Research Article |
Corresponding author: João Trovão ( jtrovaosb@gmail.com ) Academic editor: Cecile Gueidan
© 2019 João Trovão, Igor Tiago, Fabiana Soares, Diana Sofia Paiva, Nuno Mesquita, Catarina Coelho, Lídia Catarino, Francisco Gil, António Portugal.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Trovão J, Tiago I, Soares F, Paiva DS, Mesquita N, Coelho C, Catarino L, Gil F, Portugal A (2019) Description of Aeminiaceae fam. nov., Aeminium gen. nov. and Aeminium ludgeri sp. nov. (Capnodiales), isolated from a biodeteriorated art-piece in the Old Cathedral of Coimbra, Portugal. MycoKeys 45: 57-73. https://doi.org/10.3897/mycokeys.45.31799
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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.
Biodeterioration, Capnodiales , microcolonial black fungi, phylogeny, taxonomy
Microcolonial black fungi (MCBF) are a remarkably diverse fungal group characterized by unique phenotypic features, such as strongly melanized cell walls, slow growth, ability to shift from a mycelial to a meristematic state, high morphological plasticity, and predominant asexual reproduction (
Classical morphological approaches used to identify MCBF are largely inefficient due to their extremely poor differentiation and, in some cases, to the existence of polymorphic traits (
In 2013, UNESCO recognized the University of Coimbra, Alta and Sofia (Coimbra, Portugal) as a World Heritage Site. Inside this area, several monuments exhibit clear signs of biodeterioration, including microcolonial black fungi proliferation. During one experimental survey performed in the Old Cathedral of Coimbra (Sé Velha de Coimbra), an unknown slow-growing microcolonial black fungi with late melanization was retrieved. Therefore, we aim to determine, through a multi-gene analysis (ITS rDNA, LSU and rpb2) coupled with a morphological, physiological and ecological examination, the taxonomic status and position of this fungus in the order Capnodiales.
The Old Cathedral of Coimbra is the only Portuguese Romanesque cathedral from the Reconquista times to have survived relatively intact until now. The Romanesque church is located on a hillside in the historic city center and was constructed between the 12th and early 13th centuries. The single-floored cloister is arranged laterally to the south of the church and is surrounded by five chapels carved in yellow dolomitic limestone. Samples were collected using sterile scalpels by scrapping small areas (3 cm2) into a collection tube, from a deteriorated art-piece in the Santa Maria chapel (40°12'32"N, 8°25'38"W) (Suppl. material
DNA from pure fungal cultures was obtained using the Extract-N-Amp Plant PCR Kit (Sigma-Aldrich, USA) with several modifications. A small portion of the colonies was scraped from the agar surface using a sterile scalpel, submerged in 10 µl of extraction solution and incubated in an ABI GeneAmp 9700 PCR System (Applied Biosystems, USA), with the following protocol: 65 °C for 10 min, followed by 95 °C for 15 min. After the incubation, reactions were stopped by adding 10 µl of elution solution. The obtained genomic DNA was subjected to PCR amplification with a final volume of 25 µl, with 12.5 µl of NZYTaq Green Master Mix (NZYTech, Portugal), 1 µl of each primer (10 mM), 9.5 µl of ultra-pure water and 1 µl of template DNA. Primer pairs ITS1-F/ITS4 (
DNA sequences were assembled using the Geneious R11.0.02 software (https://www.geneious.com), deposited in GenBank and compared with sequences from the National Center of Biotechnology Information nucleotide databases using NCBIs Basic Local Alignment Search Tool (BLAST), with the option Standard nucleotide BLAST of BLASTN v. 2.6 (
To examine heat resistance, mycelia from grown cultures on PDA were homogenized and heated at 75 °C for 30 min, in a shaking water bath. A small aliquot of the heated suspension was plated on fresh PDA culture medium and examined periodically to evaluate fungal growth (according to
For morphological characterization, strains were cultivated on PDA (Difco, USA), Malt Extract Agar (MEA), (Difco, USA) with 10% NaCl (w/v) and Dichloran Glycerol Agar (DG-18), (Sigma-Aldrich, USA) for up to 6 months. Morphological analysis was performed directly on the cultured media plates or using the slide culture technique. Preparations were transferred into slides, observed with a light microscope (Leica DM 4000B (Leica, Germany)), and photographed (Leica DFC 490 digital camera (Leica, Germany)). At least 30 measurements per structure were considered. Representative drawings of microscopic morphological characteristics were obtained with Adobe Illustrator CC (Adobe, USA).
The phylogenetic analysis was performed using the aligned sequences of the concatenated three-gene dataset with 1301 characters (627 for LSU, 204 for rpb2 and 470 for ITS), encompassing 133 representative sequences belonging to the different families of the order Capnodiales (Fig.
Bayesian 50% majority rule consensus tree based on an LSU/rpb2/ITS concatenated alignment, containing representative sequences from the order Capnodiales. The new strains are shown in bold. Bayesian posterior probabilities (BP) ≥ 0.95 are presented at the nodes. The tree was rooted to Parastagonospora nodorum CBS 110109. The scale bar specifies 0.2 expected changes per site.
Preliminary physiological analysis comprised all the isolates obtained in this study (data not shown). However, as no significant statistical difference was observed among the isolates under the different tested conditions, the final analysis consisted only of data regarding a copy of the culture DSM 106916. No growth was observed for the fungus after exposure to the heat tolerance protocol and therefore, it was classified as non-heat tolerant and non-heat activated. Results for NaCl tolerance test are shown in Suppl. material
Asexual morph: mycelium consisting of septate, smooth hyphae, gradually becoming widen, thick-walled, darker and developing into meristematic chains of conidia. Conidia dark brown, thick-walled, smooth, rugose, globose with single central septa resulting from the differentiation of toruloid-like hyphal cells. Sexual morph: unknown.
Aeminium J. Trovão, I. Tiago & A. Portugal.
Aeminium ludgeri J. Trovão, I. Tiago & A. Portugal.
Members of Aeminiaceae encompass microcolonial black fungi occurring in deteriorated limestones and are classified as halotolerant, xerophilic, and facultative alkaliphiles. They exhibit slow growth and late melanization, derived from the late differentiation of intercalary or terminal hyphal cells into arthroconidia, that turn olivaceous brown to dark. Fully maturation of the arthroconidia occurs after at least a 2-month incubation period. Their geographical distribution seems to be confined, for now, to limestones in the Iberian Peninsula, although further sampling is necessary to fully highlight their complete geographical and ecological spectrum.
Asexual morph: mycelium consisting of septate, smooth hyphae, gradually becoming widen, thick-walled, darker and developing into meristematic chains of conidia. Conidia dark brown, thick-walled, smooth, rugose, globose with single central septa resulting from the differentiation of toruloid-like hyphal cells. Chlamydospores not observed in culture. Sexual morph: unknown.
Named after the old Latin name of Coimbra (Aeminium), the city where the strains were isolated.
Aeminium ludgeri J. Trovão, I. Tiago & A. Portugal.
Portugal, Coimbra (40°12'32"N, 8°25'38"W), isolated from a biodeteriorated limestone art piece in the Old Cathedral of Coimbra, 22 November 2016, Igor Tiago, (holotype: permanently preserved in metabolically inactive state DSM 106916).
In memory of our late colleague Ludgero Avelar.
Phylogenetic analysis based on the concatenated ITS rDNA, LSU and rpb2 dataset considered in the present study clustered the retrieved strains in a monophyletic separate lineage related to the families Neodevriesiaceae and Extremaceae. Therefore, a new family Aeminiaceae fam. nov, a new genus Aeminium gen. nov., and a new species Aeminium ludgeri sp. nov. in the order Capnodiales are here proposed.
Mycelium initially consisting of branched, septate, smooth, subhyaline to pale green, 2–3 μm wide hyphae. Hyphae moniliform, gradually becoming widen, thick-walled, darker and developing into meristematic conidial chains. Conidiophores micronematous. Arthroconidia dark brown, thick-walled, smooth, sometimes rugose, globose, measuring 3.5–6 × 4.5–6 μm, single central septa, resulting from the differentiation of intercalary or terminal toruloid-like hyphal cells. Sexual morph unknown.
Aeminium ludgeri a Colony appearance on PDA b Colony appearance on MEA+10% NaCl (w/v) c Colony appearance on DG-18 d Colony appearance on PDA after maturation e Colony appearance on MEA+10% NaCl (w/v) after maturation f Colony appearance on DG-18 after maturation g Initial simple, branched, septate hyphae becoming toruloid-like (scale 20 μm) h Differentiated, toruloid-like hyphae and mature chains of arthroconidia (scale 50 μm) i Intercalary and terminal conidial chains (scale 20 μm); j typical aspect of arthroconidia (scale 20 μm)
On 6 weeks old PDA plates, colonies growing slowly, to 8 mm in diameter, cerebriform, irregular, raised centrally, often moist, deeply immersed into agar, pale pink, with scarce velvety, pale pink-hyaline, aerial, short hyphae and well-defined, small, white, and glossy margin; pale pink on reverse. After at least 2 months, colonies become fully mature and melanized, olivaceous brown-black on top and on reverse. On 6 weeks old MEA+10% NaCl plates, colonies growing slowly, to 25 mm in diameter, flat, circular, moist, pale pink, with prominent velvety pale pink-hyaline aerial short hyphae and with undulate margin; pale pink in reverse. After at least 2 months, colonies become fully mature and melanized, with velvety grey-white aerial short hyphae, filiform margin, olivaceous brown on top and in reverse. On 6-weeks-old DG-18 plates, colonies growing slowly, to 20 mm in diameter, flat, irregular, often moist, deeply immersed into agar, with velvety yellow-pale brown aerial short hyphae and with irregular undulate white margin; yellow-pale brown in reverse. After at least 2 months, colonies become fully mature and melanized, raised, rugose, olivaceous brown-black on top and in reverse.
Portugal.
Portugal, Coimbra (40°12'32"N, 8°25'38"W), isolated from a biodeteriorated limestone art piece in the Old Cathedral of Coimbra, I. Tiago, living culture E8, ibid. living culture E11, ibid. living culture E12, ibid. living culture E13, ibid. living culture E14, ibid. living culture E15, ibid. living culture E16, ibid. living culture E17, ibid. living culture E18; ibid. living culture E19.
Here we describe Aeminium ludgeri, a new MCBF species, as well as establish a new genus and family within the order Capnodiales to accommodate this fungus. Phylogenetic analyses, based on ITS rDNA, LSU and rpb2 molecular data, revealed that the retrieved isolates cluster in a separate lineage strongly supported at a family-level, related to the families Extremaceae and Neodevriesiaceae. Although some molecular variance can be observed among the studied isolates, when dealing with MCBF, larger degrees of sequence heterogeneity for species delimitation are accepted due to the lack of sexual recombination, predominance of clonality, and perpetuation of super-adapted genotypes (
The clustering of the environmental sequences obtained by
The limestones used in the construction of the Old Cathedral of Coimbra hail from unique areas of Portugal (namely Ançã and Portunhos, near Coimbra), and similar stone structures were exported and used on several “Our Ladies of the O” statues and in the portal of the Royal Hospital in Santiago de Compostela (Spain). We hypothesize that the transportation of limestone to such places might have contributed to the dispersion of this organism and, thus, explain the detection of Aeminiaceae environmental clones in the Cathedral of Santiago de Compostela by
Physiological tests allowed us to characterize the retrieved isolates as non-heat tolerant, non-heat activated, xerophilic, halotolerant (enduring NaCl concentrations up to 20%), and facultative alkaliphiles. Ecologically, these traits are somewhat more similar to those of Extremaceae (except for pH values, as Aeminiaceae is an alkaliphile and Extremaceae is an acidophile). Regarding heat-tolerance, the isolates are somewhat more similar to Neodevriesiaceae because they do not exhibit heat tolerance. To the best of our knowledge, data regarding heat tolerance for Extremaceae is scarce and could not be further compared. Furthermore, information regarding NaCl tolerance is still poorly described for both Extremaceae and Neodevriesiaceae, but future studies related to stress tolerance in these organisms could provide a valuable character in strain typification.
Although MCBF morphology-based distinction is particularly difficult to accomplish, it can be easily verified that Aeminiaceae differs from the closely related Extremaceae and Neodevriesiaceae due to the necessary period for conidia maturation. Additionally, the verified globose arthroconidia, with single central septa, are distinct from the “subcylindrical to narrowly fusoid-ellipsoidal or obclavate conidia with rarely 1–2 transverse septa” described for Extremaceae and the “rarely septate, solitary conidia composed of a central stalk and two lateral arms with 1–2 transverse septa”, when compared to Neodevriesiaceae (
Regarding stone monuments exposed to the environment, microcolonial black fungi are one of the main culprits of stone biodeterioration and are responsible for severe aesthetic, biochemical, and biophysical alterations (
We are grateful to the Direcção Regional de Cultura do Centro (DRCC), the staff and technicians from the Old Cathedral of Coimbra (Sé Velha) and the University of Coimbra for their kind collaborations. We also thank Miguel Mesquita, for kindly providing the photographs of the sampling site. This work was financed by FEDER- Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020- Operational Programme for Competitiveness and internationalization (POCI) and by Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT) in the framework of the project POCI-01-0145-FEDER-PTDC/EPH-PAT/3345/2014. João Trovão was supported by Programa Operacional Capital Humano (POCH; co-funding by the European Social Fund and national funding by MCTES), through a “FCT- Fundação para a Ciência e Tecnologia” PhD research grant (SFRH/BD/132523/2017). Fabiana Soares was supported by POCH (co-funding by the European Social Fund and national funding by MCTES), through a “FCT- Fundação para a Ciência e Tecnologia” PhD research grant (SFRH/BD/139720/2018). Nuno Mesquita was supported by POCH (co-funding by the European Social Fund and national funding by MCTES), with a Post-Doc Research grant (SFRH/BPD/112830/2015). Catarina Coelho was supported by Portuguese funds through “FCT – Fundação para a Ciência e a Tecnologia” in project IN0756 - INV.EXPLORATORIA - IF/01061/2014. Igor Tiago acknowledges an Investigator contract reference IF/01061/2014.
Figure S1. Sampling site
Data type: species data
Explanation note: a) Cloister of the Old Cathedral of Coimbra; b) lateral view of the Santa Maria Chapel; c) Particular art-piece from where the studied fungi were retrieved (photos by Miguel Mesquita)
Table S1. Fungal strains used in the phylogenetic analysis
Data type: phylogenetic data
Figure S2. Growth of Aeminium ludgeri incubated at different NaCl concentrations after 4 weeks
Data type: statistical data
Figure S3. Growth of Aeminium ludgeri incubated at different pHs levels after 4 weeks
Data type: statistical data
Figure S4. Representative drawing of Aeminium ludgeri
Data type: species data
Explanation note: a) initial simple, branched, septate hyphae becoming toruloid-like and strongly melanized; b) differentiated, toruloid-like hyphae with chains of arthroconidia; c) and d) arthroconidia (scale 10 μm).