# Black stains of Lascaux Cave involve different melanin synthesis pathways as shown by Alkaline/H2O2 hydrolysis and UHPLC-ESI/QTOF analysis of pigmented fungal isolates and stains from cave walls

Pierre Vergne, Malik Bahsoun, Marie-Geneviève Dijoux-Franca, Gilles Comte, Jeanne Doré, Maximilien Gonçalves-Martins, Lise Alonso, Isabelle Kerzaon, Patricia Luis, Yvan Moënne-Loccoz, et al.

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Pierre Vergne, Malik Bahsoun, Marie-Geneviève Dijoux-Franca, Gilles Comte, Jeanne Doré, et al. Black stains of Lascaux Cave involve different melanin synthesis pathways as shown by Alkaline/H2O2 hydrolysis and UHPLC-ESI/QTOF analysis of pigmented fungal isolates and stains from cave walls. International Biodeterioration & Biodegradation, 2025, 204, pp.106142. 10.1016/j.ibiod.2025.106142. hal-05447952

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# Black stains of Lascaux Cave involve different melanin synthesis pathways as shown by Alkaline/H₂O₂ hydrolysis and UHPLC-ESI/QTOF analysis of pigmented fungal isolates and stains from cave walls

Pierre Vergne, Malik Bahsoun, Marie-Geneviève Dijoux-Franca, Gilles Comte, Jeanne Doré, Maximilien Gonçalves-Martins, Lise Alonso, Isabelle Kerzaon, Patricia Luis, Yvan Moënne-Loccoz, Serge Michalet* ID

Université Claude Bernard Lyon 1, CNRS, INRAE, VetAgro Sup, UMR5557 Ecologie Microbienne, F-69622, Villeurbanne, France

<figure><img src="image_2.png" /></figure>

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## ARTICLE INFO

**Keywords:**

Lascaux Cave

Fungi characterization

Melanin identification

Black stain

Cave conservation

## ABSTRACT

Lascaux cave in southwestern France is a UNESCO landmark famous for its Paleolithic engravings and paintings, but it suffered from microbial outgrowth and black strain formation following unbalanced touristic operations and chemical treatments. Pigments synthesized by black fungi isolated from these stains were previously identified as melanin, but a limited range of black fungi was assessed and black stains themselves have been investigated by surface-enhanced Raman spectroscopy, only. This study aimed at identifying the origins and chemical features of the melanin polymers produced by 10 black fungal strains isolated from Lascaux black stains. Fungi were identified by ITS2 sequencing. Mycelial pigment extracts were analyzed by UHPLC/ESI-QTOF after alkaline H₂O₂ hydrolysis, which allowed to detect simultaneously monomers of different melanin types. Analytical markers from several biosynthetic pathways of melanin were evidenced in all fungal isolates, and some strains showed the ability to produce melanin from at least five different pathways, which to our knowledge constitutes the first report of such a variety of means to achieve black pigmentation within a single fungal strain. As proof of principle, this method was applied to black stains directly collected from the cave and revealed three to four melanin types in samples from the Apse, the Nave and the Passage. Some of these melanin types corresponded to those of the fungal isolates analyzed, namely *Alternaria alternata*, *Acremonium nepalense* or *Ochroconis lascauxensis*. This is the first characterization of the types of melanin synthesized on Lascaux surfaces. Results emphasize the interest of this method to understand different pigmentation patterns in caves and to analyze melanin pigments from diverse biological matrices.

## 1. Introduction

Lascaux Cave (Montignac, France) was discovered in September 1940, classified in December 1940 and labeled UNESCO site in 1979 (Bastian et al., 2010). The cave was open to visitors from 1948 to 1963. Microbial alterations of cave walls took place during this period and in subsequent years. In the last decades, black stains have formed in various parts of the Cave (since December 2001), especially in the Passage, the Apse and the Nave (Bontemps et al., 2022). Current cave management practices have limited the extension of black stains and some of these stains are now attenuated (Bontemps et al., 2024b), but many remain and represent a source of concern for cave conservation (Bontemps et al., 2024a). Therefore, it is of prime importance to

understand the formation of these stains and the chemical nature of the black pigments present. Black stains are also a conservation issue in other UNESCO's World Heritage sites, including ancient tombs (Ma et al., 2020) and medieval buildings (Fomina et al., 2024).

As previously shown in 2012 by Surface-Enhanced Raman Spectroscopy (SERS) analysis, the origin of black stains in Lascaux could be related to fungal melanin (Martin-Sanchez et al., 2012b). In particular, the fungal species *Ochroconis lascauxensis* (syn. *Scolecobasidium lascauxense*) and *O. anomalala* (syn. *S. anomalum*) were identified from black stains of Lascaux Cave (Martin-Sanchez et al., 2012a) and both are producing black pigments. *O. lascauxensis* was mainly reported on ceilings and walls of the cave while *O. anomalala* was scarcely distributed. From these fungi, melanins were tentatively identified using SERS, ¹⁵N

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: serge.michalet@univ-lyon1.fr (S. Michalet).</footnote>
and ¹³C-NMR experiments and extended pyrolysis-GC/MS (De la Rosa et al., 2017; Saiz-Jimenez et al., 2021). No link could be established with a known chemical structure of fungal melanin, as no clear evidence of the presence of 3,4-dihydroxy-phenylalanine (DOPA)- or 1,8-dihydroxy-naphthalene (DHN)-melanins was found. Nevertheless, the presence of a pyomelanin type, derived from homogentisic acid (HGA) or other phenol was hypothesized (Saiz-Jimenez et al., 2021). However, the presence of genes that may enable the fungus to synthesize melanin *via* the DHN pathway have been described in an *in-silico* genome analysis of an *O. mirabilis* strain (Samerpitak et al., 2014; Yew et al., 2016). Thus, the chemical nature of Lascaux's black stains is still under investigation.

The objective of this work was to clarify the chemical composition of black pigments formed by fungi in black stains found on wall surfaces of Lascaux Cave. Completing previous findings (De la Rosa et al., 2017; Martin-Sanchez et al., 2012b), ten black fungal strains were isolated from black stains in Lascaux Cave and grown in the laboratory, and the chemical structure of their melanin(s) was investigated. We used alkaline hydrolysis followed by H₂O₂ oxidation (Ito et al., 2011; Szeke-ly-Klepser et al., 2005), which allows to detect simultaneously the oxidized monomers of different melanin types at the mg/g range in biological matrices (Ito et al., 2011). This method has been widely used to characterize eumelanin and pheomelanin in human and animal tissues (d'Ischia et al., 2015) and was also successfully applied to the characterization of melanin from hymenoptera (Hines et al., 2017) or even fossils (Glass et al., 2012; Tanaka et al., 2014). Though, to our knowledge, it has never been used for the analysis of melanins of fungal origin. The coupling of this method with UHPLC/HRMS analyses enabled us to detect melanin markers at lower concentrations compared to UV analysis, which was used to develop the method initially described in the literature (Ito et al., 2011). Moreover, MS/MS analyses allowed us to obtain more accurate information on structural bases of fungal melanins. This extraction and analysis protocol was also applied to the analysis of melanin content in three black stains collected from Lascaux Cave.

## 2. Materials & methods

### 2.1. Chemicals and reagents

Methanol and ethanol were purchased from VWR International (Radnor, PA, USA); HPLC-MS grade formic acid, water and acetonitrile from Fisher chemical (Pittsburg, PA, USA); synthetic DOPA-melanin (CAS: 8049-97-6, Lot #BCBN7087V), K₂CO₃ (99,95 %) from Sigma-Aldrich Corporation (St. Louis, MO, USA); Na₂CO₃ (99,8 %), H₂O₂ (30 %) from Carl Roth GmbH + Co. KG (Karlsruhe, Germany); HCl (37 %) from Merck KGaA (Darmstadt, Germany).

### 2.2. Fungal strains, media and growth conditions

The fungi were isolated from swab or rock wall samples (Fig. S1) by vortexing in 0.9 % NaCl solution, following by spread plating onto Dichloran Rose Bengal Chloramphenicol selective medium (Biokar Diagnostics, Beauvais, France). Fungal colonies were then transferred onto Complete Yeast Maltose (CYM) medium (Raper and Hoffman, 1974) and kept at 22 °C.

DNA extraction from 43 fungal mycelia was carried out as described by van Kan et al. (1991), except that freeze-dried mycelium was crushed mechanically using toothpicks prior to chemical lysis. DNA pellets obtained after extraction were washed with 70 % ethanol and resuspended with 25 µL UP H₂O. Fungal ITS2 fragments were amplified using primers ITS1-F (5'-CTTGGTCATTTAGAGGAAGTAA-3') (Gardes and Bruns, 1993) and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') (White et al., 1990) in 50-µL volumes containing 1 × buffer (Invitrogen, Carlsbab, CA), 0.15 mM of each dNTP, 1.5 mM MgCl₂, 0.4 µM of each primer, 0.25 % BSA and 0.5 U Taq Polymerase (Invitrogen). PCR was performed at 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 60 s, and 72 °C for 90 s.

PCR products were sequenced using Sanger methodology (Biofidal/Microsynth company, Lyon, France) and sequences were blasted against NCBI nr-database (Johnson et al., 2008). ITS sequences are available at NCBI (accession numbers PV400642, PV400648, PV400651, PV400653, PV400659, PV400661, PV400664, PV400665, and SUB15212079).

The growth of 10 fungi for melanin analysis was carried out using prewashed and autoclaved cellulose discs placed on the surface of CYM agar, on which two 6-mm mycelial plugs from each fungus were added. These procedures facilitated the recovery of the mycelium for the extraction of pigments.

### 2.3. Extraction and alkaline hydrogen peroxide oxidation of pigments from mycelium and black stains

After 1 month of growth at room temperature, in the dark, the mycelium was collected with a stainless-steel spatula and then freeze-dried and weighted. Pigments oxidation products were extracted following the alkaline H₂O₂ hydrolysis method according to Ito et al. (2011). Ten mg of dried mycelium or 150 mg of freeze-dried black stain were placed in a 2 mL Eppendorf tube with 2 inox beads, immersed in liquid nitrogen then grinded (QIAGEN, TissueLyser II) two times for 2 min at 30 Hz. Once grinded, 100 µL of ultrapure water, 375 µL 1 M K₂CO₃ and 25 µL 30 % (v/v) H₂O₂ were added. The tubes were sonicated (Fisherbrand, FB 15061 ultrasonic bath) for 2 h at 20 °C. The tubes were then centrifugated (Eppendorf, centrifuge 5425) at 15 000 rpm for 15 min. The supernatants were placed in 6 mL test tubes. The residual H₂O₂ was decomposed by adding 50 µL 10 % Na₂CO₃ and the mixture was acidified with 140 µL HCl (6 M). To facilitate evaporation, 500 µL EtOH were added, and the tubes were dried under vacuum (LABCONCO, CentriVap Concentrator) for 3 h. Once evaporated, 1 mL MeOH and 10 µL formic acid were added to the tubes, then vortexed, sonicated for 15 min, centrifugated for 15 min at 15 000 rpm. The supernatants were transferred to 2 mL Eppendorf tubes. The previous step was repeated, then the tubes evaporated at 15000 rpm for 2 h. Finally, 100 µL MeOH and 10 µL formic acid were added to the tubes. They were vortexed, sonicated for 15 min, centrifugated for 15 min at 15 000 rpm. Then the supernatant was transferred into UHPLC vials for UHPLC/MS/MS analysis.

### 2.4. Synthetic melanin standard preparation

For melanin standard preparation, 1 mg of synthetic DOPA-melanin was weighed in a 2 mL Eppendorf tube. Then the same steps for the extraction of mycelium pigments oxidation products were followed.

### 2.5. UHPLC/MS/MS analysis

UHPLC-DAD-ESI/QTOF analysis was performed on an Agilent Infinity 1290 system (Agilent Technologies) coupled to a UV/vis DAD detector and equipped with a QTOF 6530 (Agilent Technologies) detector controlled by MassHunter software (Agilent Technologies). Analyte separation was carried out on a Poroshell 120 EC-C18 column (100 mm × 3.0 mm, 2.7 µm). The following gradient was run using 0.4 % aqueous formic acid (solvent A) and acetonitrile (solvent B): 0 min, 1 % B; 1 min, 1 % B; 3 min, 30 % B; 10 min, 70 % B; 12 min, 70 % B; 15 min, 100 % B; 16 min, 100 % B. The flow rate and column temperature were 1 mL min⁻¹, and 50 °C, respectively and 3.0 µL of sample extracts were injected. The ESI source and spectrometer were optimized as follows: positive ionization in auto-MS/MS mode, scan spectra from m/z 100 to 1000, capillary voltage 2.5 kV, nozzle voltage 2 kV, fragmentor 100 V and the fixed collision-induced dissociation (CID) energy was set at 20 eV. Nitrogen was used as the nebulizing gas with a flow rate of 11 L/min and a temperature of 310 °C at 40 psi. EIC were extracted on MassHunter Qualitative Analysis B.07.00, with a symmetric window of 10 ppm and only ions detected at at least 3.10³ counts were considered (which
<table><caption>**Table 1**
The 10 black fungi used in the study and the melanin types they produce in vitro.</caption><thead><tr><th rowspan="2">Taxa</th><th rowspan="2">Class</th><th rowspan="2">Order</th><th rowspan="2">Family</th><th colspan="7">Melanin types</th></tr><tr><th>DOPA</th><th>DHN</th><th>CAT</th><th>GDHB</th><th>HGA</th><th>BOS</th><th>ASP</th></tr></thead><tbody><tr><td>Alternaria alternata</td><td>Dothideomycetes</td><td>Pleosporales</td><td>Pleosporaceae</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Ochroconis lascauxensis</td><td>Dothideomycetes</td><td>Venturiales</td><td>Sympoventuriaceae</td><td>+</td><td>(+)</td><td>+</td><td>+</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Chaetothyriales sp.</td><td>Eurotiomycetes</td><td>Chaetothyriales</td><td>(not identified)</td><td>+</td><td>+</td><td>++</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Exophiala angulospora</td><td>Eurotiomycetes</td><td>Chaetothyriales</td><td>Herpotrichiellaceae</td><td>+</td><td>+</td><td>++</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Exophiala castellanii</td><td>Eurotiomycetes</td><td>Chaetothyriales</td><td>Herpotrichiellaceae</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Exophiala moniliae</td><td>Eurotiomycetes</td><td>Chaetothyriales</td><td>Herpotrichiellaceae</td><td>+</td><td>+</td><td>++</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Exophiala sp.</td><td>Eurotiomycetes</td><td>Chaetothyriales</td><td>Herpotrichiellaceae</td><td>+</td><td>+</td><td>++</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Minimelanolocus sp.</td><td>Eurotiomycetes</td><td>Chaetothyriales</td><td>Herpotrichiellaceae</td><td>+</td><td>+</td><td>+</td><td>-</td><td>++</td><td>-</td><td>-</td></tr><tr><td>Acremonium nepalense</td><td>Sordariomycetes</td><td>Glomerellales</td><td>Plectosphaerellaceae</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>Doratomyces sp.</td><td>Sordariomycetes</td><td>Microascales</td><td>Microascaceae</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Black pigmented fungal strains isolated from Lascaux Cave and identified by partial ITS2 sequencing. Pictures taken 30 days after culture on CYM media at room temperature in the dark.</figcaption></figure>

correspond approximatively to three times the noise limit which was found to be around 1.10³ counts for each EIC analyzed). No traces were obtained for each EIC screened in analytical or reactional blanks. For MS/MS analysis, targeted-MS/MS was used as the auto-MS/MS mode could not allow to obtain all MS/MS spectra.

## 2.6. Analysis of black stains from Lascaux

As a proof of principle, the methodology used with black fungal isolates was also applied to assess three black stains sampled with sterile scalpels directly in Lascaux Cave. They were taken in the Apse (sample La958), the Nave (sample La959) and the Passage (sample La960) (Fig. S1).

# 3. Results & discussion

## 3.1. Identification of black microorganisms

Stains from various parts of Lascaux Cave, especially black stains, were used to isolate pigmented fungi, and 152 fungal colonies were thus obtained. Among them, 43 formed black colonies on CYM medium. All 43 were Ascomycetes from the Pezizomycotina subphylum. They were identified as Chaetothyriales species, *Minimelanolocus* sp., Herpotrichiellaceae species (this isolate was not used hereafter), *Exophiala angulospora*, *Exophiala castelanii* (renamed *E. mansonii*), *Exophiala moniliae*, and *Exophiala* sp. (all from the Chaetothyriales order), *Ochroconis lascauxensis* (from the Venturales order), *Doratomyces* sp. (from the

Microascales order) and *Acremonium* (syn. *Brunneochlamydosporium nepalense* (from the Glomerellales order) based on partial ITS2 sequencing. Information is shown for 9 representative fungi subsequently used for chemical analysis (Table 1 and S1). An additional Lascaux black fungus (*Alternaria alternata*, from the Pleosporales order) was obtained from the Laboratoire de Recherche des Monuments Historiques. Phylogenetic analysis using maximum-likelihood (PhyML) showed that this collection of 10 fungi displayed significant diversity albeit limited to four Pezizomycotina orders (Fig. S2). Pigmentation develops progressively in these fungi as fungal colonies age on plates (Fig. 1).

## 3.2. UHPLC-ESI/QTOF analysis after alkaline/H₂O₂ hydrolysis for identification of melanin types in fungi

Most known fungal melanins can be synthetized via several identified pathways, the main described ones are the DHN pathway where the precursor is malonyl-CoA and the DOPA pathway where the precursor is tyrosine or dopamine (Eisenman and Casadevall, 2012; Ito et al., 2011; Pombeiro-Sponchiado et al., 2017). Fungal melanogenesis has also been shown to occur via many other alternative pathways (Toledo et al., 2017), namely by the polymerization of (i) homogentisic acid (HGA), an intermediate of the tyrosine degradation pathway, leading to pyomelanin (Schmaler-Ripcke et al., 2009); (ii) glutaminyl 3,4-dihydroxybenzene (GDHB) obtained after oxidation of glutaminyl 4-hydroxybenzene (GHB), a process mostly described in Basidiomycetes (Jolivet et al., 1998); (iii) catechol dimers (CAT) obtained after oxidation of catechol
<figure><img src="image_4.png" /></figure>

Chemical Formula: C₁₀H₇O₂⁻
Exact Mass: 159.0452

DHN

<figure><img src="image_5.png" /></figure>

Chemical Formula: C₆H₂NO₆S⁻
Exact Mass: 215.9608

TTCA

<figure><img src="image_6.png" /></figure>

Chemical Formula: C₁₁H₁₃N₂O₅⁻
Exact Mass: 253.0830

GDHB

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

Chemical Formula: C₁₇H₁₁O₇⁻
Exact Mass: 327.0510

Dihydroxy-
aspuvilone E

<figure><img src="image_8.png" /></figure>

Chemical Formula: C₇H₄NO₆⁻
Exact Mass: 198.0044

PTCA

<figure><img src="image_9.png" /></figure>

Chemical Formula: C₉H₁₁N₂O₃⁻
Exact Mass: 195.0775

4-AHP

<figure><img src="image_10.png" /></figure>

Chemical Formula: C₆H₆NO₂⁻
Exact Mass: 124.0404

Para-amino
catechol

<figure><img src="image_11.png" /></figure>

Chemical Formula: C₈H₇O₄⁻
Exact Mass: 167.0350

HGA

<figure><img src="image_12.png" /></figure>

Chemical Formula: C₁₁H₉N₂O₅S⁻
Exact Mass: 281.0238

BTCA

<figure><img src="image_13.png" /></figure>

Chemical Formula: C₁₂H₉O₄⁻
Exact Mass: 217.0506

Catechol dimer

<figure><img src="image_14.png" /></figure>

Chemical Formula: C₁₄H₈NO₄⁻
Exact Mass: 254.0459

6-O-desmethyl-
5-desoxybostrycoidin

<caption>**Fig. 2.** Melanin biomarkers analyzed. Compounds are presented in their anionic forms in order to get their exact masses, which were used for targeted-MS/MS. Major ions were detected: DHN (RT: 6.3 min; 7.9 min); PTCA (RT: 5.4 min); HGA (RT: 6.4 min; 7.3 min); GDHB (RT: 1.1 min; 6.7 min; 11.8 min); catechol dimer (RT: 0.7 min); dihydroxyaspuvilone E (RT: 5.8 min; 6.1 min), 6-O-desmethyl-5-desoxybostrycoidin (RT: 0.9 min; 1.1 min).</caption>
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Fig. 3.** Extracted Ion Chromatograms (EIC) of PTCA [(M-H)⁻, *m/z* 198.0044] in extracts of synthetic DOPA-melanin or fungal mycelia (merged chromatograms). A Three major ions were found at three different retention times for DOPA-melanin standard and could correspond to three different isomers. B Zoom on the major ion detected at a retention time of 5.4 min.</figcaption></figure>
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 4.** Extracted Ion Chromatograms (EIC) of catechol dimer [(M-H)`, m/z 217.0506] in fungal isolate extracts (merged chromatograms). **A** One major ion was found. **B** Zoom on the major ion detected at a retention time of 0.75 min.</figcaption></figure>
(Solano, 2014); (iv) *para*-amino catechol obtained after oxidation of *para*-amino phenol (PAP); or (v) aspulvinone E, obtained by Non-Ribosomal Peptide Synthase (NRPS)-like enzymes and leading to ASP-melanin (Geib et al., 2016), and (vi) 5-deoxybostrycoidin, a derivative of hydroxynaphthalene (Frandsen et al., 2016), the two latter being more recently described.

All these melanin types generate different oxidized metabolites after alkaline hydrogen peroxide hydrolysis, and these could be used as proxy for the characterization of melanin types represented in the biological matrix used (Ito et al., 2011). Analyzed metabolites included pyrrole-2, 3,5-tricarboxylic acid (PTCA), a marker for DOPA-melanin (Ito et al., 2011); DHN for DHN-melanin; thiazole-2,4,5-tricarboxylic acid (TTCA), 4-aminohydroxyphenylalanine (4-AHP) and 6-alanyl-2-carboxy-4-hydroxybenzothiazole (BTCA) for pheomelanin (Ito et al., 2011); GDHB for GHB-melanin (Ward et al., 2008); para-amino catechol for PAP-melanin; catechol dimer for catechol-melanin (Ward et al., 2008); dihydroxy-aspulvinone E for ASP-melanin (Geib et al., 2016) and 6-O-desmethyl-5-deoxybostrycoidin, for deoxybostrycoidin-melanin (Frandsen et al., 2016). These compounds are presented in Fig. 2 in their anionic forms, and their exact masses were used for EIC acquisition. EICs obtained for each strain after laboratory growth under conditions promoting pigment synthesis were compared between strains and with synthetic DOPA-melanin in the case of PTCA.

To identify PTCA in the extracts, the MS/MS spectrum corresponding to its pseudomolecular ion at *m/z* = 198.0044 and retention time (RT) 5.4 min allowed to identify product ions at *m/z* = 111.0112 and *m/z* = 67.0208 corresponding to the loss of two or three carboxylic units, respectively (Fig. S3). The same procedure was applied for DHN pseudomolecular ion at *m/z* = 159.0452 and RT 7.9 min and a product ion at *m/z* = 92.9981, corresponding to the fragmentation between aromatic rings following a Retro Diels-Adler (RDA) type rearrangement (Fig. S4). For catechol dimer, the fragmentation of its pseudomolecular ion at *m/z* = 217.0506 and RT 0.7 min showed a complex spectrum (Fig. S5). The major product ion at *m/z* = 59.0156 could correspond to the fragmentation of the catechol ring. The MS/MS spectrum of GDHB pseudomolecular ion at *m/z* = 253.0830 and RT 1.1 min is also complex (Fig. S6). Major product ions detected at *m/z* = 108.9032 and *m/z* = 93.0363 could correspond to the catechol ion generated after the loss of glutamic acid and its dehydroxy derivative, respectively. For HGA, the fragmentation spectrum of its pseudomolecular ion at *m/z* = 167.0350 and RT 7.3 min showed a base peak at *m/z* = 108.0236 corresponding to the para-hydroquinone ion generated after the loss of the acetate moiety (Fig. S7). For dihydroxyaspuvilone E, the fragmentation of its pseudomolecular ion at *m/z* = 327.0510 and RT 5.8 min showed a base peak at *m/z* = 203.0356 corresponding to the classic benzylic fragmentation (Fig. S8). No MS/MS spectrum could be obtained for 6-O-desmethyl-5-deoxybostrycoidin.

### 3.3. Melanin type(s) according to the fungi analyzed

As shown in Fig. 3, all 10 fungal strains analyzed seem to produce DOPA-melanin in the condition tested, since PTCA was detected in all chromatograms (as shown by comparison with the main ion detected also in synthetic DOPA-melanin; Fig. 3B). Though, differences between strains could be evidenced: *A. alternata* and *A. nepalense* were those presenting the lowest amount of PTCA (which was detected at low levels), whereas in *O. lascauxensis* extract, the PTCA signal was found approximatively tree times more intense, and the other strains were ranging between these limits (Fig. 3B).

For catechol-melanin, the analysis of catechol dimer EICs revealed that all fungal strain studied produced catechol-melanin at significant levels since the signals were detected at relative high intensities (Fig. 4), especially in Chaetothyriales sp., *Exophialia* sp., *E. angulospora* and *E. moniliae* for which the signals detected were approximatively ten times more intense (around 5.10⁵ counts) than in *A. alternata* for which the smaller signal was detected (around 5.10⁴ counts) (Fig. 4B).

For DHN-melanin (Fig. S9), the analysis of both signals detected corresponding to DHN (probably isomers) revealed that most strains synthesized DHN-melanin except *A. alternata*, for which no DHN was detected. Also, *O. lascauxensis* produced very little DHN-melanin (signal at the limit of detection, Fig. S9C) compared to the other strains like *Exophialia* spp. In addition, the DHN-melanin produced by *O. lascauxensis* did not have the same structure as in the other strains analyzed, since the only DHN ion detected for this strain (at RT 6.3 min, Fig. S9C) was the one being the least represented in most other strains (Fig. S9), suggesting a different polymerization pattern.

The other types of melanin markers screened were detected only in a few fungal strains: this was the case of (i) GDHB, which was found in *A. alternata*, *Doratomyces* sp., *Exophialia* sp., *E. angulospora*, *E. moniliae* and *O. lascauxensis* (Fig. S10); (ii) HGA, which was only detected in Herpotrichelaceae sp. and to a lesser degree in *A. alternata* (with differences in the ion types detected) (Fig. S11); (iii) dihydroxy-aspulvinone E, which was only retrieved in *A. alternata* and *O. lascauxensis* (with differences in the ion types detected) (Fig. S12); and (iv) 6-O-desmethyl-5-deoxybostrycoidin, which was detected in *Doratomyces* sp. and *A. nepalense* (with differences in the ion types detected) (Fig. S13). The markers of PAP-melanin and pheomelanin were not detected in any strain (data not shown).

All the melanin types potentially synthesized by the studied fungal strains, as revealed by our analysis, are listed in Table 1. All strains were able to produce melanin at least by three different ways and up to five for some strains, namely *A. alternata*, *Doratomyces* sp. and possibly *O. lascauxensis*. It might be thus possible for these strains to use many biosynthetic pathways to produce black pigments, which would represent, to our knowledge, the first description of such a variety of ways to achieve black pigmentation within a single fungal strain. We speculate that this apparent functional redundancy might be related to variable availability of melanin precursors and/or to the synergistic effects of distinct melanin polymers for stress tolerance, hence the selection of these fungi by cave conditions.

It is interesting that the strain possessing the brighter mycelium (*A. alternata*) was also presenting paradoxically the highest number of melanin pathways detected. Though, the amounts of melanin biomarkers detected were each time low and among the lowest compared with those for the other strains, thus pointing that the method used is adapted and accurate to detect melanin polymer units even at very low levels. The fact that this fungus produces so many melanin types but at low levels should have an ecological significance and this might be related to the fact that this plant opportunistic pathogen/endophyte (depending on isolates and plant species) can resist to very extreme environmental conditions - it has been found colonising walls in Chernobyl central (Mironenko et al., 2000) - and has a great versatility and faculty of adaptation, as illustrated by its species and genus names.

Based on SERS, ¹⁵N and ¹³C NMR, and extended pyrolysis-GC/MS experiments, *De la Rosa* et al. (2017) and Saiz-Jimenez et al. (2021) concluded that the melanin structure of *O. lascauxensis* is not related to DOPA or DHN. Although these techniques are well adapted to have a general view of polymer structures, they are not sensitive enough to detect minor structures in complex polymers. In addition, some results of *De la Rosa* et al. (2017) may lead to a different interpretation, based on the presence of biphenyl (in high proportion), naphthalene (and derivatives) and indane (at low but significant amounts) in the pyrolysis-GC/MS chromatograms. With extended experiments (including temperature variation and with/without the addition of a step of acidic digestion), Saiz-Jimenez et al. (2021) found a lot of aromatic derivatives in the different chromatograms presented (i.e. pyrrole, pyridine, benzene, phenol, naphthalene, biphenyl, indole and numerous derivatives) and some were found as major peaks depending on the condition used. The authors attributed these peaks to be mainly due to proteolysis, and also hypothesized that a phenolic core similar to pyomelanin could be present in *Ochroconis* melanin. However, the presence of major peaks relative to biphenyl and naphthalene in the pyrogram
<figure><img src="image_19.png" /><figcaption>**Fig. 5.** Black stains scrubbed with a scalpel from Lascaux walls in the Apse (La958, A), the Nave (La959, B) and the Passage (La960, C) (left). Merged Extracted Ion Chromatograms (EIC) of detected markers in each stain extracts (right). ASP: dihydroxy-aspulvinone E; Bos: 6-O-desmethyl-5-deoxybostrycoidin; CAT: catechol dimer.</figcaption></figure>
obtained at 500 °C after acid hydrolysis (same conditions as in De la Rosa et al. (2017), which should enable to remove proteins) was not explained by the authors. De la Rosa et al. (2017) also used specific inhibitors of either DOPA-melanin (kojic acid) or DHN-melanin (tricyclazole) biosynthesis, to see if their presence in the growing media would affect the pigmentation of this strain. It is interesting to notice that when tricyclazole was used, a browner tint of the mycelium could be observed compared to the black tint of the non-treated strain, and this was even more pronounced with the other Ochroconis fungus tested, i.e. O. tshawytschae (De la Rosa et al., 2017), thus bringing another evidence that DHN melanin might be present. Nevertheless, our results indicate that the use of single inhibitors would not work on these strains since they are able to produce melanin by several manners. To which extent this could be generalized to other species than the ones tested remains to be further explored, but this might oblige to rethink the significance of the use of single inhibitor tests to validate (or invalidate) melanin structure type in fungal isolates. Another concern is how long a given strain would maintain the expression of different melanin biosynthetic pathways, especially in the absence of the stress or the stimuli that induced melanin production. We observed that a few fungi did not produce certain types of melanin anymore when grown after long-term storage at -80 °C (data not shown).

### 3.4. Mass spectrometry for identification of black pigments in Lascaux's black stains

As proof of principle and preliminary experiment, the same procedure was applied to three black stains scrubbed from the walls of Lascaux Cave, i.e. in the Apse (Fig. 5A), the Nave (Fig. 5B) and the Passage (Fig. 5C). From the Apse sample, which was the blackest pigmented stain, the specific markers of four melanin types were detected, namely catechol-, DOPA-, ASP- and deoxybostrycoidin-melanins (Fig. 5A). The two latter melanin types were only retrieved in some fungal isolate extracts, i.e. *Alternaria alternata* and *Ochroconis lascauxensis* for ASP-melanin and *Acremonium nepalense* for deoxybostrycoidin-melanin. RT of the signal detected in stain extract was more related to A. nepalense than *Doratomyces* sp., which also possesses the ability to produce this type of melanin. For the samples from the Nave and the Passage, melanin markers were detected but at lower intensities, and only two types were detected for the Nave (Fig. 5B) and three types for the Passage (Fig. 5C).

## 4. Conclusion

The black fungi isolated from Lascaux were phylogenetically diverse. They could produce a diverse array of melanins, whose synthesis followed at least three and up to five different pathways depending on the fungus. The analytical methodology was validated on three cave stains, which is the first characterization of the melanin polymers forming inside Lascaux Cave. The formation of black stains having taken place after significant biocide applications, it suggests that melanin synthesis was selected as an adaptation to chemical stress. Since a diverse array of melanins was evidenced, it implies that selective removal methods aiming at specific melanins only would be of limited potential for removal of cave wall black stains. Therefore, research attention should focus on the development of generic removal strategies applicable to the various kinds of melanin polymers. Finally, the analytical methodology used here should now be applied to the various black stains present in Lascaux to better understand stain formation and the link with the visual properties of these black stains, so as to help guide cave conservation efforts.

### CRediT authorship contribution statement

Pierre Vergne: Writing – original draft, Visualization, Validation, Methodology, Investigation. Malik Bahsoun: Methodology,

Investigation, Formal analysis. Marie-Genevieve Dijoux-Franca: Validation, Methodology, Investigation. Gilles Comte: Validation, Supervision, Methodology, Formal analysis, Conceptualization. Jeanne Doré: Investigation. Maximilien Gonçalves-Martins: Methodology, Investigation. Lise Alonso: Investigation. Isabelle Kerzaon: Methodology, Investigation. Patricia Luis: Validation, Methodology. Yvan Moëlle-Loccoz: Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization. Serge Michalet: Writing – original draft, Supervision, Methodology, Investigation, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

We are grateful to D. Chapulliot and T. Pommier (UMR *Ecologie Microbienne*) for help, S. Géraud, J.C. Portais, A. Rieu and M. Mauriac (DRAC *Nouvelle Aquitaine*) for key information, guidance and help, D. Henry-Lormelle and her restorer team for technical help with Lascaux sampling, Lascaux Scientific Board for helpful discussions and F. Bousta (Laboratoire de Recherche des Monuments Historiques -LRMH, Champs-sur-Marne) for providing A. alternata strain. This work was carried out at *Centre d'Etudes des Substances Naturelles* (in UMR *Ecologie Microbienne*) and funded by DRAC *Nouvelle Aquitaine* (Bordeaux, France). P.V. and M.B. contributed equally to this work.

## Appendix. ASupplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ibiod.2025.106142.

### Data availability

Data will be made available on request.

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