Three New Derivatives of Zopfinol from Pseudorhypophila Mangenotii gen. et comb. nov.

Triangularia mangenotti was analyzed for the production of secondary metabolites, resulting in the isolation of known zopfinol (1) and its new derivatives zopfinol B–C (2–4), the 10-membered lactones 7-O-acetylmultiplolide A (5) and 8-O-acetylmultiplolide A (6), together with sordarin (7), sordarin B (8), and hypoxysordarin (9). The absolute configuration of 1 was elucidated by the synthesis of MPTA-esters. Compound 1 showed antimicrobial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the fungus Mucor hiemalis. While 4 was weakly antibacterial, 3 showed stronger antibiotic activity against the Gram-positive bacteria and weak antifungal activity against M. hiemalis and Rhodotorula glutinis. We furthermore observed the cytotoxicity of 1, 3 and 4 against the mammalian cell lines KB3.1 and L929. Moreover, the new genus Pseudorhypophila is introduced herein to accommodate Triangularia mangenotii together with several species of Zopfiella—Z. marina, Z. pilifera, and Z. submersa. These taxa formed a well-supported monophyletic clade in the recently introduced family Navicularisporaceae, located far from the type species of the respective original genera, in a phylogram based on the combined dataset sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of the ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes. Zopfiella submersa is synonymized with P. marina due to the phylogenetic and morphological similarity. The isolation of zopfinols 1–4 and sordarins 7–9 confirms the potential of this fungal order as producers of bioactive compounds and suggests these compounds as potential chemotaxonomic markers.


Introduction
The genus Triangularia was recently found to be polyphyletic, and its species were scattered along the phylogenetic tree of the order Sordariales [1,2]. Two years ago, Wang et al. [3] delimited the genus to the type species, together with other species previously placed in the genera Apiosordaria, Podospora, and Zopfiella. Recently, Triangularia karachiensis was transferred to the new genus Lundqvistomyces, since it was not located in the monophyletic clade comprising Triangularia [4]. On the other hand, the genus Zopfiella could so far not be correctly delimited due to the lack of type material of the type species Z. tabulata [4]. One reference strain of this species was placed with other ones producing ascospores with septate upper cell in the family Lasiosphaeriaceae, suggesting that this is the right monophyletic lineage representing the genus [3,4]. Therefore, other species of Zopfiella not located in this lineage have been transferred to other genera, e.g., Z. longicaudata and Table 1. Strains of the order Sordariales included in the phylogenetic study. Taxonomic novelties are indicated in bold italic.

Fermentation and Extraction
The fungus was grown in yeast malt agar (YM agar; malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, agar 20 g/L, pH 6.3 before autoclaving [28]) at 23 • C. Once the fungus was grown, the cultures were cut into small pieces using a cork borer (1 × 1 cm) and five of these pieces were placed into a 200 mL Erlenmeyer flask containing 100 mL of yeast-malt extract broth (YM broth; malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, pH 6.3 before autoclaving) under shake conditions at 140 rpm at 23 • C. After 20 days, 10 flasks of 500 mL containing BRFT medium [brown rice 28 g as well as 0.1 L of base liquid (yeast extract 1 g/L, sodium tartrate 0.5 g/L, KH 2 PO 4 0.5 g/L [29])] were inoculated with 6 mL of the seed culture, and incubated for 15 days at 23 • C.
For the compound extraction, the solid cultures in BRFT were covered with acetone, and sonicated in an ultrasonic bath for 30 min at 40 • C. Paper filters were used to separate the acetone from the mycelium, and the latter was again subjected to the same sonication and separation procedure. Both acetone extracts were combined and dried in vacuo at 40 • C. The remaining aqueous residue was diluted with the same amount of ethyl acetate (EtOAc) and extracted twice. The crude extract obtained after drying in vacuo at 40 • C was solved in methanol (MeOH) and extracted twice against one-part methanol-water (distilled water, methanol 1:1) and one-part heptane. Finally, the aqueous phase was again diluted with the same amount of EtOAc and extracted twice. The extracts were combined, dried in vacuo at 40 • C and weighed. Crude extract yield was 2230 mg.
Compound 1 (6.9 mg, t R = 23.5-25 min) was obtained from purification of fraction 9, and 9 (1.92 mg, t R = 36-37 min) from purification of fraction 11 in the same HPLC system with the same solvents, using XBridge ® Prep C 18 5 µm OBD TM (250 × 19 mm, 5 µm; Waters, Milford, MA, USA) as the stationary phase with a flow rate of 15 mL/min and a fractionation of 5 mL. The HPLC gradient for the purification of fraction 9 is as follows: increase from 33% B to 43% B for 15 min, followed by an increase to 50% B in 30 min, then increase to 100% B in 10 min, and a final isocratic elution of 100% B for 5 min. The HPLC gradient for the purification of fraction 11 consists of an increase from 45% B to 50% B for 10 min, followed by an increase to 55% B in 30 min, then an increase to 100% B in 7 min, and a final isocratic elution of 100% B for 5 min.
Fraction 7 and 10 were further separated using an Agilent 1200 Infinity Series HPLC-UV system (Agilent Technologies, Santa Clara, CA, USA) with a Nucleodur 100-10 C18ec (250 × 10 mm, 10 µm; Macherey-Nagel, Düren, Germany) as the stationary phase and the following conditions: solvent A: H 2 O + 0.1% formic acid, solvent B: ACN + 0.1% formic acid; flow: 5 mL/min, fractionation: 2.5 mL. Compound 3 (1.75 mg, t R = 19.5-20.5 min) was obtained from fraction 10 with the following gradient: an increase from 30% B to 40% B for 7 min, then an increase to 60 % B in 30 min, an increase to 100% B in 7 min, and a final isocratic step of 100% B for 7 min. Compound 4 (0.8 mg, t R = 22-23 min) was obtained from fraction 7 with the following gradient: an increase from 25% B to 38% B for 7 min, followed by an increase to 43% B in 20 min, then an increase to 100% B in 7 min, and a final isocratic step of 100% B for 5 min.

Biological Testing
Isolated compounds were tested for their antimicrobial activity against five fungi (Candida albicans, Mucor hiemalis, Rhodotorula glutinis, Schizosaccharomyces pombe and Wickerhamomyces anomalus), four Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus, Mycobacterium smegmatis and Staphylococcus aureus) and three Gram-negative bacteria (Chromobacterium violaceum, Escherichia coli and Pseudomonas aeruginosa), using nystatin as a positive control against all the tested fungi and oxytetracycline against all the bacteria, except for My. smegmatis and Ps. aeruginosa, against which kanamycin and gentamycin were used, respectively. Moreover, the cytotoxicity of the compounds against two different mammalian cell lines-human endocervical adenocarcinoma KB 3.1 and mouse fibroblasts L929-were determined by the MTT method using epothilone B as the positive control. Both biological assays were performed following the protocols described by Becker et al. [33].

Phylogenetic Analysis
The lengths of the individual alignments used in the combined dataset were 634 bp (ITS), 891 bp (LSU), 972 bp (rpb2) and 618 bp (tub2), and the final total alignment was 3115 bp. The phylogentic tree obtained from the RAxML analysis of the combined dataset, including bootstrap support and Bayesian posterior probability at the nodes, is shown in Figure 1. The RAxML tree obtained agreed with the topology of the tree generated by the Bayesian analysis. The ex-type strain of Triangularia mangenotii was located in the Naviculisporaceae clade, forming a well-supported clade (100% bs/1 pp) independent from the other lineages of the family, together with the type strains of Zopfiella marina, Z. pilifera and Z. submersa. However, the monophyletic lineage representing the genus Triangularia was placed in the Podosporaceae clade, while the type species of Zopfiella, Z. tabulata was located in the Lasiosphaeriaceae clade. Therefore, the new genus Pseudorhypophila is introduced herein to accommodate these four taxa. Additionally, the close phylogenetic distance between Z. marina and Z. submersa suggested that these could indeed represent the same taxa. The nucleotide similarity of the rpb2 sequences of both taxa was 99.88%, while that of the ITS sequences was 99.78% (the only difference was due to the presence of an indeterminable base-pair in one of the sequences). The same occurred in the LSU sequence comparison, in which the similarity was only 97.43% but the differences were due to indeterminate nucleotide positions in the sequences of Z. marina. The nucleotide similarity of tub2 sequences (a fragment different from the one used in the present phylogenetic study; GenBank acc. numbers MK926951 and MK926953) was also 100%. Therefore, and in accordance with phenotype-derived data, the synonymy of both species is proposed. Etymology: Based on the phylogenetic relation to Rhypophila. Ascomata non-ostiolate or ostiolate, superficial or immersed, black, globose to subglobose, or ovate to pyriform, almost glabrous or covered by short or long, flexuous hairs; neck short, cylindrical to conical, covered with small black papillae. Asci clavate to cylindrical, stipitate, 4-8-spored, with a small apical ring sometimes indistinct. Periphyses present or absent. Paraphyses present or absent, septate, hyaline. Ascospores biseriate, two-celled; upper cell narrowly conical, acuminate towards apex and rounded at base, or ovoid to limoniform with somewhat truncate base, olivaceous brown to dark brown, with an apical or subapical germ pore, sometimes with a distinct apical appendage; lower cell remaining hyaline, or sometimes becoming pale olivaceous brown or pale brown, occasionally dark brown, cylindrical and straight or curved, or hemisphaerical, or at first broadly obconical and then becoming flattened at apex; gelatinous sheats sometimes present, hyaline, thin. Conidia holoblastic, sessile, borne singly along the vegetative hyphae, hyaline, spherical to subspherical, or ovate to elongate, smooth-walled. Notes: Pseudorhypophila is related to Gilmaniella and Rhypophila. The former genus produces the humicola-like asexual morph characterized by the production of dark brown, spherical conidia with marked apical germ pores and borne singly or in clusters of up to four [34], while the new genus Pseudorhypophila produces a chrysosporium-like asexual morph, and the asexual morph is absent in Rhypophila [4]. Rhypophila differs from Pseudorhypophila by the production of ascomata with elongate, tuberculate projections in the neck, while these are mostly non-ostiolate ascomata in the new genus. Moreover, Rhypophila is characterized by having mostly more than eight-spored asci and ascospores with lower cell as long as, or longer, than the upper cell.

Taxonomy
Zopfiella submersa was introduced by Guarro et al. [35] in 1997. These authors discussed the similarity of this taxon with Zopfiella marina, which was introduced before by Furuya and Udagawa [36]. The main differences between both species according to reference [36] were the presence of a sexual morph and ascospore with an apical pore in the upper cell in Z. marina, whereas the asexual morph is absent and the upper cell of the ascospores have a subapical pore in Z. submersa. Both taxa were isolated only from aquatic environments in Asia (China and Iraq). Whereas Z. marina was found in marine mud (in depth of 120 m), Z. submersa was reported from dead culms of Arundo donax submerged in a river. Due to the scarce molecular and morphological differences between both taxa, we proposed here their synonymy under the new combination P. marina. The other two species of the genus-P. mangenotii and P. pilifera-are also closely related to each other, but these showed only a 98.04 % nucleotide similarity of the rpb2 sequences. Both species are characterized by ascospores with conical upper cells [37], but these can be easily distinguished by the ascomata, being ostiolate in P. mangenotii [38] and non-ostiolate in P. pilifera, and by the presence of an asexual morph in the latter [39].
Key to species of Pseudorhypophila.
Compound 3 was obtained as colorless-to-white crystals. The molecular ion cluster at m/z 365.1491 [M + Na]+ in the HRESIMS spectrum indicated that the molecular formula is C 18 H 27 ClO 4 . The NMR data of 3 were highly similar to those of 2, with the key difference being the exchange of the olefinic methines 12-H/13-H by two methylenes. Therefore, we assigned 3 as 12,13-dihydrozopfinol, the name given to it being zopfinol C.
Compound 4 was obtained as a yellow oil and its molecular formula was established as C 18 H 28 O 4 according to the mass ion peak at m/z 331.1878 [M + Na] + in the HRESIMS spectrum, indicating the formal addition of two hydrogens. The key difference in the NMR spectra of 4 compared to 1 was the exchange of the olefinic methines 12-H/13-H by two methylenes. Therefore, we elucidated 4 as dechloro-12,13-dihydrozopfinol, and named it zopfinol D.
On the other hand, compound 7 and 9 showed antifungal activity against Candida albicans, even though the activity of 9 was weak. Compound 9 showed a much stronger antifungal activity against Mucor hiemalis.
Compound 1, 3 and 4 showed weak cytotoxic activity against the two different mammalian cell lines tested (Table 4).

Discussion
Lasiosphaeriaceous genera have been considered polyphyletic since their taxa were scattered in different clades along the Sordariales [1,3,18,22,26]. This was a consequence of the traditional delimitation of the genera based on the ascospore morphology, which resulted in an extremely homoplastic character not useful in predicting the phylogenetic relationships [1,22]. Recent phylogenetic studies based on the ITS, LSU, rpb2 and tub2 sequences were focused on the right delimitation of both the polyphyletic family and genera, resulting in the introduction of the monophyletic families Podosporaceae [3], Diplogelasinosporaceae, Naviculisporaceae and Schizotheciaceae [4]. Moreover, some of the genera were properly delimited, such as Podospora and Triangularia [3]. However, large genera such as Cercophora and Zopfiella still remain polyphyletic, and other species of the already delimited genera are awaiting a correct taxonomic placement. In that context, the type strain of T. mangenotii, which was located in the family Naviculisporaceae and far from the monophyletic clade of Triangularia in the Podosporaceae, is currently relocated in the new genus Pseudorhypophila, together with other species of Zopfiella far from the type species of the genus, Z. tabulata, which is located in the Lasiosphaeriaceae. This new genus is characterized by mostly non-ostiolate ascomata and a chrysosporium-like asexual morph. On the other hand, the most phylogenetically related genus, Gilmaniella, is characterized by the production of a solely humicola-like asexual morph [34].
Zopfinol (1) is a chloratinated phenol with an aliphatic side chain and was isolated before from the marine fungus Zopfiella marina [40], which we transferred in the present study to the new genus Pseudorhypophila. In addition, the strain we studied produced three new derivatives of zopfinol (2)(3)(4). Compound 1 showed weak antimicrobial activity against Mucor hiemalis and Staphylococcus aureus, and moderate antibacterial activity against Bacillus subtilis. On the other hand, the new derivative 4 showed only a weak activity against the Gram-positive bacteria, B. subtilis and S. aureus. Compound 3 was moderately active against the same two Gram-positive bacteria, and exhibited weak antifungal activity against M. hiemalis and Rhodotorula glutinis.
7-O-acetylmultiplolide A (5) and 8-O-acetylmultiplolide A (6) are 10-membered lactones, first reported from a Diaporthe sp. [30,41], which pertains to the class Sordariomycetes. Both compounds were devoid of antimicrobial activity against the microorganisms tested in the present study. However, compound 6 had shown antifungal activity against Aspergillus niger, Bipolaris maydis, Botrytis cinerea, Fusarium moniliforme, Ophiostoma minus and Talaromyces islandicus, as previously reported by Wu et al. [30]. Surprisingly, compound 5 only showed weak activity against A. niger, even though both compounds 5 and 6 differ only in the position of the acetoxy group [30]. Compound 6 was reported to have significant inhibitory activity towards acetylcholinesterase [41], and antihyperlipidemic activity equivalent to that observed in lovastatin, which was used as a positive control [44]. Other ten-membered lactones have been found in Diaporthe [30,41,44], as well as in other Sordariomycetes, i.e., Xylaria multiplex [45] and Gilmaniella humicola [46], the latter of which is also now located in the Naviculisporaceae like P. mangenotii. Some of these 10-membered lactones also showed antifungal activity, i.e., multiplolides A and B were active against Candida albicans [45]. Moreover, humilactone from Gilmaniella humicola showed strong cytotoxic activity [46], which was not observed in the other related compounds mentioned.
The last group of compounds isolated from P. mangenotii were the sordarins (7-9). Those are a class of natural antifungal agents that act at the protein synthesis level, inhibit-ing it through their interaction with the elongation factor 2 in eukaryotes (eEF2) [47,48]. This essential enzyme catalyzes the translocation of transfer RNA and messenger RNA after peptide bond formation in the translation process, leading to an inhibition of this step and promoting cell death [49,50]. What makes these compounds have a solely antimycotic activity is the high affinity for fungal eEF2 when it is compared against that of plants or mammals [50]. These compounds are mainly produced by Xylariales, but also by members of Eurotiales, Microascales and Sordariales [8]. In this last order, the taxa reported to produce these kinds of compounds are Podospora araneosa [42], Rhypophila pleiospora [7] and Z. marina [51], which is here transferred to the genus Pseudorhypophila (as P. marina), all of which are members of the family Naviculisporaceae. Therefore, the production of sordarin and related compounds could be restricted to this family. Podospora araneosa clustered in the monophyletic clade of Rhypophila, suggesting that it could belong to this genus. However, further studies including the type material of this species need to be carried out to corroborate this hypothesis. Compound 7 was found in cultures of Podospora araneosa [42] and 8 in Rhypophila pleiospora [7], while 9 was only reported before from Hypoxylon croceum [32], which is located in the Xylariales. Podospora araneosa also produced hydroxysordarin and neosordarin, which is closely related to 9, with only small differences in the aliphatic side chain acylating the hydroxyl in the 3 -position of the sordarose moiety [51]. Pseudorhypophila marina produces the sordarin derivative known as zofimarin [51], which was demonstrated to have antifungal activity against Candida albicans, C. pseudotropicalis and Crytococcus neoformans [52]. In our antimicrobial study, 8 was not active against any of the microorganisms tested. However, Weber et al. [7] observed antifungal activity against Nematospora coryli, Sporobolomyces roseus and Thelebolus nanus. In the present study, 7 was only active against C. albicans, while 9 showed weak activity against C. albicans but moderate activity against M. hiemalis. The higher antifungal activity of 9 with respect to the other sordarin or sordarin-related compounds was already observed by Davoli et al. [53]. In that work, 9 showed antifungal activity against Paecilomyces variotii, Penicillium notatum, Nematospora coryli and M. miehei, while 7 only had activity against the last two fungi. The comparison between the activities of different sordarin derivatives demonstrated that the nature of the side chain plays an important role in the antifungal activity, increasing when there is a 3 -O-acyl group and decreasing in the presence of a hydroxymethyl group in the sugar moiety [52,53].
Pseudorhypophila marina also produced salicylaldehyde and dihydroisobenzofuran derivatives [54], apart from zopfinols [40] and zopfimarin [51], mentioned before. The structures of these compounds are related to zopfinol, but most of them were not active, except for one of the salicylaldehyde derivatives, which showed weak activity against Mycobacterium tuberculosis and Bacillus cereus [54]. Other compounds with structures related to zopfinol and its derivatives are the salicylaldehyde sordarial produced by Neurospora crassa, which also belongs to the order Sordariales [55], and the pyriculols, which are phytotoxic polyketides produced by the sordariomycete phytopathogenic fungus Pyricularia oryzae [56]. Since the phytotoxic pyriculol [57] differs from 2 only in the length of its aliphatic side chain, it would be highly interesting to test the phytotoxicity of zopfinols.
The production of secondary metabolites by the new genus Pseudorhypophila could be useful as chemotaxonomic markers, since the zopfinol is produced by different species of Pseudorhypophila, but it was not reported in any other taxon. Sordarins seem to be present in different taxa belonging to the family Naviculisporaceae, also being a potential chemotaxonomic marker for this family. Chemotaxonomy could help us in the achievement of a more natural classification of the sordariaceous fungi.
Our work, together with those focused on the screening for bioactive metabolites produced by members of the Sordariales [5,6,9], confirms the potential of this fungal order as a producer of bioactive compounds. In particular, the new genus Pseudorhypophila includes species able to produce a plethora of bioactive compounds, including the widely studied antifungal sordarins.