudoreplicates with ten random taxon addition replicates per pseudoreplicate for major lineages only. The obscure family Niessliaceae was detailed based on morphological characteristics by Samuels & Barr (1997) and included in the Hypocreales (Rossman et al. 1999). However species of Niesslia have not been included in molecular phylogenetic analyses and placement within the Hypocreales has remained questionable (Kirk et al. 2001, Eriksson et al. 2004). All analyses performed in this study firmly support the placement of N. exilis, the type species of the genus Niesslia, in the Hypocreales with the Bayesian analysis of the 5-gene hypocrealean dataset (Fig. 2) suggesting a close relationship with the Bionectriaceae. The genus Melanopsamma based on the type species M. pomiformis (anamorph Stachybotrys albipes) was previously placed in the Niessliaceae (Samuels & Barr 1997, Rossman et al. 1999). The Stachybotrys lineage, including L. A. Castlebury and others 869 Stephanonectria keithii Bionectria pityrodes Bionectria ochroleuca Bionectriaceae Ochronectria calami 100 100 Hydropisphaera peziza 100 Nectriopsis violacea Stilbocrea macrostoma 69 100 99 60 Niesslia exilis Niessliaceae Niesslia exilis Haematonectria haematococca Ophionectria trichospora Nectria cinnabarina 100 96 Cosmospora coccinea Nectriaceae Leuconectria clusiae Viridispora diparietispora Pseudonectria rousseliana 100 97 Balansia henningsiana Myriogenospora atramentosa Claviceps purpurea Epichloe typhina 100 Cordyceps capitata 70 Clavicipitaceae Cordyceps ophioglossoides Cordyceps heteropoda 100 85 Cordyceps gunnii Hypomyces polyporinus Sphaerostilbella berkeleyana 100 100 100 100 Aphysiostroma stercorarium Hypocreaceae Hypocrea rufa Hypocrea lutea Myrothecium roridum Myrothecium inundatum 100 100 100 100 Myrothecium cinctum Myrothecium Myrothecium leucotrichum Myrothecium verrucaria 100 100 Didymostilbe echinofibrosa 100 Peethambara spirostriata Stachybotrys chartarum 100 Stachybotrys chartarum 100 100 99 100 Glomerella cingulata Stachybotrys echinata Stachybotrys subsimplex Stachybotrys Melanopsamma pomiformis (Stachybotrys albipes) Verticillium dahliae 0.01 substitutions/site Fig. 2. Bayesian phylogeny for 41 hypocrealean and two phyllachoralean outgroup taxa based on 4070 bp from four nuclear (nrSSU, nrLSU, RPB1, EF1-a) and one mitochondrial (ATP6) genes. The numbers above the branches indicate pooled posterior probabilities obtained from three independent Bayesian anlyses, each consisting of 500 000 Markov Chain Monte Carlo generations (GTR+C+I model), with a burnin of 100 000 generations. Numbers below the branches indicate MP bootstrap support proportions from 1000 pseudoreplicates with 10 random taxon addition replicates per pseudoreplicate. Posterior probabilities and bootstrap supports are only indicated on the internal branches leading up to the family level groupings. Thickened lines indicate branches present in strict consensus MP tree leading up to the family level groupings, except for the Stachybotrys/Myrothecium lineage. M. pomiformis, was not closely related to N. exilis in these analyses. Contrary to Samuels & Barr (1997), Melanopsamma is not a member of the Niessliaceae. The closest relatives of the Stachybotrys/Melanopsamma lineage in these analyses were species of Myrothecium, another asexually reproducing genus, Multigene phylogeny of Stachybotrys chartarum and Peethambara spirostriata, an obscure sexually reproducing fungus, and its anamorph Didymostilbe echinofibrosa (syn. Virgatospora echinofibrosa). P. spirostriata was discovered only recently, and is known from just a few collections in tropical regions on decaying plant debris (Rossman 1983). Although its asexual state is more commonly reported, D. echinofibrosa also appears to be restricted to tropical areas (Ellis 1971 as Virgatospora echinofibrosa, Rossman 1983 under Nectria spirostriata, Seifert 1990). Both Myrothecium and Peethambara were previously thought to be allied with the Bionectriaceae (Rossman et al. 2001). Most species of Myrothecium are cosmopolitan, associated with plant debris from both temperate and tropical regions (Farr et al. 2004), but species of Myrothecium have been reported as indoor air fungi (Levetin & Shaughnessy 1997). Despite their ecological success, no sexual state has been discovered for any species of Myrothecium. The genus Myrothecium is typified by Myrothecium inundatum, a species that occurs on fruit bodies of Russula (Tulloch 1972). The most commonly known species of Myrothecium, namely M. cinctum, M. roridum, and M. verrucaria, are known to degrade cellulose, and M. verrucaria has been reported as ‘ one of the most potent cellulose decomposers known’ (Domsch et al. 1980). M. verrucaria and M. roridum are plant pathogens, with M. verrucaria being considered for use in the control of noxious weeds (Domsch et al. 1980, Abbas et al. 2001, 2002, Boyette, Walker & Abbas 2002). The anamorphic genera Stachybotrys, Myrothecium, and Didymostilbe are united by morphological characteristics, such as their production of dark green conidia as well as characteristics of their conidiogenesis. The conidia tend to be elongated, fusiform, ellipsoid or cylindrical, smooth, striate, or roughened. In these genera, the conidiophores are penicillately arranged, bearing either 3–10 phialides directly, or 3–10 secondary conidiophores each of which produces a cluster of phialides; the phialides tend to be cylindrical. In M. inundatum, M. leucotrichum and M. verrucaria, the conidia are smooth. In M. cinctum and D. echinofibrosa, the conidia are distinctly striate. In most species of Stachybotrys the conidia are smooth or roughened to a greater or lesser extent. Species of both Stachybotrys and Myrothecium produce macrocyclic trichothecenes including verrucarins and roridins. Species of Myrothecium, including M. roridum and M. verrucaria, produce a series of eight macrocyclic trichothecenes identical to those formed by the trichothecene-producing isolates of S. chartarum (Jarvis, Pavanasasivam & Bean 1985, Jarvis 1991). According to Andersen et al. (2002), S. albipes, the anamorph of M. pomiformis, did not produce any of the tested trichothecene metabolites. It is not known if P. spirostriata/D. echinofibrosa produces mycotoxins similar to those of Stachybotrys or Myrothecium. 870 In all analyses of the five-gene hypocrealean dataset, Stachybotrys/Melanopsamma, Myrothecium, and Peethambara/Didymostilbe form a strongly supported, previously undiscovered sister lineage to all other families currently accepted in the Hypocreales, the Bionectriaceae, Clavicipitaceae, Hypocreaceae, Nectriaceae, and Niessliaceae. Within this lineage, Stachybotrys and Myrothecium are well supported as monophyletic anamorphic genera and the relationship of Melanopsamma pomiformis to Stachybotrys is confirmed. The connection of Melanopsamma to Stachybotrys has been made only once by Booth (1957). Although this lineage seemingly comprises a newly discovered family within the Hypocreales, additional teleomorph isolates, including the type species of Peethambara, P. sundara, are required to fully justify formally describing the new family. Perithecia of Peethambara are large, yellow, collapsed cupulate, and quite distinct from the small, black, albeit collapsed and cupulate, perithecia of Melanopsamma. This research shows that species of Stachybotrys and Myrothecium are closely related to each other and share morphological and molecular characteristics as well as the production of similar mycotoxins. Those seeking to resolve the problems posed by S. chartarum inhabiting moist cellulose-rich substrates in building interiors may also need to test for the presence of species of Myrothecium and, in tropical regions, the related D. echinofibrosa. Given the lack of systematic knowledge of tropical fungi and even of many saprophytic temperate fungi, one would expect to find additional members of this new lineage that occupy a similar niche on decaying, cellulose-rich plant material and that are capable of producing the same or similar toxic compounds. Such basic information is necessary for developing rapid identification tools (Haugland & Heckman 1998) for determining the presence of S. chartarum and related toxin-producing fungi in environmental samples. ACKNOWLEDGEMENTS We express our appreciation to Gerald Bills, Richard Haugland and Rosalind Lowen, who supplied some of the cultures used in this study. 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