Phylogenetic tree of living strains of <i>Hypoxylon pulicicidum</i> and related environmental strains and sequences deposited in GenBank inferred from Bayesian analysis of filtered ITS sequences.

<p><i>Hypoxylon fendleri</i> was designated the outgroup. Clade probability values/maximum likelihood bootstrap values are indicated respectively at the branches. Values <50 are designated by -. Bar represents 10 changes. <i>Hypoxylon pulicicidum</i> ITS1 sequence groups are identified as group1 with long repeated motifs, group 2 with intermediate repeated motifs, and group 3 with short repeated motifs. ITS1 base pair lengths are listed at the far right.</p

Phylogenetic tree of <i>Hypoxylon pulicicidum</i> and related species of the Xylariaceae inferred from Bayesian analysis of a combined gene genealogy of α-actin, β-tubulin and ITS-5.8S-ITS2 genes.

<p><i>Biscogniauxia anceps</i> was designated the outgroup. Clade probability values/maximum likelihood bootstrap values are indicated respectively at the branches. Values <50 are designated by -. Bar represents 10 changes.</p

Living strains examined and their sequences. GenBank accession codes for previous and new sequences used during this work.

a<p>Strains designated with MF are maintained at Merck Research Laboratories and at the Fundación MEDINA. Strains designated with F-xxxxx are maintained at the Fundación MEDINA.</p>b<p>Production of nodulisporic acids (NAs) were observed in previous referenced studies and/or this report.</p

The structures of nodulisporic acids unequivocally detected during this study.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046687#pone.0046687-Singh1" target="_blank">[12]</a> for a detailed description of structures of the nodulisporic acids and their stereochemistry.</p

Cultures and microscopic features of <i>Hypoxylon pulicicidum</i>, MF5954.

<p>A. from left to right, 14-d-old cultures on oatmeal agar, malt-yeast extract agar, 2% malt agar. B. Conidiophore from SNA. C. Conidiophore from SNA, note dark pigment granules. D. Conidiogenous cells and conidia from SNA, note dark pigment granules. E. Aerial view of conidiophore from SNA. F. Aerial view of conidiophore from SNA. G. Conidiogenous cells and conidia from SNA.</p

Preliminary identification of nodulisporic acids in fermentation extracts of strains of <i>Hypoxylon investiens</i> and <i>H. pulicicidum</i> by LC-MS database matching.

a<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046687#pone-0046687-t001" target="_blank">Table 1</a> for strain origins and details.</p>b<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046687#s4" target="_blank">Methods and Material</a> for medium formulations, fermentation and extraction protocols. Either fermentations harvested at 14, 21 d with nodulisporic acids, or both were considered positive.</p>c<p>Identification based on database matching of ion fragmentation patterns from authentic nodulisporic acids chromatographed fermentation extracts.</p

<i>Hypoxylon pulicicidum.</i>

<p>A, C, F, G: CLL0727; B, D, E, H: Holotype MJF 07147. A-C. Stromata. d. Close up of stromatal surfaces of a purplish mature stroma (left) and a blackish overmature stroma with slightly papillate ostioles (right). E, F. Stromata in vertical section showing perithecia with green contents (rehydrated in e). G, H. KOH-extractable pigments. Scale bar A-C = 5 mm; D-F 1 mm.</p

Detection and distribution of nodulisporic acid analogs by liquid chromatography high-resolution mass spectrometry among fermentation extracts of selected endophytic and teleomorph-derived strains of <i>Hypoxylon pulicicidum</i>.

*<p>SeeTable 1 for strain details and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046687#s4" target="_blank">Methods and Material</a> for description of fermentation media and protocols.</p><p>Calculated (Calc.) <i>m/z</i> for the known nodulisporic acids and their dehydrated and Na⁺ adducts are given above. For each strain, the retention times (RT), measured (Meas.) m/z and error in ppm are given for each detected nodulisporic acid ion and its dehydrated and Na+ adducts, respectively.</p

Cultures and microscopic features of <i>Hypoxylon pulicicidum</i>.

<p> A. MUCL 49879, from left to right, 14-d-old cultures on oatmeal agar, malt-yeast extract agar, 2% malt agar. B. MUCL 49879, conidiophores from SNA. C. MUCL 49879, conidiophores from SNA. D. MUCL 49879, conidiogenous cells and conidia from SNA. E. MUCL 49879, Aerial view of conidiophore from SNA. F. Ascus mounted in 1% SDS (MJF 07147). G. Ascus apical apparatus stained in Melzer’s reagent. H. Ascospores mounted in water (CLL 0727). Scale bar: F. 20 µm; H. 10 µm.</p

HPLC-UV chromatograms of <i>Hypoxlyon pulicicidum</i> and <i>H. investiens</i>.

<p>9a. HPLC-UV chromatograms of stromatal methanol extracts of the <i>H. pulicicidum</i> (MJF 07147, Holotype). 9b. HPLC-UV chromatograms of stromatal methanol extracts of specimen of <i>H. investiens</i> s. str. (material from Taiwan, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046687#pone.0046687.s004" target="_blank">Fig. S4</a>). 9c. UV spectra of BNT and unknown components from <i>H. pulicicidum</i>. The unknown compound B in <i>H. pulicicidum</i> has a MW of 506 DA. 9d. UV spectra of components from <i>H. investiens</i> including daldinone A and another unknown compound D.</p