Antonie van Leeuwenhoek (2007) 91:217–227 DOI 10.1007/s10482-006-9111-9

ORIGINAL PAPER

Microcolonial fungi from antique marbles in Perge/Side/Termessos (Antalya/Turkey) Hacer (Bakır) Sert Æ Hu¨seyin Su¨mbu¨l Æ Katja Sterflinger

Received: 11 April 2006 / Accepted: 2 August 2006 / Published online: 2 November 2006
Springer Science+Business Media B.V. 2006

Abstract In this study rock surfaces of archaeological sites in Antalya were investigated with a focus on black fungi for the first time. Black, meristematic fungi were isolated from surfaces of antique marble monuments in Antalya (Side, Perge, Termessos). Their morphology was characterized, their diversity was documented and the taxonomy and phylogeny of new isolates was clarified based on molecular methods, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer (ITS) regions. From a total of around 250 samples 99 different fungal strains were isolated. In most of 99 strains the rDNA sequencing data and the lack of homologies in ‘‘Genbank’’ gave strong evidence that these strains have to be described as new species/subspecies and/or genera. All of them, however, clustered within the ascomycete orders of Dothideales, Chaetothyriales, and Pleosporales. Field studies show that these organisms cause color changes, black spots, crater shaped

H. (Bakır) Sert Æ K. Sterflinger Austrian Center of Biological Resources and Applied Mycology (ACBR), University of Natural Resources and Applied Life Sciences, Muthgasse 18, A 1190 Vienna, Austria H. Su¨mbu¨l Æ H. (Bakır) Sert (&) Faculty of Arts and Sciences, Department of Biology, Akdeniz University, TR-07058 Antalya, Turkey e-mail: [email protected]

lesions as well as chipping and exfoliation of the rock surfaces in Antalya and thus they have to be regarded as a serious threat to the antique cultural heritage in Turkey. Keywords Biopitting Æ Bio-deterioration Æ Historical monuments Æ Molecular taxonomy Æ Rock-inhabiting fungi

Introduction Fungi are noteworthy members in many terrestrial and aquatic ecosystems and contribute significantly to their biodiversity. About 75,000 species of fungi have been described, but it is estimated that 1–1.5 million species exist (Hawksworth et al. 1995). These interact in various ways with their substrates, with their hosts, with their competitors, and with abiotic variables of their environment. They exist in almost every conceivable habitat where organic carbon is available: in fresh water and the sea, in soil, litter, in dung, in living plants and decaying remains of plants and animals (Dix and Webster 1995), in marble and other calcareous rock types in nature and on monuments (Sterflinger and Krumbein 1997; Wollenzien et al. 1995; Turian 1977; Staley et al. 1982; Taylor-George et al. 1983). Many new fungal species exist on Mediterranean rock, as clearly shown in earlier publications e.g.,

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Sterflinger et al. (1997), Wollenzien et al. (1997), de Leo et al. (1999), Ruibal et al. (2005) and Bills et al. (2004). In terms of geomicrobiology fungi play an important role in the alteration and weathering of rock. A recent research (Decrouez et al. 1992) has evidenced that especially a very slow-growing group of black fungi and yeast-like fungi plays an important role in the deterioration of marbles and other rocks (Urzi and Krumbein 1994). Black fungi are colonizers of bare rock surfaces in hot deserts (Staley et al. 1982) and in semi-arid climatic regions such as the Mediterranean (Gorbushina et al. 1994) but are also part of the cryptoendolithic community of the Antarctica (Selbmann et al. 2004). All those rock inhabiting fungi have something in common in that they form cauliflower-like microcolonies on and in rock, they have very thick, multilayered cell walls and are incrusted with melanins giving them a dark, blackish brown appearance (Fig. 1, 2). Generally, meristematic morphology is interpreted as a response to multiple stress factors (oligotrophic nutrient conditions, elevated temperatures, UV-radiation, osmotic stress) supporting temperature tolerance and decreasing the rate and speed of desiccation by keeping the volume–surface ratio optimal (Wollenzien et al. 1995). The combined influence of these stress factors exerts a high selective pressure on the microbial community and as a consequence black yeast and meristematic fungi are rarely found in complex microbial populations but solitary or in spatial association with

Fig. 1 Part of a column (from the ruins of Perge (near the theater)

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Fig. 2 Macrophotograph of microcolonies on marble surface

comparably stress resistant organisms such as lichens and cyanobacteria in very special habitats (Sterflinger 2005). Fungi were first considered to be agents of carbonate deterioration by Krumbein (1969). The mechanical action of fungal growth affects building materials such as brick and concrete (Gravesen et al. 1994) and marble, limestone and sandstone (Sterflinger and Krumbein 1997). Their deteriorating effect is due to mechanical and chemical actions as penetration of materials with deep-reaching deteriorating effects, such as swelling and deflation as physical effects, constant microvibrations through micromotility and acid production (Urzi and Krumbein 1994). Fungi also play a major role in the color change of rock surfaces. Sterflinger et al. (1998) demonstrated that there is a direct correlation between orange pigmentation (patination) of granite and sandstone and rock inhabiting fungi. In relatively few cases the epilithic fungal communities on marble, limestone, sandstone and other rock surfaces were completely inventoried. Still relatively few species have been described and characterized phylogenetically. The aim of this study was to document the biodiversity of microcolonial fungi (MCF) on historical marble monuments using molecular data and to clarify their deterioration-potential. The three ancient cities Perge, Side and Termessos (Antalya/Turkey) in Mediterranean area were chosen, due to their outstanding historic and artistic value and the large variety of marble monuments.

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Material and methods Studied areas The marble samples for this study were collected from three different ancient cities in Antalya, located in the southern Anatolia region of Turkey: (1) Perge is located 18 km east of Antalya between the Du¨den and Aksu streams. This is reached along the Antalya–Alanya highway, turning north at Aksu, going 2 km further on. It lived through the Hellenistic, the Roman and the Byzantine epochs; (2) Side is reached by turning south 3 km before Manavgat on the Antalya– Alanya highway. From inscriptions it appears that Side dates back to the Hittite Period. The city was constructed on a peninsula and was a Hellenistic and Roman town, protected by city and seawalls; (3) The Termessos National Park is 30 km along the Antalya–Korkuteli highway. Termessos is perhaps the most interesting ancient city in Antalya region. It is a Psidian city built at a height of 1050 m in the Taurus Mountains. Termessos constitutes an unusual synthesis of a large number of rare plants and animal species, which are under protection in the Termessos National Park. Sample collection A total of 250 marble samples was taken from antique monuments with the help of chisel, hammer and scalpel (previously cleaned 70% ethyl alcohol), transferred to paper bags and stored at room temperature. Samples were processed for isolation within 1 week. Samples were collected in January, April, August, and in October between 2003 and 2005. The isolated strains were conserved in the ACBR (Austrian Center of Biological Resources and Applied Mycology, http://www.biotec.boku.ac.at/acbr.html) culture collection for further investigation. The origin of the isolation of the strains is available from the culture collection website. Isolation and morphological characterization The fungi were isolated from the samples at the stereo microscope by carefully picking fungal colonies using needles (according to Sterflinger

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1995). The black colonies were transferred to petri dishes and two different media were used: (1) 2% malt extract agar (MERCK, Darmstadt, Germany), (2) dichloran rose bengal agar (growth inhibiting medium to limit growth of contaminants; MERCK, Darmstadt, Germany). The plates were incubated at room temperature. The isolates were purified in two or three steps by transfer to fresh medium (malt extract agar and czapek agar; MERCK, Darmstadt, Germany); purity was checked and maintained light microscopically. For morphological characterization mounts of the mycelium were prepared and observed light microscopically. Molecular characterization Fungi were grown on 2% malt extract agar. About 1 cm2 mycelium, which was scraped from the plates with a scalpel, was transferred into 1.5 ml Eppendorf tubes containing 500 ll lysing buffer and 200 ll glass beads (425–600 mm, MERCK, Darmstadt, Germany). The mixture was shaken in cell disrupter (Thermo Savant FastPrep, FP120, Holbrook, USA) at full speed for 3 min. After incubation for 1 h at 65C the mixture was shaken again at full speed for 3 min and then centrifuged for 15 min at 14,000 rpm. The supernatant was transferred to a new eppendorf tube. About 2.5 ll RNAse was added, and the mixture was incubated for 1.5 h at room temperature. The DNA was prepared as a 1:150 dilution to be used in the PCR reactions. DNA was determined on 1% agarose gels with TAE buffer (40 mM Tris–acetate, 1 mM EDTA, pH 8.0) and visualized with ethidium bromide stain (0.5 mg/ml). For sequencing 18S and ITS I/5.8S/ITS II PCR was performed in 50 ll volumes containing 12.6 ll of template DNA, 30.57 ll distilled water, 5.5 ll buffer (1.1 ll MgSO4, 2.75 ll dNTP), 0.35 ll of each primer and 0.44 ll Taq DNA Polymerase (Genekraft, Mu¨nster, Germany). Primers NSO (TAT CTG GTT GAT CCT GCC) and ITS4 (TCC TCC GCT TAT TGA TAT GC) were used. The mixture was amplified by 40 cycles in a MJ Research PTC 200 thermocycler, as follows: denaturation (94C, 30¢¢), annealing (55C, 30¢¢), extension (72C, 1¢), final extension

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(72C, 5¢). Amplicons were electrophoresed in 1.2% agarose gels (Cambrex Bio Science SeaKem LE Agarose) contanining ethidium bromide for 0.5 h at 120 V in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3) and photographed for further analyses. The PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen, Hilden Germany). The cycle sequencing reactions were performed in 20 ll volumes containing 10 ll distilled water, 6 ll buffer (Big Dye Buffer), 2 ll Big Dye RR Mix (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), 1 ll PCR product and 1 ll primer. Amplification was done in a MJ Research PTC 200 thermocycler as follows: (96 C, 30¢¢), (50C, 15¢¢), (60C, 4¢) (25 cycles). The products were purified using an Amersham Bioscience AutoSeqTM G-50 Purification Kit (Amersham Biosciences, Freiburg, Germany). The Sequencing was performed on an ABI Prism automatic sequencer (Applied Biosystems, CA, USA). Sequence assembly was done using the Seqman program (Dnastar Inc., Madison, USA). The sequences were then compared with the database of the National Centre for Biotechnology Information, Bethesda, Md. (BLAST-search, http:// www.ncbi.nlm.nih.gov/BLAST/). The resultant sequences were aligned using the Megalign package (DNAStar). The tree was constructed with the TREECON software package (Van de Peer and de Wachter 1994) using the neighborJoining method (Saitou and Nei 1987) based on a dissimilarity matrix corrected for multiple mutations at any given site in the molecule (Jukes and Cantor 1969). A total of 100 bootstrap replicates were used for analysis. Sequences were deposited in the EMBL databank (Cambridge, http:// www.ebi.ac.be) for small ribosomal subunit RNA.

Results and discussion Black meristematic fungi intensively inhabit monument surfaces in Antalya. From every monument observed, it becomes quite clear that the alterations and material losses are progressive. Moreover, the growth of the fungi is not restricted to the surface as fungal colonies are found in the depth of the samples.

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Out of a total of around 250 samples 99 fungal strains were isolated, and could be assigned to 11 different genera. It was extremely difficult to describe in classical morphological terms of isolates because of the scarcity of diagnostic features such as colony morphology and conidiogenesis. Therefore, fungi were characterized by sequencing SSU and ITS I, 5.8S and ITS II regions of the rDNA. All isolates clustered within the ascomycete orders of Dothideales, Chaetothyriales, and Pleosporales. Consensus tree of 5.8S/ITS I and II rDNA of Chaetothyriales, and Pleosporales are shown in Fig. 3a, b. The genus Coniosporium First Coniosporium species were found on rotten wood. Link’s (1809) original specimen of Coniosporium olivaceum Link:Fr was isolated from Pinus maritima. Later, Coniosporium apollinis, Coniosporium perforans (from Greece), and Coniosporium uncinatum (from Italy) were isolated from marble monuments. In addition to aesthetical spoiling of monuments, C. perforans is assumed to have a very high mechanical destruction potential towards rock (Sterflinger and Krumbein 1997). In this study the most frequently isolated genus was Coniosporium with 25 different isolates. Only one of the strains (MA 4956) had a 100% ITSI+II, 5.8S homology with C. perforans and thus could be clearly assigned to this species. The others could not be clearly identified at the species level because of their lack of homology with Coniosporium species in Genbank. However, all strains are morphologically highly similar and the ITSI region is known to be highly variable in black yeasts and meristematic fungi. For this reason most strains found were clustered in the group of species C. perforans or C. apollinis, most probably representing subspecies variations of these taxa. The strains MA 4597 and MA 4608 show close similarity to a melanized ascomycete that was isolated by Ruibal et al. from limestone in Mallorca (strain TRN131 according to ITS I/5.8S/ITS II sequences Ruibal et al. in 2005, data available on http:// www.ncbi.nlm.nih.gov). Based on SSU phylogeny data MA 4597 has 99% similarity to Coniosporium sp. (CBS 665.80, EMBL No: Y11712) (Fig. 4).

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While the sequencing of the SSU is suitable for placement into the right classes and genera, ITS I is said to be a taxonomic tool for identification of species (Sterflinger 2005; de Hoog et al. 1999). However, the results of this study show that the

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ITS I region alone has a variability indicating subspecies variations within the Coniosporium species. The 5.8S and part of ITS II region can close a gap between the species level (ITS I) and higher taxonomic levels although the 5.8S gene

Fig. 3 Consensus tree of 5.8S/ITS I and II rDNA of: (a) Chaetothyriales (L. arundinis was used as outgroup); (b) Pleosporales (C. perforans was used as outgroup.) (sequences published for the first time are printed in bold letters)

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Fig. 3 continued

sometimes indicates a higher heterogeneity than the ITS I region (de Hoog et al. 1999).

MA 4760 (selected strain from TRN 74 cluster) is similar to S. petricola (Fig. 4).

The genus Sarcinomyces

The genus Capnobotryella

Sarcinomyces species described to date were found on several substrates. Sarcinomyces crustaceus was isolated from wood, Sarcinomyces phaeomuriformis (new comb.: Exophiala phaeomuriformis) was isolated from an infected tissue. Sarcinomyces petricola developing on antique marble monuments is similar to our Sarcinomyces species. S. petricola was originally found in Mediterranean environments (Greece, Italy, Wollenzien et al. 1997), but also in Vienna (Sterflinger and Prillinger 2001). In this study 15 different strains of Sarcinomyces were isolated. Six strains are clustered according to their ITS I/ 5.8S/ITS II rDNA sequences as S. petricola. Nine strains have close similarity to the strain TRN 74 from Mallorca based on ITS I/5.8S/ITS II sequences, whereas according to SSU sequences

Hitherto only one species has been described within the genus Capnobotryella. Sugiyama and Amano (1987) found Capnobotryella renispora J. Sugiyama invariably in association with Capnobotrys neesii Hughes on Abies veitchii branches is Japan. Later it was found on roof tiles between the thalli of Lichenotelia cf. convexa Henssen (Titze and de Hoog 1990). Members of the genus Capnobotryella appeared 17 times in this study. Most of the ITS I/5.8S/ITS II sequences of Capnobotryella strains isolated from Antalya show close similarity to the strains from calcareous rock in Mallorca. Judging from SSU phylogeny data all these strains have close similarity to C. renispora (Fig. 4). Only strains MA 4565 and MA 4642 corresponds with C. renispora (97%, 93%) (ITS I/5.8S/ITS II). The strain MA 4674

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Fig. 4 The SSU rDNA tree showing representatives of the genera Sarcinomyces, Capnobotyrella and related taxa (sequences published for the first time are printed in bold letters)

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shows 98% similarity with C. apollinis based on ITS I/5.8S/ITS II sequences, whereas according to partial 18S sequence it is close to C. renispora. The genus Phaeococcomyces The three widely accepted species of the genus Phaeococcomyces are Phaeococcomyces nigricans (M.A. Rich & A.M. Stern) de Hoog, Phaeococcomyces catenatus de Hoog & Herm.-Nijh., and Phaeococcomyces chersonesos Bogolomova & Minter. Phaeococcomyces exophialae (de Hoog) de Hoog was combined as Exophiala exophialae (de Hoog et al. 2003). Two different strains were isolated from Antalya. The strain MA 4712 is genetically (based on ITS I/5.8S/ITS II sequences) close to P. catenatus, whereas MA 4938 was closely related to P. chersonesos isolated from marble in Chersonesus (Crimea, Ukraine) (Bogolomova and Minter 2003). The genus Rhinocladiella The genus Rhinocladiella is naturally found in soil and on woody plant material. Moreover it is a principal agent of chromoblastomycosis, a rare infection of the skin. In this study Rhinocladiella strains were isolated from rock for the first time. The ITS I/5.8S/ITS II sequences of the strain MA 4765 did not correspond with any sequence from GenBank. However, the 18S sequence data shows similarity 96% with Rhinocladiella atrovirens (Fig. 4). The strain MA 4653 has 12% difference from R. aquaspersa according to ITS I/5.8S/ITS II sequences.

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Unidentified fungi Of the total sample, 27 strains did not correspond with any sequence deposited in public databases, suggesting they could represent undescribed genera and species. On the other hand many socalled meristematic or ‘‘dematiaceous’’ genera as Trimmatostroma, Taeniolella, Bispora, Intralichen (Ellis 1971; Hawksworth 1979) and others are not well defined and cannot be found in Genbank because of a complete lack of DNA-sequence data. Based on morphological investigation of our isolates it is likely that many relations to existing genera could be found. However, much molecular work is still outstanding on the genera described above. According to the literature, the number of new fungal taxa, especially found in dry and Mediterranean climates, is notably high. It is also the same in our study which has an abundance and variety of isolates. Figure 5 shows hyphal formations for 1-month-old culture of a Capnobotryella species, which could not to be clearly identified. The sequencing data obtained in our study show that there is an extraordinary high diversity in the rock inhabiting genera Coniosporium, Sarcinomyces and Capnobotryella, albeit on the subspecies level. Similar to the humanassociated genus Exophiala that was described as an evolutionary hot-spot with Chaetotyriales (de Hoog et al. 1999), it seems that also that rock inhabiting black fungi and black yeasts are strongly evolving with respect to their genetical diversity. Some MCF lost their ability to form

Lichenicolous fungi Additionaly, lichenicolous fungi belonging to genus Mycocalicium were isolated in this study. Lichenicolous fungi form colonies that are similar or alike to MCF but, in close spatial association with lichen thalli. Because the rock samples from Antalya were also inhabited by epi- and endolitic lichens, lichenicolous fungi are readily isolated together with free MCF. The strain MA 4790 shows high similarity to Mycocalicium victoriae. Morphologically the in-situ growth of lichenicolous fungi is alike to free MCF.

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Fig. 5 Hyphal formation in 1-month-old culture of Capnobotryella sp. (MA 4902) ( · 3000)

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hyphal mycelia completely whereas others still exhibit hyphal growth under laboratory conditions albeit being completely meristematic in nature. Some genera—as Cladosporium—form mycelia but, in few cases the genus was reported to grow microcolonial and yeast-like. These observations, together with the fact that microcolonial growth develops in at least three orders of ascomycetes suggest that MCF developed from originally mycelial ancestors coming from more moderate natural niches. Once adapted morphologically to grow in extreme environments high mutation rates due to extreme UV-radiation and other hostile environmental factors cause a rapid genetical diversification of the fungi sometimes combined with the loss of the ability to switch back to mycelial growth. The genera Lophiostoma, Massarina and Monodyctis Three Pleosporaceous genera, of which growth is not microcolonial, have been described based on ITS I/5.8S/ITS II sequences, the genera Lophiostoma, Massarina and Monodyctis. Hitherto only one meristematically species has been described from Pleosporales, Botryomyces caespitosus. Members of the genus Lophiostoma appeared to be further examples. The strain MA 4558 has 96% similarity with Lophiostoma arundinis. MA 4611 did correspond with Massarina papulosa (98%), and the strain MA 4784 is 99% similar to Massarina rubi. One strain was isolated from the genus Monodyctis. The strain MA 4647 shows 91% sequence similarity (ITS I/5.8S/ITS II) to Monodyctis castaneae. The genus Cladosporium Cladosporium species are ubiquitous and can readily be isolated from air and solid substrata (Sterflinger and Prillinger 2001). More than 500 species have been described, although most of them should probably be reduced to synonymy (Takeo et al. 1995). There is evidence that the organism plays little part in bio-deterioration processes (de la Torre et al. 1993). In this study four strains of Cladosporium were identified at the genus level. All exhibited microcolonial growth

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in situ, but changed into mycelial growth when exposed to laboratory conditions. According to their ITS I/5.8S/ITS II sequences the strains are closely related to Cladosporium sphaerospermum, Cladosporium cladosporioides and Cladosporium elatum. The genus Phoma Phoma species have frequently been isolated from rock surfaces (Anagnostidis et al. 1992). Three species of Phoma, of which growth is not microcolonial, were isolated from rock; Phoma exigua var. foveata (Foister) Boerema, Phoma glomerata (Corda) Wollenweber & Hochapfel and Phoma macrostoma Montagne. In our study area two epilithic species (MA 4621 and MA 4794) of this genus were found, and they have close similarity to Phoma herbarum (according to ITS I/5.8S/ITS II and partial 18S sequences).

Conclusion In this study fungal strains were isolated from rock samples, which were collected from three different ancient cities in Antalya (Perge, Side and Termessos). The biodiversity of the fungi in Side was higher than in Perge and Termessos. Sterflinger and Prillinger (2001) explained that this difference could be attributed to the elevated organic pollution in the city. Coniosporium was the most frequently isolated genus in the study, and it developed on nearly all monuments. After Coniosporium, the genera Capnobotryella and Sarcinomyces were abundant. The black spots on marble monuments have been clearly determined as fungal colonies by transferring single spots to suitable media where they generated mycelia within 2–8 weeks. The observations of the rock surfaces clearly demonstrate that there is a positive correlation between the fungi and the alteration of the rock surfaces. In this study, a sugaring, crumbling or pitted surface has never been found free of black fungal colonies and the fungi have never been found on rock surfaces that do not show any granular disintegration or pitting. The sizes of the pits observed in Perge, Side, and Termessos biopitting

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were in a size of 0.2–2 cm in diameter. They can clearly be assigned to the destructive activity of MCF (Fig. 2). Sterflinger and Krumbein (1997) clearly demonstrated that the fungi are penetrating the rock actively without initiative work of other organisms. During recent years, when it has become more and more obvious that the activity of fungi as rock invaders was underestimated for a long time, the discussion on destruction mechanisms was raised again and it was stated that MCF with yeast-like growth patterns may exert mechanical destructive forces on marbles (Krumbein and Urzi 1993; Urzi et al. 1993). The theory of mechanical attack is further supported by publications of Gorbushina et al. (1994), Wollenzien et al. (1995), Diakumaku et al. (1995), Sterflinger and Krumbein (1997) and Sterflinger (2000). In summary, what we have found in this study is that the alteration and deterioration in ancient cities Perge, Side, and Termessos have strong relationship with microcolonies of dematiaceous fungi, which go through the rock and generates colonies in and on it. In addition to this, it is clear that there is a need for more research on rockinhabiting fungi due to the high taxonomic diversity of dematiaceous fungi and novelty of the species in the area where this study took place. Acknowledgments We would like to thank Ministry of Culture/Turkey for allowing the sampling and field experiments on Perge, Side and Termessos, and Institution ¨ .K) for PhD-Scholarship for H.S. of High Education (Y.O This study was financed by Austrian Center of Biological Resources and Applied Mycology (ACBR) and Akdeniz University Scientific Research Projects Unit.

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