# Metabolomic analysis of macromycetes from French Guiana revueals chemodiversity in Xylariales order

Jonathan Sorres, Juliette Segret, Elsa Van-Elslande, David Touboul,

Véronique Eparvier

► To cite this version:

Jonathan Sorres, Juliette Segret, Elsa Van-Elslande, David Touboul, Véronique Eparvier. Metabolomic analysis of macromycetes from French Guiana revueals chemodiversity in Xylariales order. Phytochemistry, 2026, 242, pp.114709. 10.1016/j.phytochem.2025.114709. hal-05333878

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# Metabolomic analysis of macromycetes from French Guiana reveals chemodiversity in Xylariales order.

Jonathan Sorres<sup>a,§</sup>, Juliette Segret<sup>a,§</sup>, Elsa Van-Elsande<sup>a</sup>, David Touboul<sup>b</sup>, Véronique Eparvier<sup>a,*</sup>

<sup>a</sup> Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, 91198 Gif-sur-Yvette, France.

<sup>b</sup> LCM, CNRS, Ecole Polytechnique, Institut Polytechnique de Paris, Route de Saclay, 91120 Palaiseau, France

*Corresponding author. E-mail address: [veronique.eparvier@cnrs.fr](mailto:veronique.eparvier@cnrs.fr). Postal address: 1 avenue de la terrasse, 91198, Gif-sur-Yvette, France, Tel.: +33-169-823-679 (V.E.)

§ These authors contributed equally to this work

## Abstract

An original approach was undertaken to explore the metabolomic variability of macroscopic fungi collected in French Guiana. Extracts from 160 specimens, representing 13 fungal orders, were analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The resulting metabolomic profiles enabled the construction of chemotaxonomic dendrograms, which successfully grouped most samples assigned to the order Xylariales and highlighted chemodiversity at both the genus and species levels within this lineage. Principal Component Analysis based on molecular family presence/absence revealed 35 discriminant clusters, including specific markers distinguishing *Thamnomyces chordalis* and *Phylacia* sp.. Five previously undescribed natural products were isolated and structurally characterized: four polyketides—prochordapyrone (1) and chordapyrones A–C (2–4)—and one terpenoid, chordapyranone (5). Structural elucidation was accomplished through a combination of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism and single-crystal X-ray diffraction. Based on structural and mechanistic considerations, a putative biosynthetic pathway was proposed for the formation of the polyketide compounds.

## Keywords

*Thamnomyces chordalis*, *Phylacia* sp., Xylariales, Fungi, metabolomics, chemomarkers

## 1. Introduction

Fungi are integral components of ecosystems, functioning as primary decomposers and playing pivotal roles in complex ecological networks through their interactions with plants, animals, and other microorganisms. Consequently, they have evolved the capacity to produce an exceptional array of specialized metabolites, many of which mediate communication or serve defensive functions (Bérdy, 2005; Kluger et al., 2015; Lind et al., 2017, Helaly et al.,
41 2018). This extensive metabolic repertoire positions fungi as a prolific source of both biological
42 and chemical diversity.

43 In order to study the full range of specialized metabolites produced by macroscopic fungi,
44 recent technological advancements as molecular networking has emerged as a powerful
45 method for the dereplication of complex metabolomic data, enabling the annotation and
46 visualization of high-resolution tandem mass spectrometry (HR-MS/MS) datasets based on
47 spectral similarity (Wang et al., 2016). The integration of data processing tools such as MZmine
48 (Katajamaa et al., 2006; Pluskal et al., 2010; Schmid et al., 2023) with molecular networking
49 platforms like GNPS (Nothias et al., 2020; Wang et al., 2016) and MetGem (Olivon et al., 2018,
50 Barthélemy et al., 2020) now facilitates metabolimic analyses on an unprecedented scale.

51 In the present study, we assessed the variability of metabolites in macroscopic fungi collected
52 in French Guiana, a biodiversity hotspot (Henkel et al., 2011; Matheny et al., 2003; Stadler et
53 al., 2004; Vasco-Palacios and Franco-Molano, 2013) that is home to many endemic fungal
54 taxa (Fournier et al., 2023; Fournier and Lechat, 2015; Henkel et al., 2004). Our objective was
55 to identify specific and unique metabolites of certain taxa. To do this, we used a contemporary
56 dereplication strategy based on molecular networking, which organizes structurally related
57 metabolites into molecular families based on their spectral characteristics. Although
58 environmental factors can influence the biosynthesis of specialized metabolites in wild fungi,
59 taxonomically related species often share conserved biosynthetic pathways that produce
60 structurally similar compounds.
## 2. Results and discussion

### 2.1. Macromycetes bio- and chemo-diversity

A total of 160 fungal samples were collected and identified, representing 13 orders, 30 families, and 57 genera, with a strong predominance of Polyporales, Xylariales, and Agaricales (85% of the collection, Table S1). All samples were extracted in a standardized manner with ethyl acetate and analyzed by LC-HRMS/MS, generating 30,773 molecular signals, 70% of which were specific to a fungal order (Table S2). Molecular networking analysis identified 1,551 clusters, of which 737 spectral families were specific to an order (Fig. S1). The greatest chemical diversity was observed within the Xylariales, which accounted for 316 unique clusters across 37 samples (Fig. 1). We therefore focused our efforts on this order to identify novel metabolites. To further investigate the role and specificity of specialized metabolites within Xylariales, we focused on compounds potentially involved in differentiation at the genus and species levels.

FIGURE 1

### 2.2. Metabolic diversity of Xylariales

A data processing was performed exclusively on samples taxonomically classified within the order Xylariales (Fig. S2 and S3). Principal component analysis (PCA) enabled the discrimination of the genera *Thamnomyces* and *Phylacia* from other members of the order (Fig. S4). This separation was driven by nine distinct molecular clusters (clusters 23, 26, 34, 73, 103, 110, 128, 184, and 273) (Fig. S5). Seven of these clusters (23, 73, 103, 110, 128, 184, and 273) appeared to belong to the same molecular family, as evidenced by a shared major fragment ion at *m/z* 125.0233 (calculated for C~6~H~5~O~3~+, error -0.8 ppm) (Fig. S5-S10). The remaining two clusters (26 and 34) also exhibited high spectral similarity, including a characteristic fragment at *m/z* 147.1168 (calculated for C~11~H~15~+, error +0.7 ppm) (Fig. S6 and S11), suggesting affiliation with a distinct molecular family. Despite these findings, no annotation could be assigned to any of the clusters using MetGem or other available online databases. These compounds represent the main metabolites produced by the fruiting bodies of *Thamnomyces chordalis*.

### 2.3. Isolation and characterization of Xylariales chemomarkers

A chemo-guided isolation approach of this fungus led to the identification of five previously unreported metabolites: compounds **1–3**, associated with cluster 23, and compounds **4** and **5**, corresponding to clusters 273 and 34, respectively (Fig. 2 and S5).

FIGURE 2

For compound **1**, molecular formula was determined to be C~20~H~30~O~5~ by HRESIMS ([M + H]⁺ peak at *m/z* 351.2167 calcd for C~20~H~31~O~5~⁺ at *m/z* 351.2167, err. 0.28 ppm) suggesting 6 degrees of unsaturation. The ¹H spectra revealed three doublets (δ~H~ 1.10, 1.03 and 0.97 ppm), one multiplet (δ~H~ 4.39 ppm) suggests it is a hydroxylated methylene, one singlet (δ~H~ 3.80 ppm),
5 multiplets at $\delta_H$ 2.62, 2.18, 1.99, 1.40 and 1.31 ppm, and five olefinic protons at 6.46, 6.18, 5.45, 5.43 and 5.33 ppm (Table 1). From the ¹³C NMR spectrum 3 olefinic quaternary carbon were observed at $\delta_C$ 164.3, 159.6, and 128.9 ppm, together with one carbonyl carbon at $\delta_C$ 171.5 ppm. The ¹H–¹³C correlations in the HSQC spectrum confirmed the presence of four methyl carbons ($\delta_H/\delta_C$ 3.80/55.9, 1.10/19.8, 1.03/20.9, and 0.97/15.4 ppm), four methylene carbons ($\delta_H/\delta_C$ 4.39/57.1, 1.99/32.6, 1.40/36.4, and 1.31/27.4 ppm), and eight methine carbons ($\delta_H/\delta_C$ 6.46/145.0, 6.18/98.7, 5.45/88.4, 5.43/131.3, 5.33/132.3, 3.66/71.1, 2.62/33.0, and 2.18/43.7 ppm). ¹H–¹H correlations observed in COSY between H-20/H-16, H-19/H-15 and H-18/H-9 permitted to place the 3 methyl groups. The interpretation of the COSY spectrum led to build a polyketidic spin system with protons H-10 through to H-16 but also by ¹H–¹³C correlation observed in HMBC between H-19/C-16 and C-14 and H-18/ C10 and C8 (Fig. 4 and Fig. S12–23). ¹H–¹³C correlations until H-17 and C-8 and C-5 permitted also to place the hydroxymethyl group. Finally, the HMBC correlations between methyl at $\delta_H$ 3.80 ppm and methine at $\delta_H$ 6.18 and $\delta_H$ 5.45 ppm with carbon at $\delta_C$ 171.5 ppm but also proton at $\delta_H$ 6.18 ppm with carbons at $\delta_C$ 88.4, 128.9, 159.6 ppm allowed to determine a methoxypyranone ring. HMBC correlation of H-17 with C-7 allowed for the placement of the hydroxylated methylene group. This structure was confirmed by the chemical shift of doublets C-4 and C-2 ($\delta_C$ 98.7 and 88.4 ppm) and their low coupling constant ($J=2$ Hz) consistent with a *meta* coupling of two olefinic protons in an aromatic ring with heteroatoms. These results were corroborated by the NMR data of solanapyrones (Schmidt et al., 2007) which possess a similar methoxypyranone moiety. We observed characteristic chemical shifts for carbons C-1 through C-5 with the exception of carbon C-2 which is bonded to an aldehyde in solanapyrones. Correlation between the H-8 proton and the H-17 protons were not observed on NOESY NMR analyses, suggesting an possible *E* configuration of the double bond. This previously undescribed compound **1** was named prochordapyrone (Fig. 2).

TABLE 1

Analysis of ¹H and ¹³C NMR spectra of compounds **2–4** (Table 1) showed that they possess the same characteristic chemical shift of the methoxypyranone moiety as molecule **1**: $\delta_C$ around 172 ppm for C-3, 165 ppm for C-1, 100 ppm for C-4 and 88 ppm for C-5 as well as the isolated methyl group in C-6 with a chemical shift $\delta_C$ of 56 ppm and $\delta_H$ of 3.80 ppm. For compound **2**, molecular formula was determined to be C₂₀H₂₈O₅ by HRESIMS (peak at *m/z* 331.1912, [M – H₂O + H]⁺, calcd for C₂₀H₂₆O₄H⁺, 331.1904, err. 2.5 ppm) suggesting 7 degrees of unsaturation. The ¹H NMR spectrum revealed six doublets ($\delta_H$ 6.21, 5.41, 4.08, 3.49, 2.36, and 0.95 ppm), one broad triplet ($\delta_H$ 2.55 ppm), three singlets ($\delta_H$ 3.78, 1.62, and 1.16 ppm), seven multiplets ($\delta_H$ 1.82, 1.79, 1.71, 1.64, 1.59, 1.56, and 1.18 ppm), and three olefinic protons: two doublets ($\delta_H$ 6.21 and 5.41 ppm) and one broad singlet ($\delta_H$ 5.08 ppm). From the ¹³C NMR spectrum, one olefinic quaternary carbons ($\delta_C$ 134.1 ppm), one carbonyl carbon ($\delta_C$
135 172.1 ppm), and two non-olefinic quaternary carbons (δC 50.0 and 74.0 ppm) were observed.
136 The chemical shift at δC 74.0 ppm suggests a hydroxylated carbon. HSQC spectrum (Fig. S27
137 and S28) confirmed the presence of one methoxymethyl (δH/δC 3.78/55.7) and three methyls
138 (δH/δC 0.95/18.1, 1.62/22.0 and 1.16/30.3 ppm), 4 methylene (δH/δC 2.36/45.0, 1.82 and
139 1.18/34.2, 1.71 and 1.64/22.1, 1.79 and 1.56/43.2 ppm) including a hydroxylated methylene
140 (δH/δC 4.08 and 3.49/63.4 ppm). We also detect five methine (δH/δC 5.41/87.5, 6.21/100.8,
141 5.08/124.9, 2.55/32.4, 1.59/47.5 ppm). With the methoxypyranone and the detected olefinic
142 carbons we only account for 5 degrees of unsaturations which suggest the presence of two
143 secondary rings. Furthermore, HMBC correlations (Fig. 3) from H-18 to C-17, C-16 and C-5
144 suggests the methoxypyranone moiety and the hydroxylated methylene C-18 are both linked
145 to quaternary carbon C-17 and HMBC correlation from H-12 and C-10 suggests both are linked
146 to quaternary carbon C-17 as well. From then on, as we observe ¹H-¹H COSY correlation (Fig.
147 3) between protons H-14 through to H-10 and an HMBC correlation from H-8 to C-9 we were
148 able to build a two-ring system. The methyl C-20 was determined to be carried by olefinic
149 quaternary carbon C-15 by its HMBC correlation to the second olefinic carbon C-8 and methyl
150 C-19 was linked to hydroxylated quaternary carbon C-19 through its HMBC correlation to C-10.
151 The NOESY spectrum (Fig. 3) showed correlation between protons H-17/H-19 and H-20,
152 therefore all of these seem to be in the same plan. A crystal of compound **2** was obtained
153 under slow evaporative condition which confirmed what was determined by NOESY and
154 showed the absolute configuration to be C-7*R*, C-8*R*, C-9*R*, C-13*S*, C-16*R* (Fig. 4). This
155 compound was named chordapyrone A.

156 FIGURE 3

157 For compound **3**, molecular formula was determined to be C₂₀H₂₈O₄ by HRESIMS (peak at *m/z*
158 333.2068, [M + H]⁺, calcd for C₂₀H₂₈O₄H⁺, 333.2060, err. 2.3 ppm) suggesting again 7 degrees
159 of unsaturation. In comparison to compound **2** ¹H NMR only shows the supplementary
160 presence of a methine group (δH 1.68/δC 22.4 ppm) (Table 1, Fig. S35–S46) and the absence
161 of a quaternary carbon in ¹³C NMR with a shift up-field of C-9 (δC 38.3 ppm) (Table 1) which
162 suggests it is no longer linked to a hydroxyl group. The NOESY spectra was similar to that of
163 compound **2** (Fig. 3) with an added correlation between H-13 and H-18, this suggests similar
164 relative configuration to compound **2**. A crystal of compound **3** was also obtained and
165 confirmed the absolute configuration to be the same as to compounds **2**: C-7*S*, C-8*R*, C-9*S*,
166 C-13*S*, C-16*R* (Fig. 4). This molecule was named chordapyrone B.

167 For compound **4**, molecular formula was determined to be C₂₀H₂₆O₄ by HRESIMS (peak at *m/z*
168 331.1913 [M + H]⁺, calcd for C₂₀H₂₇O₄⁺, 331.1904, err. 2.5 ppm). By comparison to compound
169 **3** the only visible difference in the ¹H and ¹³C NMR spectra is a signal consistent with the
170 presence of an aldehyde (δH 9.92/δC 200.3 ppm) (Table 1). This signal was detected instead
171 of the doublet of doublet usually corresponding to the hydroxylated methylene in position 18
for compounds **1–3**. The NOESY spectrum of compound **4** (Fig. 3) shows differences to compound **3** as H-18 does not correlate with proton H-9 but instead with protons H-17, which was determined the be on the opposite plan to H-13 in compound **2** and **3**. Therefore, here we expect H-18 to be on the same plan with protons H-8, H-20 and H-19. We were not able to obtain a crystal of compound **4** therefore circular dichroism (Fig. S55) was performed on compounds **1–4** to compare with crystallographic data (Fig. 4). Overall, compounds **2–4** shared similar CD spectra with a positive cotton effect at $\lambda_{max}$ 207 nm and a negative cotton effect at $\lambda_{max}$ 280 nm suggests a similar relative configuration to compounds **2** and **3**. It is also important to note that compounds **2** and **3** also share a similar CD spectrum to that of Solanapyrone A (Kasahara et al., 2010) despite having different absolute configurations, therefore we consider this circular dichroism assay to be inconclusive for compound **4**. We assume that the absolute configuration of the molecule named chordapyrone C, is identical to compounds **2** and **3** and be C-7R, C-8R, C-9S C-13S, C-16R (Fig. 2).

FIGURE 4

Finally, for compound **5**, molecular formula was determined to be C₃₀H₄₈O₅ by HRESIMS (peak at *m/z* 489.3579, [M + H]⁺, calcd for C₃₀H₄₉O₅⁺, 489.3575, err. 0.9 ppm). ¹H NMR and ¹³C NMR spectra of compound **5** showed no similarities with those of compounds **1–4** (Table 2). Analysis of the ¹H NMR data showed the presence of two doublets of doublets (δH 4.20 and 3.86 ppm), two broad doublets (δH 4.53 and 4.40 ppm), one broad triplet (δH 2.18 ppm), five singlets (δH 1.65, 1.54, 1.47, 1.43, and 1.40 ppm), eleven multiplets (δH 2.95, 2.51, 2.50, 2.35, 2.13, 2.12, 2.11, 2.09, 2.07, 1.93, and 1.83 ppm), and four olefinic protons, including one triplet (δH 6.05 ppm) and three multiplets (δH 5.33, 5.29, and 5.25 ppm) (Table 2). From the ¹³C NMR spectrum, one carbonyl carbon (δC 166.2 ppm), four olefinic quaternary carbons (δC 166.2, 142.4, 135.1, and 126.4 ppm), and two non-olefinic quaternary carbons (δC 79.5 and 70.9 ppm) were observed. The latter chemical shifts suggest a direct linkage to an oxygen atom. The HSQC spectrum confirmed the presence of six methyl carbons (δH/δC 1.65/16.5, 1.65/16.4, 1.54/28.1, 1.47/20.4, 1.43/25.9, and 1.40/26.9 ppm) and eleven methylene carbons (δH/δC 2.95/28.8, 2.51/29.5, 2.50 and 2.35/32.3, 2.13 and 1.93/35.4, 2.18/39.6, 2.09/40.1, 2.12/28.9, 2.11/28.9, 2.07/26.2, 1.83/24.1, and 4.53 and 4.40/63.9 ppm). We also detect six methine (δH/δC 5.33/125.6, 5.29/125.3, 6.05/146.6, 5.25/123.3, 4.20/86.7 and 3.86/77.4 ppm) (Table 2). For this compound, we first observed signal consistent with the presence of an ester or carboxylic acid (C-2, δC 166.5 ppm), then with HMBC correlation from C-1'' to C-1 and to C-3 and from C-3 to C-2 and COSY correlation between C-3, C-4 and C-5 we were able to build a pyranone ring. This ring is substituted in position 6' with a propane moiety as we observe HMBC correlations from H-5 to C-7 and from H₃-3''' to C-6, C-1''' and C-5. Chemical shift of C-2''' (δC 70.9) suggest this propane moiety is hydroxylated. COSY correlation between protons H-1'', H-2'' and H-3'' and HMBC correlation between H₂-3'', H-5'' and C-13'' led us to
identify an isoprenic unit linked to the pyranone through olefinic carbon C-1'' (Fig. 5). Through COSY correlation of H-5'' through to H-8'' we deduced that the isoprenic moiety is linked to another isoprenic moiety in a head-to-head fashion through carbons C-6'' and C-7''. We also identified the presence of an oxepan ring, as HSQC spectra showed that carbons H-5'a, H-5'b are carried by a single methene carbon (C-4'), similarly for H-3'a and H-3'b (C-3') and H-2''a and H-6'b (C-6'), which therefore suggest they are cyclic carbons. The broad doublet of protons H-6' (Fig. S66) suggested they are linked to the oxygen atom as does the δ ppm (79.5) of C-1'. COSY correlation helped us construct the ring as we observed COSY correlation between protons H-2', H-3' and H-4'. Finally, HMBC correlations between H-2' and C-1' as well as between protons H-6'/H-4' and C-5' confirmed the presence of both these quaternary carbons within the oxepan ring. We were also able to place two methyl (C-8' and C-7') as substituents in position 1' due to their HMBC correlation with C-1' while the δ ppm (77.4) of C-2' suggests it is substituted with a hydroxyl group. HMBC correlation showed that the oxepan ring is linked to the isoprenic chain through olefinic carbon C-12'' which is connected to C-11'' as we see through the COSY spectrum. Relative configuration of the double bonds between C-5' and C-12'' and C2 and C-1'' were determined from the NOESY correlation observed between H-5' and C-12'' and H-3 and H-1''. For the other alkenes, relative configuration could not be determined. They were represented as they are in Figure 4 as the head-to-head isoprenic unit linkage seems to suggest biosynthetic origins linked to squalene, therefore we represented the relative configuration of squalene. Relative configuration of carbons C-1' and C-2' were determined due to NOESY correlation between protons H-8' and H-3'. The absolute configuration for C-2' could have been determined through a Mosher ester analysis (Hoye et al., 2007), however, the isolated quantity of compound **5** was too small to attempt it. This compound was name Chordapyranone.

TABLE 2

FIGURE 5

## 2.4. Biosynthetic origin of chordapyrones

Compounds **1–4** exhibit significant structural resemblance to solanapyrones, a class of metabolites previously isolated from phytopathogenic fungi such as *Alternaria solani*, *Ascochyta rabiei*, and *Mycosphaerella rabiei* (Alam et al., 1989; Oikawa et al., 1998; Schmidt et al., 2007). Prior investigations into solanapyrone biosynthesis have proposed the involvement of a Diels–Alderase-catalyzed [4+2] cycloaddition step, which prompted us to consider the linear structure of compound **1** as a plausible biosynthetic precursor to the three cyclic derivatives. In this context, chlordapyrone B (**3**) could originate from compound **1** (Fig. 6) via the sequential enzymatic action of a dehydroxylase followed by a Diels–Alderase, a mechanism analogous to that described for solanapyrone biosynthesis, where polyketide
intermediates undergo intramolecular cycloaddition (Kasahara et al., 2010; Berlink & Skellam, 2024; Oikawa, 2010). Compounds **2** and **4** are postulated to result from downstream enzymatic modifications of compound **3**, specifically through hydroxylation and dehydrogenation, mediated by hydroxylases and dehydrogenases, respectively. Drawing parallels with solanapyrones, and based on the observed stereochemical configurations of compounds **2** and **3**, an *endo*-selective cycloaddition appears to be favored. This is further supported by the exclusive isolation of these specific isomers, suggesting their dominance in the reaction equilibrium and sufficient abundance for chromatographic purification. Moreover, the proposed biosynthetic route reinforces the assigned configuration of compound **4**. These findings suggest that the biosynthetic gene cluster present in *T. chordalis* is distinct from those previously characterized for solanapyrone biosynthesis (Berlink and Skellam, 2024).

FIGURE 6

It is noteworthy that although compounds **1–4** are biosynthesized via a polyketide pathway (Kasahara et al., 2010; Berlink and Skellam, 2024), and compound **5** is of terpenoid origin, all five share a common pyran-2-one core. In addition to compounds **1–4**, further chemotaxonomic markers were identified. Specifically, two major groups of biosynthetic clusters were detected: clusters 23, 73, 103, 110, 128, 184, and 273, all associated with chordapyrones and present in both *Thamnomyces chordalis* and *Phylacia* sp. Notably, among the isolated compounds, only chordapyrones A and C were observed in both genera. In contrast, clusters 26 and 34 correspond to a distinct molecular family, apparently unique to *T. chordalis* (Fig. S5). From this latter group, compound **5** was isolated, characterized by the presence of a pyranone moiety and a structurally related isoprenoid side chain.

Previous studies on the *Thamnomyces* genus, including investigations of stromata from *T. chordalis*, did not report these specific metabolites. However, compounds belonging to the same chemical family were tentatively detected based on similarities in UV spectra, suggesting the presence of non-isolated analogues of chordapyrones (Stadler et al., 2010). Interestingly, Stadler et al. (2011) reported only one compound in common between *Phylacia poculiformis* and *T. chordalis*, although they identified several non-isolated yet shared compounds between *P. poculiformis* and *T. dendroidea*. Comprehensive comparative studies involving multiple species from both *Thamnomyces* and *Phylacia* would be required to uncover additional shared metabolites and further assess the potential of chordapyrones and chordapyranone derivatives as chemotaxonomic markers for these taxa.

3. Conclusion

In this study, the analysis of 160 macroscopic fungal specimens through molecular network-based metabolomics enabled the classification of taxa according to their production of
280 specialized metabolite families. This approach successfully distinguished members of the
281 Xylariales from those of other fungal orders based on their unique chemical signatures.
282 Focusing on the Xylariales, we identified novel compounds specific to the genera
283 *Thamnomyces* and *Phylacia*, including several representatives of the chordapyrone series and
284 a structurally distinct metabolite featuring a chordapyranone scaffold. These compounds show
285 strong potential as diagnostic chemomarkers for the taxonomic classification of fungal
286 carpophores.

287 From a biosynthetic perspective, chordapyrones (1–4) display notable structural and
288 mechanistic similarities to solanapyrones, a well-characterized class of polyketides produced
289 by phytopathogenic fungi. The proposed involvement of a Diels–Alderase in the formation of
290 their polycyclic cores supports the hypothesis that the linear precursor 1 may act as a key
291 biosynthetic intermediate. Additionally, the biosynthetic gene cluster associated with these
292 metabolites in *T. chordalis* exhibits significant divergence from canonical solanapyrone
293 pathways, underscoring the evolutionary uniqueness of this biosynthetic route.

294 Collectively, these findings contribute new insights into fungal polyketide biosynthesis and
295 highlight the value of specialized metabolites in resolving taxonomic relationships. This work
296 further exemplifies the integrative potential of metabolomics to bridge natural product
297 chemistry and fungal systematics.

## 4. Material and method

### 4.1. Fungal samples collection and identification.

300 All fungal samples were collected in French Guiana near Sinnamary, Paracou, Remire-
301 Montjoly, Cacao and Tonnegrande between 2019 and 2022 (ABSCH-IRCC-FR-2348782-1).

302 Sample identification was first performed on phenotypic characteristics of macromycetes by
303 comparison with existing databases (GBIF, EOL) and literature. Further identification was
304 conducted externally by Microsynth AG (Balgach, Switzerland) through the amplification of the
305 ITS1 and ITS4 regions. The sequences were aligned with DNA sequence from GenBank
306 (NCBI) using BLAST+ 2.15.0 and the sequences were deposited on Genbank for accession
307 numbers (Table S2).

### 4.2. General experimental procedures

308 Optical rotations were measured at 20°C in methanol using an Anton Paar MCP300
309 polarimeter in a 100-mm-long 350 µL cell. UV spectra were recorded at 20°C in methanol using
310 a PerkinElmer Lambda 5 spectrophotometer. Electronic circular dichroism spectra were
311 acquired at 20°C in methanol on a JASCO J-810 spectropolarimeter. NMR spectra were
312 recorded on Bruker 500 and 700 MHz spectrometers (Bruker, Rheinstetten, Germany). The
313 chemical shifts (δ) are reported as ppm based on the solvent signal, and coupling constants
314 (J) are in hertz. Preparative HPLC was conducted with a Gilson system equipped with a 322
315
pumping device, a GX-271 fraction collector, a 171 diode array detector and a prep ELSII. For separation we used a Sunfire C18 column (10 x 150 mm, 5 µm, Waters, France). All solvents were HPLC grade, purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

## 4.3. LC-MS/MS data

### 4.3.1. Sample preparation

Macromycetes were stored at -80 °C after collection. The entire fruiting body was then crushed and macerated in ethyl acetate (EtOAc) on a rotary shaker at room temperature for 12 hours. Extracts were subsequently filtered and dried under reduced pressure.

### 4.3.2. Data analysis

Crude extracts were diluted to 1 mg.mL⁻¹ in methanol (MeOH) and filtered on a PTFE 0.45µm membrane before analysis. LC-MS/MS experiment were performed on a 1260 prime HPLC (Agilent Technologies, Waldbronn, Germany) coupled with an Agilent 6546 Q-ToF (Agilent Technologies, Waldbronn, Germany) tandem mass spectrometer. LC separation was achieved on an Poroshell RP-MS column (150 x 2.1mm, 2.7 µm, Agilent, Les Ulis, France). Mobile phase was comprised of H₂O with 0.1% of formic acid and acetonitrile (ACN) with a gradient from 5% ACN to 100% ACN in 20 minutes and the flow rate was fixed at 0.4 mL.min⁻¹. The column oven was set at 45°C and the injection volume for all analyses was 5 µL. Analysis was performed in positive mode, the electrospray source parameters were set as follows: drying gas flow rate 10 L.min⁻¹, gas temperature 325°C, nebulizer pressure 30 psig, sheath gas temperature 350°C, sheath gas flow 10 L.min⁻¹, capillary voltage 3500 V, nozzle voltage 500 V, fragmentor voltage 110 V, skimmer voltage 45 V and the octopole 1 RF voltage 750 V. Internal calibration was achieved with Agilent API-TOF Reference Mass Solution (purine, C₅H₄N₄, *m/z* 121.0509, and HP-921 (hexakis-(1H,1H,3H-tetrafluoropentoxy)phosphazene), C₁₈H₁₈O₆N₃P₃F₂₄, *m/z* 922.0098) and external calibration was achieved with the Agilent (Agilent, Les Ulis, France) LC/MS tuning mix for ESI. The data-dependent MS/MS events were acquired for the five most intense ions detected by full-scan MS above an absolute threshold of 1000 counts. Selected precursor ions were fragmented at a collision energy of 20 eV.

### 4.3.3. Data processing

Data were converted from .d to .mzML using MSConvert part of the ProteoWizard 3.0 package. All data was processed using MZmine v3.2.8. Mass detection was performed with MS1 noise level set at 10000 and MS2 noise level set at 100. The ADAP chromatogram builder was used with a minimum of 3 consecutive scans with a minimum intensity of 5000 and a minimum absolute height of 5000, the *m/z* tolerance was set at 0.008 or 20 ppm. Deconvolution was performed with the ADAP resolver with a signal to noise ratio of 10, a minimum feature height of 2000, a coefficient/area threshold set at 10, a peak duration range of 0 to 1 min and the *t*R wavelet range set between 0.00 and 0.05. The isotope peak grouper was used with the *m/z*
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tolerance set at 0.005 or 10 ppm, a $t_R$ tolerance of 0.1 min and the maximum number of charges set at 2. Peaks were filtered using the Feature list row filter in order to only select features with MS2 scans. The join aligner was used to align peak lists, the m/z tolerance was set at 0.008 or 20 ppm, weight for m/z as well as for $t_R$ was set at 50 and $t_R$ tolerance was set at 0.1 minute. The aligned feature list was exported as an .mgf file and metadata were generated using the molecular networking file option.

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Metgem (1.3.6) was used to calculate cosine scores and to generate molecular networks. Parameters were set as follows: m/z tolerance 0.04, minimum matched peaks 3. Molecular networks were generated with a maximum number of neighbors for a node set at 20, minimal cosine score to create an edge set at 0.7 and a maximum number of connected elements set at 1000. A cluster column was added using the "numberize cluster" function.

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Cluster grouping and all Figures were generated using R v4.3.2, Rstudio v12.0 with the packages ape (Paradis and Schliep, 2019), factoextra and RandomForest (Breiman et al., 2002). Secondary metabolite profiles of fungal samples were compared based on the presence or absence of metabolites in all registered clusters of the molecular network with a binary matrix, comparison was based on Euclidean distances and clustering was performed using Ward's agglomerative criterion. Dendrogram representation was visualized and adapted using Figtree v1.4.4.

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## 4.4. Isolation and characterization of compounds.

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Whole crude extract (1.2 g) of the fruiting body of *Thamnomyces chordalis* was fractionated by reverse-phase flash chromatography (BUCHI Pure, BUCHI SARL, Villebon-sur-Yvette, France) using a 120 g, C18 column, at a flow rate of 80 mL.min⁻¹. A gradient of (A) H₂O/formic acid (99.9/0.1) and (B) acetonitrile (ACN) was used, we gradually increased (B) percentage from 5% to 30% in 3 column volumes (CV), then 30% (B) for 3 CV followed by another gradual increase to 50% (B) in 1.7 CV, and another plateau at 50% (B) for 5.5 CV, again, we gradually increased (B) percentage to 65% in 1 CV and maintained this percentage for 3 more CV, before reaching 100% (B) in 3 CV and maintained this for 3 more CV. We generated 13 fractions (F1-F13), three (F6, F9 and F10) of them were submitted to reverse-phase preparative chromatography (Gilson, Villiers-le-bel, France). Fractionation of F6 on a (A) H₂O/formic acid (99.9/0.1) and (B) acetonitrile (ACN) gradient from 20% (B) to 100% (B) in 18 minutes with a 4.6 minutes 50% (B) plateau led to the isolation of compounds **1** (0.5 mg, $t_R$ = 7.27 min) and **2** (0.8 mg, $t_R$ = 7.62 min). Fractionation of F9 on a (A) H₂O/formic acid (99.9/0.1) and (B) acetonitrile (ACN) gradient from 30% (B) to 100% (B) in 30 minutes with a 6 minutes 50% (B) plateau led to the isolation of compound **3** (1.7 mg, $t_R$ = 15.15 min) and Fractionation of F10 on a (A) H₂O/formic acid (99.9/0.1) and (B) acetonitrile (ACN) gradient from 20% (B) to 100% (B) in 19 minutes with a 4.5 minutes 70% (B) plateau led to the isolation of previously undescribed compounds **4** (0.3 mg, $t_R$ = 9.51 min) and **5** (0.5 mg, $t_R$ = 10.88 min).

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**4.4.1. Prochordapyrone (1):** Yellow oil; [α]<sup>20</sup><sub>D</sub> -375° (c 0.8 g.L<sup>-1</sup>, ACN), UV (MeOH) λmax (ε), 224 nm (62 Lmol<sup>-1</sup>.cm<sup>-1</sup>), UV (MeOH) λmax (ε), 308 nm (21.5 Lmol<sup>-1</sup>.cm<sup>-1</sup>), CD (MeOH) λmax (Δε) 225 (-10.5), 200 (+4.9), 311 (+4.8) <sup>1</sup>H NMR (700 MHz, CDCl<sub>3</sub>), <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) (Table 1, Fig. S12–S21), ESI–HRMS *m/z* [M + H]<sup>+</sup> 351.2167 (calcd for C<sub>20</sub>H<sub>30</sub>O<sub>5</sub>H<sup>+</sup>, 351.2166, err. 0.3 ppm).

**4.4.2. Chordapyrone A (2):** Yellow oil; [α]<sup>20</sup><sub>D</sub> -250° (c 1.2 g.L<sup>-1</sup>, ACN), UV (MeOH) λmax (ε), 208 nm (26.7 Lmol<sup>-1</sup>.cm<sup>-1</sup>), UV (MeOH) λmax (ε), 285 nm (8.87 Lmol<sup>-1</sup>.cm<sup>-1</sup>), CD (MeOH) λmax (Δε) 205 (+27.1), 278 (-2.9), <sup>1</sup>H NMR (700 MHz, CDCl<sub>3</sub>), <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) (Table 1, Fig. S22–S32, S59, ESI–HRMS *m/z* [M – H<sub>2</sub>O + H]<sup>+</sup> 331.1912 (calcd for C<sub>20</sub>H<sub>26</sub>O<sub>4</sub>H<sup>+</sup>, 331.1904, err. 2.5 ppm).

**4.4.3. Chordapyrone B (3):** Yellow oil; [α]<sup>20</sup><sub>D</sub> -333° (c 1.8 g.L<sup>-1</sup>, ACN), UV (MeOH) λmax (ε), 208 nm (274.4 Lmol<sup>-1</sup>.cm<sup>-1</sup>), UV (MeOH) λmax (ε), 285 nm (92.9 Lmol<sup>-1</sup>.cm<sup>-1</sup>), CD (MeOH) λmax (Δε) 207 (+8.9), 280 (-1.5), <sup>1</sup>H NMR (500 MHz, MeOD), <sup>13</sup>C NMR (100 MHz, MeOD), (Table 1, Fig. S33–S43), ESI–HRMS *m/z* [M + H]<sup>+</sup> 333.2068 (calcd for C<sub>20</sub>H<sub>28</sub>O<sub>4</sub>H<sup>+</sup>, 333.2060, err. 2.3 ppm).

**4.4.4. Chordapyrone C (4):** Yellow powder; [α]<sup>20</sup><sub>D</sub> -100° (c 1.0 g.L<sup>-1</sup>, ACN), UV (MeOH) λmax (ε), 208 nm (90.9 Lmol<sup>-1</sup>.cm<sup>-1</sup>), UV (MeOH) λmax (ε), 203 nm (2.39 Lmol<sup>-1</sup>.cm<sup>-1</sup>), CD (MeOH) λmax (Δε) 207 (+8.9), 272 (-0.2), <sup>1</sup>H NMR (700 MHz, Pyr), <sup>13</sup>C NMR (125 MHz, Pyr) (Table 1, Fig. S44–S54), ESI–HRMS *m/z* [M + H]<sup>+</sup> 331.1913 (calcd for C<sub>20</sub>H<sub>26</sub>O<sub>4</sub>H<sup>+</sup>, 331.1904, err. 2.4 ppm).

**4.4.5. Chordapyranone (5):** Brown oil; [α]<sup>20</sup><sub>D</sub> 56° (c 1.8 g.L<sup>-1</sup>, CHCl<sub>3</sub>), UV (MeOH) λmax (ε), 211 nm (1206 Lmol<sup>-1</sup>.cm<sup>-1</sup>), CD (MeOH) λmax (Δε) 204 (+0.1), 305 (+0.7), 231 (-0.9), <sup>1</sup>H NMR (700 MHz, Pyr), <sup>13</sup>C NMR (125 MHz, Pyridine-d<sub>5</sub>) (Table 2, Fig. S56–S66), ESI–HRMS *m/z* [M + H]<sup>+</sup> 489.3579 (calcd for C<sub>30</sub>H<sub>48</sub>O<sub>5</sub>H<sup>+</sup>, 489.3575, err. 0.9 ppm).

**4.5. X-ray structure determination of compound 2 and 3.**

A crystal of compound **2** was obtained under slow evaporative condition in a 50:50 mix of ACN and chloroform at room temperature and the crystal of compound **3** was also obtained under slow evaporative condition in MeOH. For the re-crystallized sample of both compound **2** and **3** a suitable crystal noted X1 was selected and placed in a little Paratone® oil, to be set and mounted on a nylon loop before being positioned on the goniometer of the apparatus. X-ray Diffraction (XRD) analysis and associated crystallographic data were collected at room temperature (292K) using ω redundant scans on RIGAKU's "XtaLabPro" diffractometer, dedicated to the analysis of micro-crystals of small organic molecules. This diffractometer is equipped with a molybdenum microsource (Mo Kα) and an HPAD PILATUS3 R 200K detector. CrysAlisPro software (Rigaku OD, 2015) was used for data recording and processing, with empirical absorption correction using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. Using Olex2 software (Dolomanov et al., 2009), the so-called
intrinsic phasing method (SHELXT (Sheldrick, 2015a)) and least-squares refinement using SHELX (Sheldrick, 2015b). Non-hydrogen atoms have been anisotropically refined. Hydrogen atoms, where possible, were identified and positioned by analyzing electron density maps while fixing their distance and Uiso=x Umean(C,O) with x = 1.2 or 1.5. To determine the absolute configuration of the structure, we referred to the Flack parameter, calculated by the quotient method [(I+)-(I-)]/[(I+)+(I-)] (Flack, 1983). The value obtained is close to zero, and the probabilities P2 and P3 derived from Bijvoet's a-posteririo pair analysis (Parsons et al., 2013) are also favorable (equal to 1), but both compounds are composed mainly of light elements, we preferred to confirm these values with a second analysis of the same crystals at room temperature (293K) on Rigaku's "MM007" diffractometer, fitted with a rotating copper anode (Cu Kα) and equipped with a RAPID 2 curved IP detector. These data were integrated and reduced using the Fs_process software implemented in CrystalClear 2.0 (Rigaku, 2014) and corrected for radiation absorption using the Abscor multi-scan option. Data solution and refinement were carried out in the same way as before, and the Flack parameter was unambiguously confirmed (Table S3 and S4).

Finally, all molecular structure representations were obtained using Mercury 2023.2.0 software (Macrae et al., 2006).

444

## Declaration of competing interest

The authors declare no conflict of interest.

447

## Appendix A. Supplementary data

Supplementary data to this article can be found online

Crystallographic data of the 4 structures have been deposited in the Cambridge

Crystallographic Data Centre database (deposition numbers 2441159-2441162). Copies of the data can be obtained free of charge from the CCDC at www.ccdc.cam.ac.uk.

453

## Funding

This work was supported by an "Investissement d'Avenir" grant (CEBA, ref ANR-10-LABX-0025) managed by the French National Research Agency (ANR). Juliette Segret thanks the ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation (MESRI) for her PhD fellowship.

459

## Acknowledgements

We would like to thank Céline Moriou-Chesnet (ICSN-CNRS) for her help to register CD spectra.

463
464 Author contributions

465 Conceptualization: V. Eparvier, D. Touboul; Data Curation: J. Segret, E. Van Elsande, J.

466 Sorres, V. Eparvier; Formal analysis: E. Van Elsande, J. Segret, J. Sorres; Funding acquisition:

467 V. Eparvier, D. Touboul; Investigation: J. Segret, J. Sorres; Methodology: D. Touboul, V.

468 Eparvier; Project administration: V. Eparvier, D. Touboul; Resources: V. Eparvier, D. Touboul;

469 Supervision: V. Eparvier, D. Touboul; Visualization: J. Segret, J. Sorres; Writing - original draft:

470 V. Eparvier; and Writing - review & editing: V. Eparvier.

471

472 References

473 Alam, S.S., Bilton, J.N., Slawin, A.M.Z., Williams, D.J., Sheppard, R.N., Strange, R.N., 1989.

474 Chickpea blight: Production of the phytotoxins solanapyrones A and C by Ascochyta

475 rabiei. Phytochem. 28, 2627-2630. https://doi.org/10.1016/S0031-9422(00)98054-3.

476 Barthélemy, M., Guérineau, V., Genta-Jouve, G., Roy, M., Chave, J., Guillot, R., Pelissier, L.,

477 Wolfender J.-L., Stien, D., Eparvier, V. 2020. Identification and dereplication of

478 endophytic *Colletotrichum* strains by MALDI TOF mass spectrometry and molecular

479 networking. Sci. Rep. 10, 19788. https://doi.org/10.1038/s41598-020-74852-w.

480 Bérdy, J., 2005. Bioactive microbial metabolites. J. Antibiot. 58, 1-26.

481 https://doi.org/10.1038/ja.2005.1.

482 Berlinck, R.G.S., Skellam, E., 2024. Discovery, biosynthesis, and biological activities of

483 solanapyrones: [4 + 2] cycloaddition-derivatated polyketides of fungal origin. J. Nat. Prod.

484 87, 2892-2906. https://doi.org/10.1021/acs.jnatprod.4c00818

485 Breiman, L., Cutler, A., Liam, A., Wiener, M., 2002. RandomForest: Breiman and Cutler's

486 Random Forests for classification and regression 4.7-1.1.

487 https://doi.org/10.32614/CRAN/package.randomForest.

488 Dolomanov, O.V., Bourhis, L.J., Gildea, R.J., Howard, J.A.K., Puschmann, H., 2009. OLEX2:

489 a complete structure solution, refinement and analysis program. J. Appl. Crystallogr.

490 42, 339-341. https://doi.org/10.1107/S0021889808042726.

491 Flack, H.D., 1983. On enantiomorph-polarity estimation. Acta Crystallogr. A 39, 876-881.

492 https://doi.org/10.1107/S0108767383001762.

493 Fournier, J., Hsieh, H.-M., Lechat, C., Ju, Y.-M., Chaduli, D., Favel, A., 2023. Five new

494 *Camillea* (Xylariales) species described from French Guiana. Bot. Stud. 64, 31.

495 https://doi.org/10.1186/s40529-023-00397-6.

496 Fournier, J., Lechat, C., 2015. *Phylacia korfii* sp. nov., a new species of *Phylacia* (Xylariaceae)

497 from French Guiana, with notes on three other *Phylacia* spp. Persoonia - Mol.

498 Phylogeny Evol. Fungi 25, 11-21. https://doi.org/10.3767/003158510X524231.
499 Helaly, S.E., Thongbai, B., Stadler, M., 2018. Diversity of biologically active secondary
500 metabolites from endophytic and saprotrophic fungi of the ascomycete order Xylariales.
501 Nat. Prod. Rep. 35, 992–1014. https://doi.org/10.1039/C8NP00010G.

502 Henkel, T.W., Aime, M.C., Chin, M., Andrew, C., 2004. Edible mushrooms from Guyana. 503 Mycologist 18, 104–111. https://doi.org/10.1017/S0269915X04003027.

504 Henkel, T.W., Aime, M.C., Largent, D.L., Baroni, T.J., 2011. The Entolomataceae of the 505 Pakaraima Mountains of Guyana 5: new species of *Alboleptonia*. Mycotaxon 114, 115– 506 126. https://doi.org/10.5248/114.115.

507 Hoye, T.R., Jeffrey, C.S., Shao, F., 2007. Mosher ester analysis for the determination of 508 absolute configuration of stereogenic (chiral) carbinol carbons. Nat. Protoc. 2, 2451– 509 2458. https://doi.org/10.1038/nprot.2007.354.

510 Kasahara, K., Miyamoto, T., Fujimoto, T., Oguri, H., Tokiwano, T., Oikawa, H., Ebizuka, Y., 511 Fujii, I., 2010. Solanapyrone synthase, a possible Diels-Alderase and iterative type I 512 Polyketide Synthase encoded in a biosynthetic gene cluster from *Alternaria solani*. 513 ChemBioChem 11, 1245–1252. https://doi.org/10.1002/cbic.201000173.

514 Katajamaa, M., Miettinen, J., Orešič, M., 2006. MZmine: toolbox for processing and 515 visualization of mass spectrometry based molecular profile data. Bioinformatics 22, 516 634–636. https://doi.org/10.1093/bioinformatics/btk039.

517 Kluger, B., Lehner, S., Schuhmacher, R., 2015. Metabolomics and secondary metabolite 518 profiling of filamentous Fungi, in: Zeilinger, S., Martín, J.-F., García-Estrada, C. (Eds.), 519 Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites, Volume 2, 520 Fungal Biology. Springer New York, New York, NY, pp. 81–101. 521 https://doi.org/10.1007/978-1-4939-2531-5_6.

522 Lind, A.L., Wisecaver, J.H., Lameiras, C., Wiemann, P., Palmer, J.M., Keller, N.P., Rodrigues, 523 F., Goldman, G.H., Rokas, A., 2017. Drivers of genetic diversity in secondary metabolic 524 gene clusters within a fungal species. PLOS Biol. 15, e2003583. 525 https://doi.org/10.1371/journal.pbio.2003583.

526 Macrae, C.F., Edgington, P.R., McCabe, P., Pidcock, E., Shields, G.P., Taylor, R., Towler, M., 527 Van De Streek, J., 2006. *Mercury*: visualization and analysis of crystal structures. J. 528 Appl. Crystallogr. 39, 453–457. https://doi.org/10.1107/S002188980600731X.

529 Matheny, P.B., Aime, M.C., Henkel, T.W., 2003. New species of *Inocybe* from *Dicymbe* forests 530 of Guyana. Mycol. Res. 107, 495–505. https://doi.org/10.1017/S0953756203007627.

531 Nothias, L.-F. et al., Petras, D., Schmid, R., Dührkop, K., Rainer, J., Sarvepalli, A., Protsyuk, 532 I., Ernst, M., Tsugawa, H., Fleischauer, M., Aicheler, F., Aksenov, A.A., Alka, O., Allard, 533 P.-M., Barsch, A., Cachet, X., Caraballo-Rodriguez, A.M., Da Silva, R.R., Dang, T., 534 Garg, N., Gauglitz, J.M., Gurevich, A., Isaac, G., Jarmusch, A.K., Kameník, Z., Kang, 535 K.B., Kessler, N., Koester, I., Korf, A., Le Gouellec, A., Ludwig, M., Martin H., C.,
536 McCall, L.-I., McSayles, J., Meyer, S.W., Mohimani, H., Morsy, M., Moyne, O.,
537 Neumann, S., Neuweger, H., Nguyen, N.H., Nothias-Esposito, M., Paolini, J., Phelan,
538 V.V., Pluskal, T., Quinn, R.A., Rogers, S., Shrestha, B., Tripathi, A., van der Hooft,
539 J.J.J., Vargas, F., Weldon, K.C., Witting, M., Yang, H., Zhang, Z., Zubeil, F.,
540 Kohlbacher, O., Böcker, S., Alexandrov, T., Bandeira, N., Wang, M., Dorrestein, P.C.,
541 2020. Feature-based molecular networking in the GNPS analysis environment. Nat.
542 Methods 17, 905-908. https://doi.org/10.1038/s41592-020-0933-6.

543 Oikawa, H., Yokota, T., Sakano, C., Suzuki, Y., Naya, A., Ichihara, A., 1998. Solanapyrones,
544 Phytotoxins produced by *Alternaria solani*: biosynthesis and isolation of minor
545 components. *Biosci. Biotechnol. Biochem.* **62**, 2016-2022.
546 https://doi.org/10.1271/bbb.62.2016.

547 Oikawa, H. 8.08 - Diels-Alderases, Ed. : Hung-Wen (Ben) Liu, Lew Mander, Comprehensive
548 Natural Products II, Elsevier, 2010, 277-314. https://doi.org/10.1016/B978-008045382-
549 8.00639-0.

550 Olivon, F., Elie, N., Grelier, G., Roussi, F., Litaudon, M., Touboul, D., 2018. MetGem Software
551 for the generation of molecular networks based on the t-SNE algorithm. *Anal. Chem.*
552 90, 13900-13908. https://doi.org/10.1021/acs.analchem.8b03099.

553 Paradis, E., Schliep, K., 2019. ape 5.0: an environment for modern phylogenetics and
554 evolutionary analyses in R. *Bioinformatics* **35**, 526-528.
555 https://doi.org/10.1093/bioinformatics/bty633.

556 Parsons, S., Flack, H.D., Wagner, T., 2013. Use of intensity quotients and differences in
557 absolute structure refinement. *Acta Crystallogr. Sect. B Struct. Sci. Cryst. Eng. Mater.*
558 69, 249-259. https://doi.org/10.1107/S2052519213010014.

559 Pluskal, T., Castillo, S., Villar-Briones, A., Orešič, M., 2010. MZmine 2: Modular framework for
560 processing, visualizing, and analyzing mass spectrometry-based molecular profile
561 data. *BMC Bioinformatics* 11, 395. https://doi.org/10.1186/1471-2105-11-395.

562 Raja, H.A., Miller, A.N., Pearce, C.J., Oberlies, N.H., 2017. Fungal identification using
563 molecular tools: a primer for the natural products research community. *J. Nat. Prod.*
564 80, 756-770. https://doi.org/10.1021/acs.jnatprod.6b01085.

565 Rigaku, 2014. *CrystalClear-SM Expert 2.0 r16* [software], Rigaku Corporation, Tokyo, Japan.

566 Rigaku OD, 2015. *CrysAlis PRO*. Rigaku Oxford Diffraction [software], Yarnton, Oxfordshire,
567 England.

568 Schmid, R., Heuckeroth, S., Korf, A., Smirnov, A., Myers, O., Dyrlund, T.S., Bushuiev, R.,
569 Murray, K.J., Hoffmann, N., Lu, M., Sarvepalli, A., Zhang, Z., Fleischauer, M., Dührkop,
570 K., Wesner, M., Hoogstra, S.J., Rudt, E., Mokshyna, O., Brungs, C., Ponomarov, K.,
571 Mutabdžija, L., Damiani, T., Pudney, C.J., Earll, M., Helmer, P.O., Fallon, T.R.,
572 Schulze, T., Rivas-Ubach, A., Bilbao, A., Richter, H., Nothias, L.-F., Wang, M., Orešič,
M., Weng, J.-K., Böcker, S., Jeibmann, A., Hayen, H., Karst, U., Dorrestein, P.C., Petras, D., Du, X., Pluskal, T., 2023. Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nat. Biotechnol. 41, 447-449.
https://doi.org/10.1038/s41587-023-01690-2.

Schmidt, L.E., Gloer, J.B., Wicklow, D.T., 2007. solanapyrone analogues from a hawaiian fungicolous fungus. J. Nat. Prod. 70, 1317-1320. https://doi.org/10.1021/np070251m

Sheldrick, G.M., 2015a. SHELXT – Integrated space-group and crystal-structure determination. Acta Crystallogr. Sect. Found. Adv. 71, 3-8.
https://doi.org/10.1107/S2053273314026370.

Sheldrick, G.M., 2015b. Crystal structure refinement with SHELXL. Acta Crystallogr. Sect. C Struct. Chem. 71, 3-8. https://doi.org/10.1107/S2053229614024218.

Stadler, M., 2011. Importance of secondary metabolites in the Xylariaceae as parameters for assessment of their taxonomy, phylogeny, and functional biodiversity. Curr. Res. Environ. Appl. Mycol. 1, 75-133. https://doi.org/10.5943/cream/1/2/1.

Stadler, M., Fournier, J., Læssøe, T., Chlebicki, C. Lechat, F. Flessa, Rambobold, G., Peršoh, D., 2010. Chemotaxonomic and phylogenetic studies of *Thamnomyces* (Xylariaceae). Mycoscience 51, 189-207. https://doi.org/10.1007/S10267-009-0028-9.

Stadler, M., Ju, Y.-M., Rogers, J.D., 2004. Chemotaxonomy of *Entonaema*, *Rhopalostroma* and other Xylariaceae. Mycol. Res. 108, 239-256.
https://doi.org/10.1017/S0953756204009347.

Vasco-Palacios, A.M., Franco-Molano, A.E., 2013. Diversity of Colombian macrofungi (Ascomycota – Basidiomycotas). Mycotaxon 121, 499.

Wang, M., Carver, J.J., Phelan, V.V., Sanchez, L.M., Garg, N., Peng, Y., Nguyen, D.D., Watrous, J., Kapono, C.A., Luzzatto-Knaan, T., Porto, C., Bouslimani, A., Melnik, A.V., Meehan, M.J., Liu, W.-T., Crüsemann, M., Boudreau, P.D., Esquenazi, E., Sandoval-Calderón, M., Kersten, R.D., Pace, L.A., Quinn, R.A., Duncan, K.R., Hsu, C.-C., Floros, D.J., Gavilan, R.G., Kleigrewe, K., Northen, T., Dutton, R.J., Parrot, D., Carlson, E.E., Aigle, B., Michelsen, C.F., Jelsbak, L., Sohlenkamp, C., Pevzner, P., Edlund, A., McLean, J., Piel, J., Murphy, B.T., Gerwick, L., Liaw, C.-C., Yang, Y.-L., Humpf, H.-U., Maansson, M., Keyzers, R.A., Sims, A.C., Johnson, A.R., Sidebottom, A.M., Sedio, B.E., Klitgaard, A., Larson, C.B., Boya P, C.A., Torres-Mendoza, D., Gonzalez, D.J., Silva, D.B., Marques, L.M., Demarque, D.P., Pociute, E., O'Neill, E.C., Briand, E., Helfrich, E.J.N., Granatosky, E.A., Glukhov, E., Ryffel, F., Houson, H., Mohimani, H., Kharbush, J.J., Zeng, Y., Vorholt, J.A., Kurita, K.L., Charusanti, P., McPhail, K.L., Nielsen, K.F., Vuong, L., Elfeki, M., Traxler, M.F., Engene, N., Koyama, N., Vining, O.B., Baric, R., Silva, R.R., Mascuch, S.J., Tomasi, S., Jenkins, S., Macherla, V., Hoffman, T., Agarwal, V., Williams, P.G., Dai, J., Neupane, R., Gurr, J., Rodríguez,
610 A.M.C., Lamsa, A., Zhang, C., Dorrestein, K., Duggan, B.M., Almaliti, J., Allard, P.-M.,
611 Phapale, P., Nothias, L.-F., Alexandrov, T., Litaudon, M., Wolfender, J.-L., Kyle, J.E.,
612 Metz, T.O., Peryea, T., Nguyen, D.-T., VanLeer, D., Shinn, P., Jadhav, A., Müller, R.,
613 Waters, K.M., Shi, W., Liu, X., Zhang, L., Knight, R., Jensen, P.R., Palsson, B.Ø.,
614 Pogliano, K., Linington, R.G., Gutiérrez, M., Lopes, N.P., Gerwick, W.H., Moore, B.S.,
615 Dorrestein, P.C., Bandeira, N., 2016. Sharing and community curation of mass
616 spectrometry data with Global Natural Products Social Molecular Networking. Nat.
617 Biotechnol. 34, 828–837. https://doi.org/10.1038/nbt.3597.

618
<table><caption>Table 1. ¹H and ¹³C data of compounds 1–4</caption><thead><tr><th rowspan="2">Position</th><th colspan="2">1<sup>a</sup></th><th colspan="2">2<sup>a</sup></th><th colspan="2">3<sup>b</sup></th><th colspan="2">4<sup>c</sup></th></tr><tr><th>¹H<br>δ (ppm), J (Hz)</th><th>¹³C<br>δ (ppm)</th><th>¹H<br>δ (ppm), J (Hz)</th><th>¹³C δ (ppm)</th><th>¹H<br>δ (ppm), J (Hz)</th><th>¹³C<br>δ (ppm)</th><th>¹H :<br>δ (ppm), J (Hz)</th><th>¹³C<br>δ (ppm)</th></tr></thead><tbody><tr><td>1</td><td>-</td><td>164.3, C</td><td>-</td><td>164.5, C</td><td>-</td><td>173.7, C</td><td>-</td><td>164.4, C</td></tr><tr><td>2</td><td>5.45, d (2.0)</td><td>88.4, CH</td><td>5.41, d (2.1)</td><td>87.5, CH</td><td>5.57, d (2.2)</td><td>88.4, CH</td><td>5.81, d (2.1)</td><td>89.2, CH</td></tr><tr><td>3</td><td>-</td><td>171.5, C</td><td>-</td><td>172.1, C</td><td>-</td><td>174.9, C</td><td>-</td><td>171.6, C</td></tr><tr><td>4</td><td>6.18, d (2.1)</td><td>98.7, CH</td><td>6.21, d (2.1)</td><td>100.8, CH</td><td>6.64, d (2.2)</td><td>102.0, CH</td><td>5.91, d (2.1)</td><td>101.6, CH</td></tr><tr><td>5</td><td>-</td><td>159.6, C</td><td>-</td><td>168.2, C</td><td>-</td><td>172.6, C</td><td>-</td><td>163.6, C</td></tr><tr><td>6</td><td>3.80, s</td><td>55.9, CH<sub>3</sub></td><td>3.78, s</td><td>55.7, CH<sub>3</sub></td><td>3.87, s</td><td>57.0, CH<sub>3</sub></td><td>3.70 s</td><td>56.7, CH<sub>3</sub></td></tr><tr><td>7</td><td>-</td><td>128.9</td><td>-</td><td>50, C</td><td>-</td><td>50.7, C</td><td>-</td><td>60.4, C</td></tr><tr><td>8</td><td>6.46, d (10.3)</td><td>145.0, CH</td><td>2.36, d (10.7)</td><td>45.0, CH</td><td>1.98, dd (9.9; 9.9)</td><td>44.7, CH</td><td>2.14, dd (10.1;10.1)</td><td>43.8, CH</td></tr><tr><td>9</td><td>2.62, m</td><td>33.0, CH</td><td>-</td><td>74.0, C</td><td>1.62, m</td><td>38.3, CH</td><td>1.78, m</td><td>37.0, CH</td></tr><tr><td>10</td><td>1.40, m</td><td>36.4, CH<sub>2</sub></td><td>a 1.79, m<br>b 1.56, m</td><td>43.2, CH<sub>2</sub></td><td>a 1.68, m<br>b 1.27, m</td><td>39.7, CH<sub>2</sub></td><td>a 1.57, m<br>b 1.12, m</td><td>38.4, CH<sub>2</sub></td></tr><tr><td>11</td><td>1.31, m</td><td>27.4, CH<sub>2</sub></td><td>a 1.71, m<br>b 1.64, m</td><td>22.1, CH<sub>2</sub></td><td>a 1.76, m<br>b 1.48, m</td><td>28.3, CH<sub>2</sub></td><td>a 1.60, m<br>b 1.29, m</td><td>27.2, CH<sub>2</sub></td></tr><tr><td>12</td><td>1.99, m</td><td>32.6, CH<sub>2</sub></td><td>a 1.82, m<br>b 1.18, m</td><td>34.2, CH<sub>2</sub></td><td>a 1.82, m<br>b 1.22, m</td><td>36.1, CH<sub>2</sub></td><td>a 1.68, m<br>b 1.13, m</td><td>35.1, CH<sub>2</sub></td></tr><tr><td>13</td><td>5.43, m</td><td>131.3, CH</td><td>2.55, bt (11.1)</td><td>34.2, CH</td><td>1.90, m</td><td>42.8, CH</td><td>1.71, m</td><td>42.0, CH</td></tr><tr><td>14</td><td>5.33, m</td><td>132.3, CH</td><td>5.08, bs</td><td>124.9, CH</td><td>5.13, bs</td><td>126.5, CH</td><td>5.07, m</td><td>126.6, CH</td></tr><tr><td>15</td><td>2.18, m</td><td>43.7, CH</td><td>-</td><td>134.1, C</td><td>-</td><td>136.5, C</td><td>-</td><td>135.6, C</td></tr><tr><td>16</td><td>3.66, m</td><td>71.1, CH</td><td>1.59, m</td><td>47.5, CH</td><td>2.17, dd (14.2; 7.1)</td><td>43.3, CH</td><td>2.51, dd (14.3; 7.2)</td><td>42.8, CH</td></tr><tr><td>17</td><td>1.10, d (6.4)</td><td>19.8, CH<sub>3</sub></td><td>0.95, d (7.0)</td><td>18.1, CH<sub>3</sub></td><td>0.90, d (7.1)</td><td>18.1, CH<sub>3</sub></td><td>1.05, d (7.2)</td><td>17.0, CH<sub>3</sub></td></tr><tr><td>18</td><td>4.39, m</td><td>57.1, CH<sub>2</sub></td><td>a 4.08, d (13.0)<br>b 3.49, d (13.0)</td><td>63.4, CH<sub>2</sub></td><td>a 3.81, d (11.8)<br>b 3.96, d (11.8)</td><td>63.0, CH</td><td>9.92, s</td><td>200.3, CH</td></tr><tr><td>19</td><td>1.03, d (6.6)</td><td>20.9, CH<sub>3</sub></td><td>1.16, s</td><td>30.3, CH<sub>3</sub></td><td>0.71, d (6.8)</td><td>21.3, CH<sub>3</sub></td><td>1.00, d (6.8)</td><td>21.8, CH<sub>3</sub></td></tr><tr><td>20</td><td>0.97, d (6.8)</td><td>15.4, CH<sub>3</sub></td><td>1.62, s</td><td>22.0, CH<sub>3</sub></td><td>1.68, bs</td><td>22.4, CH<sub>3</sub></td><td>1.70, s</td><td>22.4, CH<sub>3</sub></td></tr></tbody></table>

a recorded in CDCl₃ at 700 MHz (¹H) and 175 MHz (¹³C)

b recorded in MeOD at 500 MHz (¹H) and 125 MHz (¹³C)

c recorded in Pyridine d₅ at 700 MHz (¹H) and 175 MHz (¹³C)
<table><caption>**Table 2.** NMR data (700 MHz) and ¹³C NMR data (175 MHz) in Pyridine-d₅ of compound 5.</caption><thead><tr><th>Positio<br>n</th><th>¹H<br>δ (ppm), J (Hz)</th><th>¹³C : δ (ppm)</th></tr></thead><tbody><tr><td>1''</td><td>6.05, t (7.1)</td><td>146.6, CH</td></tr><tr><td>2''</td><td>2.95, m</td><td>28.8, CH<sub>2</sub></td></tr><tr><td>3''</td><td>2.18, t (7.6)</td><td>39.6, CH<sub>2</sub></td></tr><tr><td>4''</td><td>-</td><td>135.1, C</td></tr><tr><td>5''</td><td>5.33, m</td><td>125.6, CH</td></tr><tr><td>6''</td><td>2.11, m</td><td>28.9, CH<sub>2</sub></td></tr><tr><td>7''</td><td>2.12, m</td><td>28.9, CH<sub>2</sub></td></tr><tr><td>8''</td><td>5.29, m</td><td>125.3, CH</td></tr><tr><td>9''</td><td>-</td><td>135.4, C</td></tr><tr><td>10''</td><td>2.09, m</td><td>40.1, CH<sub>2</sub></td></tr><tr><td>11''</td><td>2.07, m</td><td>26.2, CH<sub>2</sub></td></tr><tr><td>12''</td><td>5.25, m</td><td>123.3, CH</td></tr><tr><td>13''</td><td>1.65, s</td><td>16.5, CH<sub>3</sub></td></tr><tr><td>14''</td><td>1.65, s</td><td>16.4, CH<sub>3</sub></td></tr><tr><td>1''</td><td>-</td><td>166.2, C</td></tr><tr><td>2</td><td>-</td><td>126.4, C</td></tr><tr><td>3</td><td>2.51, m</td><td>29.5, CH<sub>2</sub></td></tr><tr><td>4</td><td>1.83, m</td><td>24.1, CH<sub>2</sub></td></tr><tr><td>5</td><td>4.20, dd (2.7, 11.5)</td><td>86.7, CH</td></tr><tr><td>6</td><td>-</td><td>70.9, C</td></tr><tr><td>1'</td><td>-</td><td>79.5, C</td></tr><tr><td>2'</td><td>3.86, dd (9.7, 3.9)</td><td>77.4, CH</td></tr><tr><td>3'</td><td>a 2.13, m<br>b 1.93, m</td><td>35.4, CH<sub>2</sub></td></tr><tr><td>4'</td><td>a 2.50, m<br>b 2.35, m</td><td>32.3, CH<sub>2</sub></td></tr><tr><td>5'</td><td>-</td><td>142.4, C</td></tr><tr><td>6'</td><td>a 4.53, bd (15.8)<br>b 4.40, bd (15.8)</td><td>63.9, CH<sub>2</sub></td></tr><tr><td>7'</td><td>1.54, s</td><td>28.1, CH<sub>3</sub></td></tr><tr><td>8'</td><td>1.47, s</td><td>20.4, CH<sub>3</sub></td></tr><tr><td>1'''</td><td>1.43, s</td><td>25.9, CH<sub>3</sub></td></tr><tr><td>2'''</td><td>1.40, s</td><td>26.9, CH<sub>3</sub></td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

628

629

630
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

631

632

633
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>

634
635
636
<figure><img src="image_6.png" /><figcaption>Figure 5</figcaption></figure>

637

638

639
<figure><img src="image_7.png" /></figure>

<caption>Figure 6</caption>
643 **Fig. 1.** Dendrogram generated with a binary matrix of "molecular families" (clusters) produced
644 by the 160 analyzed fungal samples, colored according to taxonomic affiliation to the Order
645 level, distance scale is indicated on the graph. Names are indicated for any sample identified
646 beyond genus level.

647
648 **Fig. 2.** Structure of previously undescribed isolated compounds **1–5**

649 **Fig. 3.** Key ¹H–¹H COSY (bold lines), NOE correlations (dashed arrows) and ¹H-¹³C HMBC
650 (blue arrows) correlations of compounds **1–4**.

651 **Fig. 4.** Ortep view of compound **2** (left) and **3** (right).

652 **Fig. 5.** Key ¹H–¹H COSY (bold lines), NOE correlations (dashed arrows) and ¹H-¹³C HMBC
653 (blue arrows) correlations of compounds **5**.

654 **Fig. 6.** Proposed biosynthetic pathways of chordapyrones from prochordapyrone (1) by a)
655 dehydroxylase and b) Diels-alderase conducing to chordapyrone B (3)

656
657 **Graphical abstract**
658 Previously undescribed compounds and chemodiversity of Xylariales order