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With an impressive ability to survive in harsh environments, black fungi are an ecological group of melanized fungi that are widely recognized as a major contributor to the biodeterioration of stone cultural heritage materials. As part of the ongoing efforts to study the fungal diversity thriving in a deteriorated limestone funerary art piece at the Lemos Pantheon, a national monument located in Águeda, Portugal, two isolates of an unknown microcolonial black fungus were retrieved. These isolates were thoroughly studied through a comprehensive analysis based on a multi-locus phylogeny of a combined dataset of ITS rDNA, LSU, and rpb2, along with morphological, physiological, and ecological characteristics. Based on the data obtained from this integrative analysis, we propose a new genus, Saxispiralis gen. nov., and a new species, Saxispiralis lemnorum sp. nov., in the recently described Aeminiaceae family (order Mycosphaerellales). Prior to this discovery, this family only had one known genus and species, Aeminium ludgeri, also isolated from deteriorated limestone. Additionally, considering the isolation source of the fungus and to better understand its potential contribution to the overall stone monument biodeterioration, its in vitro biodeteriorative potential was also evaluated. This work represents a significant contribution to the understanding of the fungal diversity involved in the biodeterioration of limestone heritage. [ABSTRACT FROM AUTHOR]

In this study, we explored the biodiversity and abundance of culturable fungi in four samples associated with different biodeterioration outlines collected from the Lemos Pantheon, a limestone-built artwork in Portugal. We compared the results from prolonged standard freezing with those previously obtained from fresh samples to analyze differences in the obtained community and assess the effectiveness of the standard freezing incubation protocol in uncovering a different segment of culturable fungal diversity. Our results showed a slight decrease in culturable diversity, but over 70% of the obtained isolates were not present in the previously studied fresh samples. We also identified a high number of potential new species with this procedure. Moreover, the use of a wide variety of selective culture media positively influenced the diversity of the cultivable fungi obtained in this study. These findings highlight the importance of developing new protocols under varying conditions to accurately characterize the culturable fraction in a given sample. The identification and study of these communities and their possible contribution to the biodeterioration process is crucial knowledge for formulating effective conservation and restoration plans to prevent further damage to valuable cultural heritage assets. [ABSTRACT FROM AUTHOR]

Featured Application: This work expands the current knowledge of fungal agents and their role in the biodeterioration of limestone rocks, while providing tools for the development of better and more adequate strategies for the conservation, restoration, and rehabilitation of the heritage. Although the information presented here is related to a single monument in Portugal, the data can be extrapolated to other monuments of similar lithology. Fungal organisms are considered one of the most relevant stone colonizers, and biodeteriogens. They are ubiquitous heterotrophs, metabolically versatile, ranging from generalist to extremophiles. Limestone, a sedimentary rock characterized by high levels of calcium carbonate, has low compressive strength and hardness and high porosity. These features make it highly susceptible to fungal colonization and an exceptional target for biodeterioration. Understanding the mycobiome composition associated with different biodeterioration scenarios is key for the development of effective guidelines and strategies for preventive conservation and viable maintenance of our cultural heritage. In this work, a thorough analysis of the fungal community composition on the Lemos Pantheon, a limestone-built artwork located in Portugal, was performed using high-throughput sequencing complemented with culture-based methods. The combined results allowed a detailed characterization of the fungal communities of each analyzed spot, revealing highly diverse and dissimilar communities according to the type of biodeterioration observed. In addition, we verified that both cultivation and metagenomics methodologies should be employed synergistically to tackle inherent limitations. [ABSTRACT FROM AUTHOR]

Stone monuments and relics are prone to biodeterioration processes prompted by microbial proliferation and activity. Among the distinct microbes capable of stone colonization, fungi are known to strongly contribute to stone biodeterioration. During the ongoing efforts aiming to study fungi thriving in dolomitic limestone walls of the Coimbra's hypogean Roman cryptoporticus (Portugal), two unknown Bionectriaceae isolates were retrieved. The aim of this work was to depict the molecular and phenotypic characteristics of these microorganisms. The phylogenetic analyses revealed that the studied strains could not be assigned to any of the currently known Bionectriaceae genera. Moreover, the isolates exhibited distinctive and peculiar characteristics, such as the packing of conidia by surrounding hyphal segments and the formation of rope-like microsclerotia with a textura globose. Taking into account all the data obtained, a novel genus and species, Circumfusicillium cavernae gen. et sp. nov. in Bionectriaceae (Hypocreales), is proposed here. [ABSTRACT FROM AUTHOR]

When colonizing stone monuments, microcolonial black fungi are considered one of the most severe and resistant groups of biodeteriorating organisms, posing a very difficult challenge to conservators and biologists working with cultural heritage preservation. During an experimental survey aimed to isolate fungi from a biodeteriorated limestone art piece in the Old Cathedral of Coimbra, Portugal (a UNESCO World Heritage Site), an unknown microcolonial black fungus was retrieved. The isolated fungus was studied through a complete examination based on multilocus phylogeny of a combined dataset of ITS rDNA, LSU and rpb2, in conjunction with morphological, physiological, and ecological characteristics. This integrative analysis allows for the description of a new family, Aeminiaceae fam. nov., a new genus Aeminium gen. nov., and a new species, Aeminium ludgeri sp. nov., in the order Capnodiales. [ABSTRACT FROM AUTHOR]

Dolomitic limestones are carbonate rocks with an inherent high intrinsic bioreceptivity, being especially prone to microbial colonization. Nonetheless, fungal dolomitic limestone bioreceptivity and biodeterioration still remains scarcely characterized. As such, the aims of this work were to: evaluate the biodeteriorative abilities of previously isolated fungi thriving at the Old Cathedral of Coimbra; access fungal limestone bioreceptivity in a laboratory simulated oligotrophic and copiotrophic environment; and to characterize the biodeterioration impacts of these organisms. The obtained results allowed to determine that a significant number of the tested species displayed in vitro biodeteriorative abilities. Inoculation of these species in dolomitic limestone replicas for one year revealed that, with a few exceptions, fungal proliferation was slightly superior in oligotrophic rather than copiotrophic conditions and that, in general, Penicillium chrysogenum , Periconia byssoides and Parengyodontium album displayed the highest average colonization rates. Moreover, most of the tested species produced noticeable alterations in the form of mineral disaggregation, displacement and possible dissolution. The obtained results also allowed to detect calcite mineralization in or near some species hyphae, suggesting that after one-year fungal stone diagenesis and secondary biomineralization was already occurring. This study contributed to further elucidate the nefarious effect resulting from fungal proliferation on dolomitic limestone. • In vitro analyses of fungi and dolomitic limestone interactions. • Fungal CaCO 3 dissolution, calcium oxalates and mineralization screening. • Dolomitic limestone bioreceptivity to fungal colonization analysis. • After one-year incubation period various species produced alterations. • Mineral-chemical analysis highlighted fungal induced carbonate mineralization. [ABSTRACT FROM AUTHOR]

• Portuguese Ançã limestone bioreceptivity to fungal colonization analysis. • Penicillia growth is favored in copiotrophic environments. • Aspergilli and Aeminium ludgeri growth is only slightly superior in oligotrophic environments. • After one-year incubation period all fungal species produced alterations. • Aspergillus westerdijkiae induced calcium carbonate mineralization. Fungal deteriorative action on historical stone objects often leads to important and incalculable cultural heritage losses. The Ançã stone is a unique limestone type widely used in some of the most important and emblematic Portuguese monuments. Being a soft and porous calcium carbonate limestone, this substrate is known to have a significant bioreceptivity to microbial colonization often resulting in notorious biodeterioration. Nonetheless, fungal bioreceptivity and biodeterioration of this lithotype remains scarcely characterized. With this in mind, the main aims of this work were to evaluate these aspects in a controlled laboratory environment. Inoculation of several fungal species in stone replicas revealed that Penicillia growth occupied larger areas under rich micro-nutrient environments, while Aspergilli and the black fungal species Aeminium ludgeri only denoted a slightly more accentuated growth under depleted nutrient conditions. Moreover, all species produced noticeable aesthetic alterations. In addition, we were able to verify A. ludgeri endolithic growth, Aspergillus glaucus putative hyphae penetration into calcite layers and mineralization phenomena occurring around A. westerdijkiae hyphae. By itself, this study provides a key update regarding fungal biodeterioration of this ancient, emblematic and remarkable limestone. [ABSTRACT FROM AUTHOR]

Fungi are powerful biodeteriogens and when colonizing stone monuments are often responsible for severe physical, chemical and aesthetical modifications. Recently the old cathedral of Coimbra (Portugal) was awarded the UNESCO World Heritage Site distinction as an integrant part of the "University of Coimbra- Alta and Sofia" area. The limestone walls in this cathedral show clear visual signs of biological colonization and biodeterioration. The aim of this work was to obtain an extensive fingerprint of the biodeteriorating fungal agents in this affected ancient limestone through the application of traditional cultivation and modern Next Generation Sequencing techniques. Due to high diversity of distinctive biodeterioration phenomena affecting this site, a rapport between the identified fungal populations and the different biodeterioration types found was also established and their importance for the monument preservation discussed. The results obtained through the application of this approach, not only allowed a robust characterization of the fungal communities colonizing this monument, but also elucidated that distinct fungal communities are dissimilar according to the type of biodeterioration analyzed. In addition, we verified that both cultivation and metagenomics methodologies should be employed synergistically in limestone biodeterioration studies in order to tackle inherent limitations regarding both techniques. • Analysis of fungal populations in the walls of the old cathedral of Coimbra. • Distinct fungal populations associated with distinct biodeterioration phenomena. • Predominance of Sordariomycetes over Dothideomycetes and Eurotiomycetes. • Both cultivation and metagenomics methodologies should be employed synergistically. [ABSTRACT FROM AUTHOR]

During the ongoing characterization of limestone biodeterioration in the UNESCO World Heritage site of "University of Coimbra – Alta and Sofia" (Coimbra, Portugal), it was brought to our attention that the limestone relieves adorning the tomb of D. Afonso I (First Portuguese King), displayed several signs of distinctive biodeterioration patterns. According to the ICOMOS Illustrated glossary on stone deterioration patterns, these could be characterized as visual mould proliferation, presence of black crusts, stone erosion and disintegration. Due to the invaluable nature of this monument, a multi-disciplinary approach (biological and analytical) to fully characterize these phenomena was applied. The results obtained allowed the characterization of the fungal diversity colonizing this monument, as well as the identification of various deterioration products. The results highlighted that the detected indoor black crusts displayed various characteristics from polluted environments, although lacking the typical association with gypsum. The black crusts were also characterized by the presence of lead oxides, damaging salts and carbonaceous particles. In addition, it could be verified that the erosion phenomena found are a result from fungal mediated calcium carbonate dissolution and calcium oxalate formation. The establishment of different fungal populations coupled with the deposition of environmental particles contributed to the development of the distinct phenomena detected through differential biodeterioration mechanisms. • Application of a multi analytical approach to study D. Afonso I tomb pathologies. • High fungal contamination associated with all biodeteriorated areas. • Black crusts holding lead oxides, anhydrite, syngenite and carbonaceous particles. • Stone erosions with calcium oxalates. • Biodeterioration by chromatic changes, stone dissolution and calcium oxalate formation. [ABSTRACT FROM AUTHOR]