A preliminary metabarcoding analysis of Portuguese raw honeys.

The microbial diversity in Portuguese raw honeys remains largely uncharacterized, constituting a serious knowledge gap in one of the country's most important resources. This work provides an initial investigation with amplicon metabarcoding analysis of two Lavandula spp. from different geographical regions of Portugal and one Eucalyptus spp. honey. The results obtained allowed to identify that each honey harbors diverse microbiomes with taxa that can potentially affect bee and human health, cause spoilage, and highlight bad bee-hive management practices. We verified that prokaryotes had a tendency towards a more marked core bacterial and a relative homogenous taxa distribution, and that the botanical origin of honey is likely to have a stronger impact on the fungal community. Thus, the results obtained in this work provide important information that can be helpful to improve this critical Portuguese product and industry.

Draft genome sequence and comparative genomic analysis of Penicillium pancosmium MUM 23.27 isolated from raw honey.

The draft genome sequence and main genomic features of Penicillium pancosmium MUM 23.27, isolated from Portuguese raw honey are reported. The genome size is 34.82 Mb, containing a 48.99% GC content, 11,394 genes, with 39 rRNAs and 147 tRNAs/tmRNAs. Twenty-six BGCs were predicted with four exhibiting significant similarities with YWA1, chaetoglobosin A/chaetoglobosin C, squalestatin S1, and nidulanin A. Moreover, the whole-genome sequencing and in silico genomic analysis, allowed to further understand some aspects of this species habitat, resistance, and evolutionary genomic events. Altogether, the results obtained also allow to dwell deeper on particular Penicillia biological characteristics and genomic traits, permitting them to thrive in these honey substrates. In addition, this resource represents the first genome for the species and one of the first for raw honeys filamentous fungi.

Introducing Petrachlorosaceae fam. nov., Petrachloros gen. nov. and Petrachloros mirabilis sp. nov. (Synechococcales, Cyanobacteria) Isolated from a Portuguese UNESCO monument.

The Synechococcales is a large cyanobacterial order comprising both unicellular and filamentous forms, with parietal thylakoid arrangement. Previously, this order has been the subject of taxonomic revisions with new families being erected. During studies of the phototrophic communities on the limestone walls of the Old Cathedral of Coimbra (UNESCO monument), a coccoid Aphanocapsa-like cyanobacterium was isolated. It was characterized using a polyphasic approach, based on morphology, 16S rRNA phylogenetic and phylogenomic analyses, internal transcribed spacer (ITS) secondary structure, and ecology. The 16S rRNA phylogenetic analyses showed that this strain is placed in a separate and highly supported family-level clade, as part of a large group comprising the families Prochlorococcaceae and Prochlorotrichaceae, with Lagosinema as the closest (although quite distant) taxon. Additionally, the phylogenomic analysis also placed this strain in a separate lineage, situated distantly apart from the family Thermosynechococcaceae, but with strains assigned to Acaryochloris marina MBIC 11017 and Aphanocapsa montana BDHKU210001 as the closest taxa. Based on these data, as well as on the results from the secondary ITS structure, morphology, and ecology, we here propose the establishment of Petrachlorosaceae fam. nov., along with the description of Petrachloros gen. nov. and Petrachloros mirabilis sp. nov. We also address additional considerations regarding some cyanobacterial taxa within the order Synechococcales, which we believe deserve further revisions.

Phototrophic and fungal communities inhabiting the Roman cryptoporticus of the national museum Machado de Castro (UNESCO site, Coimbra, Portugal).

Caves are oligotrophic environments, characterized by constant temperatures, high humidity and low natural light. However, microbial shifts can still happen in such environments, especially with the increase in tourist activity and implementation of artificial lights, making caves even more susceptible to environmental changes. As a result, proliferation of phototrophic organisms can increase dramatically, leading to their settlement on stone surfaces, which in turn facilitates the development of heterotrophic organisms, such as fungi and bacteria. The Roman Cryptoporticus of the National Museum Machado de Castro, erected by the Romans in the 1st or second century, is one of the most emblematic buildings in the city of Coimbra. However, the majority of the rooms that constitute this monument show signs of biodeterioration by microalgae and cyanobacteria as well as of fungi. The aim of this study was to characterize the phototrophic and fungal communities at this site, employing culture-dependent and-independent methodologies. Culture-dependent results showed that the phototrophic communities were mainly composed of green microalgae, whereas the culture-independent showed that cyanobacteria were the most dominant. As to the fungal communities, both approaches identified various entomopathogenic fungal species. In addition, the culture-independent analysis also allowed to verify the presence of animal reads, suggesting the hypothesis that animal vectored dispersion can play an important role in the development of fungi at this environment.

Talaromyces saxoxalicus sp. nov., isolated from the limestone walls of the Old Cathedral of Coimbra, Portugal.

Fungi are one of the main agents of stone biodeterioration worldwide, since they strongly interfere with its integrity, aesthetical and structural natural properties. During an experimental survey aimed to isolate fungal species contributing to the biodeterioration of the limestone walls of the Old Cathedral of Coimbra (Portuguese unesco World Heritage site), a Talaromyces species that could not be identified to any currently known species in this genus was isolated. Molecular phylogenetic analysis of the internal transcribed spacer, ß-tubulin and RNA polymerase II subunit 2, placed this fungus in Talaromyces sect. Purpurei, while also pointing at its phylogenetic distinction from the remaining species in this section. Thus, a novel species, Talaromyces saxoxalicus sp. nov., is here proposed. Moreover, considering the isolation source of this fungus and in an attempt to understand its contribution to the overall stone monument biodeterioration, the species's in vitro biodeteriorative potential was also evaluated. The results highlighted that the species exhibited an in vitro biodeteriorative ability (calcium oxalate crystal formation), highlighting its potential deteriorative profile.

First Genome Sequence of the Microcolonial Black Fungus Saxispiralis lemnorum MUM 23.14: Insights into the Unique Genomic Traits of the Aeminiaceae Family.

Saxispiralis lemnorum MUM 23.14 is an extremotolerant microcolonial black fungus, originally isolated from a biodeteriorated limestone artwork in Portugal. This recently introduced species belongs to the Aeminiaceae family, representing the second member of this monophyletic clade. This fungus exhibits a unique set of characteristics, including xerophily, cold tolerance, high UV radiation tolerance, and an exceptional ability to thrive in NaCl concentrations of up to 30% while also enduring pH levels ranging from 5 to 11. To gain insights into its genomic traits associated with stress resistance mechanisms, specialization, and their potential implications in stone biodeterioration, we conducted a comprehensive genome sequencing and analysis. This draft genome not only marks the first for the Saxispiralis genus but also the second for the Aeminiaceae family. Furthermore, we performed two comparative genomic analyses: one focusing on the closest relative within the Aeminiaceae family, Aeminium ludgeri, and another encompassing the genome of different extremotolerant black fungi. In this study, we successfully achieved high genome completeness for S. lemnorum and confirmed its close phylogenetic relationship to A. ludgeri. Our findings revealed traits contributing to its extremophilic nature and provided insights into potential mechanisms contributing to stone biodeterioration. Many traits are common to both Aeminiaceae species and are shared with other black fungi, while numerous unique traits may be attributed to species-specific characteristics.

Exploring Differences in Culturable Fungal Diversity Using Standard Freezing Incubation-A Case Study in the Limestones of Lemos Pantheon (Portugal).

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.

Expanding the Microcolonial Black Fungi Aeminiaceae Family: Saxispiralis lemnorum gen. et sp. nov. (Mycosphaerellales), Isolated from Deteriorated Limestone in the Lemos Pantheon, Portugal.

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.

Circumfusicillium cavernae gen. et sp. nov. (Bionectriaceae, Hypocreales) Isolated from a Hypogean Roman Cryptoporticus.

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.

Bacterial and Archaeal Structural Diversity in Several Biodeterioration Patterns on the Limestone Walls of the Old Cathedral of Coimbra.

The "University of Coimbra-Alta and Sofia" area was awarded the UNESCO World Heritage Site distinction in 2013. The Old Cathedral of Coimbra, a 12th-century limestone monument located in this area, has been significantly impacted during the last 800 years by physical, chemical, and biological processes. This led to the significant deterioration of some of its structures and carvings, with loss of aesthetical, cultural, and historical values. For this work, deteriorated spots of the walls of three semi-open chapels from the cloister of the Cathedral were sampled to ascertain their bacterial and archaeal structural diversity. Based on Next-Generation Sequencing (NGS) result analysis, we report the presence of microbial populations that are well adapted to an ecosystem with harsh conditions and that can establish a diverse biofilm in most cases. While it was possible to determine dominant phylogenetic groups in Archaea and Bacteria domains, there was no clear connection between specific core microbiomes and the different deterioration patterns analyzed. The distribution of these archaeal and bacterial communities within the analyzed biodeterioration spots suggests they are more influenced by abiotic factors (i.e., water availability, salinity, etc.), although they influence (and are influenced by) the algal and fungal population composition in this ecosystem. This work provides valuable information that can assist in establishing future guidelines for the preservation and conservation of this kind of historic stone monuments.

High-Quality Draft Genome Sequences of Three Cyanobacteria Isolated from the Limestone Walls of the Old Cathedral of Coimbra, Portugal.

The recently described species Myxacorys almedinensis and two other cyanobacteria were isolated from the limestone walls of the Old Cathedral of Coimbra, Portugal (UNESCO World Heritage Site). The high-quality genome sequences presented here will be essential for characterization purposes and description of the novel taxa.

Description of Aeminiaceae fam. nov., Aeminium gen. nov. and Aeminiumludgeri sp. nov. (Capnodiales), isolated from a biodeteriorated art-piece in the Old Cathedral of Coimbra, Portugal.

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, Aeminiumludgeri sp. nov., in the order Capnodiales.

High-Quality Draft Genome Sequence of the Microcolonial Black Fungus Aeminium ludgeri DSM 106916.

Aeminium ludgeri is an extremotolerant microcolonial black fungus isolated from a biodeteriorated limestone art piece in the Old Cathedral of Coimbra, Portugal (a UNESCO World Heritage Site). The high-quality draft genome sequence of Aeminium ludgeri presented here represents the first sequenced genome for both the recently described fungal family Aeminiaceae and the genus Aeminium.