Species-level assessment of secondary metabolite diversity among Hamigera species and a taxonomic note on the genus

Secondary metabolite phenotypes in nine species of the Hamigera clade were analysed to assess their correlations to a multi-gene species-level phylogeny. High-pressure-liquid-chromatography-based chemical analysis revealed three distinctive patterns of secondary metabolite production: (1) the nine species could be divided into two groups on the basis of production of the sesquiterpene tricinonoic acid; (2) the tricinonoic acid-producing group produced two cyclic peptides avellanins A and B; (3) the tricinonoic acid-non-producing group could be further divided into two groups according to the production of avellanins A and B. The chemical phenotype was consistent with the phylogeny of the species, although metabolite patterns were not diagnostic at the species level. In addition, the taxonomy of the Hamigera clade was updated with the new combination Hamigera ingelheimensis proposed for Merimbla ingelheimensis, so that all species in the clade are now in the same genus.


Introduction
The Eurotiales include the important genera Penicillium, Aspergillus, Talaromyces, Monascus, Paecilomyces and other lesser known genera, e.g., Hamigera. Species of Hamigera are widespread and common soil fungi, and one species is associated with beetles (Peterson et al. 2010). A hallmark feature of many species of the Eurotiales is their rich secondary metabolism, which is evident by their biosynthesis of many harmful mycotoxins and important metabolites used as medicines and as chemical probes in cell biology. The genetic basis of this rich secondary metabolism is now known to be the result of the high number of secondary metabolite gene clusters present in their genomes. Some species, e.g., Aspergillus nidulans and A. terreus, have been estimated to have as many as 50-70 individual core biosynthetic genes for non-ribosomal peptide synthases (NRPS), polyketide synthases (PKS), terpene cyclases and prenyl transferases (Inglis et al. 2013).
Fungi of the Eurotiales have served as model species for developing chemosystematic classification methods in the higher fungi, a classification approach that employs data from standardized high-pressure liquid chromatography (HPLC) separation methods linked to UV and mass spectrometer (MS) detectors to generate profiles of metabolites characteristic of species and species complexes (Nielsen and Smedsgaard 2003;Frisvad et al. 2008). This approach of joining orthogonal data from metabolite patterns with morphology and phylogenetics to accurately diagnosis contaminating fungi has been pioneered at the Danish Technical University in conjunction with investigations of food, materials and environmental contaminants (Overy et al. 2003;Frisvad et al. , 2007. The methods have been incorporated into extensive monographic works on many groups of Aspergillus, Penicillium, Talaromyces and Monascus and have consistently demonstrated that species definitions based on ribosomal and protein gene phylogenies correlate closely not only with the fungal morphological phenotype but also with the secondary metabolite phenotypes of individual strains belonging to a species Larsen et al. 2005;Frisvad et al. 2009;Nielsen et al. 2009;Samson et al. 2009;Slack et al. 2009). As of yet, an analysis of secondary metabolites has not been applied to the genus Hamigera.
At least five metabolite families have been reported from Hamigera avellanea (Figure 1). The anthraquinone pigments emodin, ω-hydroxyemodin and emodic acid have been characterized from H. avellanea (Natori et al. 1965;Cehulová et al. 1996;Isaka et al. 2010). Hamigerone and dihydrohamigerone are polyketide antifungal metabolites (Breinholt et al. 1997). A compound known as 87-250904-F1 (=radicicol analogue A) is a resorcylic acid lactone that inhibits interleukin 1β and tumour necrosis factor-α secretion from the human monocytic leukemic cells (Mak et al. 2007) and has been found in H. avellanea along with a series of biosynthetically related nonaketide macrolactones named hamigeromycins A-G (Isaka et al. 2008(Isaka et al. , 2010. The same strain also produced a pair of novel cyclopropyl diketones designated hamavellones A and B, which were moderately toxic to Plasmodium falciparum and some human cancer cell lines. The strain co-produced pseurotin A, a competitive inhibitor of chitin synthase and an inducer of nerve cell proliferation, that is, the product of a hybrid NRPS-PKS gene cluster (Maiya et al. 2007). A family of cyclic hexapeptides and a pentapeptide, PF1171A-E, were described in a Japanese patent application from a strain of H. avellanea (FERM P-16173) (Umagome et al. 1999) for inhibition of the production of apolipoprotein B, the principal component of low-density lipoprotein. Based on amino acid sequences, these cyclic peptides are likely biosynthetically related to the pentapeptides avellanins A and B. Intravenous administration of avellanins caused vasoconstriction and increased blood pressure leading to death in mice (Yamazaki et al. 1987).
The genus Hamigera was recently revised by employing phylogenies constructed from rDNA and protein gene sequences. The analysis demonstrated that Hamigera comprises a monophyletic lineage of eight ascomata-forming species and one asexual species, with H. avellanea as the type species (Peterson et al. 2010). One of the main conclusions of this study was that the high variability observed in H. avellanea was the result of inadequate discrimination among a series of six newly recognized cryptic species. Furthermore, the asexual species Merimbla ingelheimensis, often thought to be the conidial state of H. avellanea was a distinct species within the Hamigera clade. The genus has recently been reclassified in the family Aspergillaceae and is a sister clade to Aspergillus and Monascus (Houbraken and Samson 2011).
This recent revision and the consolidation of a collection of all the newly described species from Europe, Asia, Africa, Micronesia, Australia, South America and North America at the Northern Regional Research Laboratory Culture Collection (NRRL) afforded an opportunity to test the hypothesis that these newly described taxa would contribute significant new variations in secondary metabolism beyond those previously reported compounds. In this report, representative strains of the nine species (one undescribed) were grown under a set of fermentation conditions, extracted with an organic solvent, and their extracts profiled by HPLCdiode array detector (DAD) to detect major UV-absorbing metabolites. We therefore describe the first genuswide assessment of chemical diversity in Hamigera including reports of metabolites not previously encountered from species of this genus.

Strains and phylogeny
Strains (Table 1) were obtained from the NRRL Collection and cultured on potato dextrose agar (PDA) at 20°C prior to generation of fermentation extracts. DNA sequences Bootstrap statistical support for the nodes were determined from 500 bootstrap iterations and are indicated on the tree. The tree diagram was formatted for publication using CorelDraw X6.

Extraction and HPLC analysis
Equal volumes of 1-butanol were added to fermentations, and flasks were agitated for 60 min and then contents were centrifuged to separate fungal cells from the supernatant. A portion (1 mL) of the organic layer was concentrated under reduced pressure to give a crude extract that was then dissolved in dimethlysulfoxide (DMSO, 0.5 mL) for HPLC analysis on an Agilent HP1200 system equipped with a photodiode array detector. Spectral data were collected in the range from 200 to 600 nm.

Characterization of known and new compounds
The crude culture extract was fractionated by silica gel and ODS column chromatography and was further purified by preparative HPLC. Isolated compounds were subjected to spectroscopic analysis including nuclear magnetic resonance (NMR) and MS for structure determination.  (Peterson et al. 2010) showed that they are distinct species. Under the auspices of article 59 from the nomenclatural code prior to 2012, the anamorphic M. ingelheimensis was placed in a distinct genus from its sister species H. avellanea. The Melbourne Code (Mcneill et al. 2012) changed the rules for naming pleiomorphic fungi and allowed only one valid name for a fungus, even if it had distinct morphologies that could occur independently and were named in different genera. All of the Hamigera species have a Merimbla-state in association with the teleomorphic state in Hamigera. Hamigera ingelheimensis has the Merimbla-state, but the teleomorphic state has not been observed. Because the Melbourne Code allows only a single name for a fungus and because intuitively we do not expect members of different genera to be more closely related that members of a single genus, we have formed the new combination Hamigera ingelheimensis to reflect the phylogenetic position of the species.

Secondary metabolite analysis
Metabolite production was analysed by HPLC after 4, 6 or 10 days of cultivation with three strains (Hamigera insecticola NRRL35442, NRRL35443 and NRRL58093) in A-3M, A-11M and A-16 to establish the fermentation period for chemical phenotype pattern comparison. These strains produced tricinonoic acid and avellanins A and B as the major metabolites (Figure 2). Production of these three compounds was observed after 4 days, but no significant increase in production level was observed after 6 or 10 days of fermentation. Static culture was also tested but the period for production was substantially delayed (3-4 weeks) and did not significantly change metabolite patterns ( Figure 3). Therefore, a 4-day fermentation period was fixed for the subsequent experiments.
Nineteen strains from the Hamigera clade (Table 1) were fermented in six different liquid media for 4 days on a rotary shaker. The whole culture broth was extracted with 1-butanol and the solvent extracts were analysed by HPLC-DAD. Major peaks were purified by consecutive fractionation on a silica gel and an ODS column chromatography, and final purification was done by preparative HPLC if necessary. The structures of isolated metabolites were analysed by using NMR, MS and UV techniques. From the 19 strains tested, 3 biosynthetically different classes of metabolites were identified (Table 2): tricinonoic acid, which is a sesquiterpene; avellanins and PF1171s, which are cyclic pentapeptides and hexapeptides; and anthraquinones, which are aromatic polyketides.
The most commonly produced metabolites were avellanins A and B ( Table 2). Fifteen of the 19 strains Notes: *New compound. **Aromatic polyketide (UV λ max = 445 nm); structure not determined.
representing 8 of the 9 species produced these cyclic pentapeptides. Three strains of H. ingelheimensis did not. The second most common metabolite was tricinonoic acid, a sesquiterpene first isolated from Fusarium tricinctum (Bashyal and Leslie Gunatilaka 2010). To the best of our knowledge, production of tricinonoic acid has not been reported from Hamigera or Merimbla. The production pattern of this terpene compound divided nine Hamigera species into two groups.  Figure 4). Details on structure determination and biological properties of avellanin C will be reported in our forthcoming paper. It is noteworthy that avellanin C was also produced by H. ingelheimensis NRRL29060 and was the only compound detected from this strain ( Figure 5).
Production of anthraquinone-class polyketides that displayed typical UV λ max spectral profile with the absorption maximum around 445 nm (Figure 4) was noticed only with H. paravellanea NRRL35714 although their structures were not fully elucidated yet. Strain NRRL35714 was the most productive strain tested in this study, which produced the full range of cyclic pentapeptides and hexapeptides and aromatic polyketides. Genomic sequencing has not been completed for any Hamigera species to date; therefore, data on genome size and numbers and distribution of secondary metabolite gene clusters in these fungi remain unknown. Based on secondary metabolome studies of other species of the Eurotiales, it is likely that we have only sampled a portion of the secondary metabolites encoded in the genomes of these fungi. Our assessment is preliminary because we have not exhaustively probed media and fermentation techniques for stimulating metabolite expression, and our  primary detection method was based on UV absorbance. Perhaps, mass-based detection methods or sensitive biological assays would have revealed additional metabolite complexity.
Our data did not provide species-level diagnostic patterns. Nonetheless, the major metabolites reported here were consistent with the phylogeny of the species; tricinonoic acid production was a definitive character for the clade containing H. terricola, H. pallida, H. fusca, H. inflata and H. insecticola (Figure 6). Other metabolites such as PF1171B and PF1171C were variably present in strains of species from each of the main clades of Hamigera. This pattern could be explained by presence of their pathways in the most recent common ancestor followed by loss of function in some descendent species or simply lack of expression in some of the strains and species during our study. Most studies (e.g. ) that use chemotaxonomy as part of the identification scheme use a broader range of metabolites to reliably identify species. That would appear to be the case in Hamigera also. Statistically convincing chemotaxonomy must await a more thorough mining of the secondary metabolites produced by the species and isolates and use of more chemical markers.