Success in the Isolation and Axenic Culture of Anaptychia Ciliaris (Physciaceae, Lecanoromycetes) Mycobiont

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Full paper Success in the isolation and axenic culture of Anaptychia ciliaris (Physciaceae, Lecanoromycetes) mycobiont

M. Carmen Molina a,*, Pradeep K. Divakar b, Natalia Gonzalez a a Departamento de Biologıá y Geologıá (Area de Biodiversidad y Conservacion), ESCET, Universidad Rey Juan Carlos, Mostoles, 28933 Madrid, Spain b Departamento de Biologıá Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain article info abstract

Article history: The aim of this study was to evaluate the optimum conditions for culturing Anaptychia Received 30 July 2014 ciliaris mycobionts in vitro and analyzing their ontogenetic development while in culture. Received in revised form After the testing of two germination and four development media, results of germination 29 October 2014 and growth were evaluated. We here recommend the use of basal medium without addi- Accepted 29 October 2014 tional carbon source for germination process. Mycobiont growth was possible in all organic Available online 23 February 2015 media tested. The maximum mycobiont growth was observed in Lilly and Barnet medium enriched with 3% glucose. Keywords: © 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Aposymbiotic cultures Development Lichen Morphogenetic differences Ontogeny

dyes for cotton and wood since the Middle Ages. Additionally, 1. Introduction lichens have frequently been used in the fragrance industry, in medical and pharmacology applications, as biomonitors A lichen is defined as an obligate symbiosis of a fungus (usu- and bioindicators of air quality, and as bioattenuation agents ally Ascomycota) and a unicellular or filamentous green alga (e.g., Luo et al. 2010; Aberoumand 2011; Favelo-Longo and (around 85% of all lichen species), a cyanobacterium (around Piervittori 2012; Wang et al. 2012; Kosanic et al. 2013). These 10%), or both (about 5%; Showman and Flenniken 2004). Li- are just a variety of examples of past, current, and potential chens are often poorly investigated in fields such as biotech- applications of these complex organisms. nology; however, lichens have traditionally been used by It is difficult to find out precisely how and where lichen humans (Muggia et al. 2009; Molnar and Farkas 2010). Chem- materials are collected and what species are collected for ical substances from lichens have been a source of important commercial purposes due to lack of documentation and public

* Corresponding author. Tel.: þ34 914887090; fax: þ34 916647490. E-mail address: [email protected] (M.C. Molina). http://dx.doi.org/10.1016/j.myc.2014.10.003 1340-3540/© 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. 352 mycoscience 56 (2015) 351e358

information, given that the collection of these materials is cultured in vitro (Crittenden et al. 1995), but little is known often illegal. It is estimated that several thousand tons of li- about its development in aposymbiotic culture or how to in- chens are processed every year worldwide (Joulain and crease biomass yield for more extensive studies. Tabacchi 2009a, b). Such extensive exploitation, as well as The primary aim of this study was to analyze the ontoge- deterioration or destruction of habitat and environmental air netic development of the A. ciliaris mycobiont from sporula- pollution are the main causes of major loss of lichen diversity tion to formation of aposymbiotic aggregates (mycobiont (Upreti et al. 2005) and could be a threat to lichen species like colonies) and to find optimal protocols to find favorable con- Anaptychia ciliaris. ditions for mycobiont development in vitro cultures. The In order to increase the production of lichen substances for attainment of this goal will allow us to obtain total DNA suf- commercial applications, provide a sustainable harvestable ficient for complete genome sequencing of this fungal source, and reduce as much as possible the impact on natural mycobiont. communities, several new research areas and production methods have been investigated: whole and aposymbiotic culture (Yamamoto et al. 1987a; Yoshimura et al. 1989; 2. Materials and methods Stocker-Worg€ otter€ 2007), in vitro biosynthesis (Stocker- Worg€ oter€ and Elix 2006), bioreactor systems (Blanco et al. 2.1. Lichen materials 2002), and the cloning and expression of PKS (polyketide synthase) gene, a key enzyme in the synthesis of phenols Three well-developed Anaptychia ciliaris thalli growing on (Gagunashvili et al. 2009). However, in most cases these Fagus sylvatica were collected at Hayedo de Montejo, Madrid, biotechnology techniques have not gone beyond the experi- Spain (N 41 040 6300 W03 290 6500, alt. 1290 m) on Oct 15th, 2010. mental stages, since problems such as no spore discharge or Apothecia density on thalli was calculated as number of germination, difficulties in the selection of suitable culture apothecia by cm2, using total surface of all thalli. medium, and very slow growth of mycobionts under aposymbiotic conditions have been frequent (Crittenden et al. 2.2. Isolation and culture 1995; McDonald et al. 2013). Contaminations have also been particularly problematic if they are from endolichenic fungi. Twenty apothecia from one thallus (MAF-Lich 17758) were Different composition of mycobiont substances in culture and selected to be analyses. The apothecia were used immediately in nature (Molina et al. 2003) and stagnation in the early stages after their collection, because freshness is an important factor of development in experimental resynthesized lichen and in dispersal and germination success of spores (Sangvichien limited ability to keep whole-lichen thalli and aposymbiotic et al. 2011). The apothecia had intermediate diameters culture alive for an extended time are drawbacks frequently (about 5 mm in diameter) to ensure, as far as possible, sexual described (McDonald et al. 2013). In spite of these common maturity (Molina et al. 1997). All apothecia were washed and complications, an important number of in vitro cultures (be- carefully cleaned following the protocols published in Molina tween 30 and 40% of species that have been tried) from crus- and Crespo (2000). The fungi were grown from discharged tose and foliose lichens have been successfully obtained but spores following the inversed Petri dish methods of only to the early stages of development (Crittenden et al. 1995; Ahmadjian (1993). Ten clean ascomata were attached to the Jeon et al. 2009; Sangvichien et al. 2011). Therefore, it is inner side of Petri dish lids with petroleum jelly. The bottom necessary to improve techniques and protocols for artificial part of the Petri dishes contained inorganic Basal Bold me- production of lichens for scientific as well as economic pur- dium [BBM; Deason and Bold (1960)]. The other ten ascomata, poses, and also to expand the number of species available as were arranged similarly for organic media, 1% glucose BBM aposymbiotic cultures in order to obtain standardized large- (w/v) [1% G-BBM; Behera and Makhija (2001)]. The Petri dishes scale production of compounds unique to lichens. were inverted over the lids and the ascospores were allowed In this experimental study we used A. ciliaris as a model to discharge upwards onto the medium for 24 h at 18e20 Cin due to its broad applications. This is a fruticose lichen that the dark (Molina and Crespo 2000). After this time, the reproduce sexually by ascospores and it is commonly growing apothecia were removed to avoid contaminations and new as an epiphyte in Europe. It belongs to the family Physciaceae lids were placed. in Ascomycota (Divakar et al. 2007). This species has been To estimate the percentage of apothecia capable of ejecting used in biomonitoring of atmospheric pollution (Borg and ascospores, we used all the apothecia. However, to assess Aronsson 2004), in traditional alcoholic production of brandy germination in BBM and 1% G-BBM, we could only use three (see Smith 1921), and as a potential natural source of antiox- apothecia for each medium, after eliminating those apothecia idant and antibiotic (Rankovic et al. 2010). It is also a model for contaminated by bacteria or fungi. Seven days after germi- the study of symbiotic systems in algae, and therefore it is of nation, 36 randomly selected and isolated uncontaminated particular interest to get enough biomass of this mycobiont multispores from BBM media were transported to and sub- available for future experimental studies on symbiotic sys- cultured on four organic different media: 2% glucose BBM (w/ tems. Trebouxia decolorans (the A. ciliaris algal photobiont) v) [2% G-BBM; Behera and Makhija (2001)]; Lilly and Barnet genome has recently been sequenced to analyze horizontal medium enriched with 3% glucose (w/v) [3% G-LBM; according gene transfer from fungi to lichen algae (Beck et al., pers. to Lilly and Barnett (1951) as modified by Lallemant (1985)]; comm.). The next step of this ongoing project is to acquire the 0.2% glucose malt-yeast extract (w/v) [0.2% G-MY; modified in fungal genome for comparison both two complete genome this study from Ahmadjian (1993) as follows: 5 g of malt (photobiont and mycobiont). Anaptychia ciliaris has been extract, 2 g of glucose, 0.25 g of yeast extract, and 15 g of agar mycoscience 56 (2015) 351e358 353

in 1 L of D.W.], and corn meal agar (CMA) following manu- and editing sequence followed protocols according to Molina facturer's instructions (Difco, Detroit, MI, USA). In parallel, we et al. (2011). The sequence were blasted against GenBank continued to study the spore development in germination database (http://www.ncbi.nlm.nih.gov/genbank/) following media (inorganic BBM and organic 1% G-BBM). Differences in the methodology outlined in Sayers et al. (2011) to approach growth rates were studied in a total of six media (two germi- the species identity as A. ciliaris. The culture reported here nation and four development media) after 50, 100, and 300 from CMA was submitted to Fungal culture Bank of Korean days. Lichen Research Institute (Suncheon National University, To avoid phototrophic organism contamination, cultures Suncheon, South Korea) after ITS nuDNA was sequenced. were incubated in darkness at 18e20 C(Molina and Crespo Accession number of this culture is KOLABI 021278 (http:// 2000). Mycobionts were examined periodically using an Axi- kolabic.knrrc.or.kr). oskop (Zeiss, Oberkochen, Germany) and a Nikon Eclipse 80 The amount of total genomic DNA was estimated from 10 microscope with a magnifier glass. For photography, an cultures grown in 3G-LBM after six months. The assessment of automatic ring flash system was attached to the camera lens the total amount of DNA was carried out by comparison with a (SPOT Insight Wide-field 2 MP). Photographs used white light standardized molecular weight marker whose amount of DNA or Nomarsky interference contrast. When necessary, the col- per band is known (Catalog number 170-8352, Bio-Rad, Rich- onies were slightly stained with lactophenol cotton blue, mond, CA, USA). especially when colony growth was compact. The Munsell color system was used as color criteria in description of the mycobiont cultures after 300 days of growth (Munsell 1912) 3. Results and it is indicated in parentheses. The parameters used to assess the ontogenetic develop- The ITS sequences obtained from mycobiont culture were ment of A. ciliaris in culture were: spore ejection, spore identified as A. ciliaris by GenBank blast comparison, con- germination, hyphae growth and pigment synthesis. For the firming the species ID (100% similar). The sequence generated analysis of spore ejection, we evaluated two variables: 1) for this study was deposited in GenBank under accession percentage of apothecia able to propel spores upward (sexu- number KC559095. ally mature) and 2) the number of spores ejected from each The field-collected Anaptychia ciliaris showed abundant apothecia. For spore germination, the percentage of spores apothecia (1.6 ± 0.2 apothecia/cm2 (n ¼ 3) and all investigated that germinated was calculated 15 days after ejection. For apothecia ejected spores from their mature asci. In most growth, the globular colony growth after 300 days on different cases, the meiospores were attached to the media in the media was estimated, where globular growth was defined as upside-down petri dish bottom as single spores (Fig. 1A). The the diameter of the central part of the cultured mycobiont, number of group of ascospores ejected per apothecia always which forms colonies with globose cells and very compact exceeded 100, with an average of 151.33 ± 41.15 (n ¼ 20). The growth (Molina et al. 2013). Lastly, to approximate the meta- percentage of germinated spores per apothecia depended on bolic capacity to produce pigments, the percentage of colonies the media used: the 1% G-BBM media showed a germination that showed color after 50 days on different organic media rate of 25%e45% (35.66 ± 10.06, n ¼ 3) and BBM media resulted was calculated. Thirteen of 36 colonies per media (a total of 78 in 47%e70% germination (56.66 ± 12.28, n ¼ 3). The ascospores colonies) were tested. were subglobular and uniseptated with a brown external ornamented capsule (Fig. 1B). The first spores ejected were 2.3. Statistical analysis seen at 12 h, with high production at about 24 h. The first germ tubes were observed 6e7 days after discharge, exhibiting bi- In order to compare if the germination percentage between polar germination (Fig. 1C). media BBM and 1% G-BBM (3 replicates by each growth media) Germination percentage was higher on basal medium were statistically different, an analysis of variance (ANOVA) (BBM) than on enriched medium (1% G-BBM) though the dif- was performed. The variances were checked for homogeneity ferences are not statistically significant. Germinated asco- using the Cochran's test. Data were considered significantly spores exhibited hyphae with short and septated cells in both different when p < 0.05. KruskaleWallis test was used when inorganic (BBM) and organic (1% G-BBM) media (Fig. 2). After the data were not strictly parametric. All statistical tests were one month, the colonies in organic media (1G-BBM) consisted performed with the software Statistica 6.0 for Windows of well-developed radial mycelia with globular cells in the (StatSoft, Tulsa, OK, USA). center and more elongated cells distally (Fig. 2A). The cultured mycobiont maintained in inorganic media (BBM) showed a 2.4. Molecular identity and estimation of DNA amount low globular growth pattern. The hyphae in the inorganic media had a filamentous growth with weak radial mycelia and One fragment from a mycobiont culture from CMA was pre- generated colonies were poorly developed (Fig. 2B). In all cases pared from fresh culture were ground into powder with sterile the BBM colonies were white and no synthesis of pigments plastic pestles. Total genomic DNA was extracted using the was detected. ¼ ¼ DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Fungal nu- Growth rate was significantly different (H5 63.5; n 78; clear ribosomal internal transcribed spacer (ITS) was used as p < 0.001) between media after 50 days with higher and lower molecular marker because this region has the highest proba- values at CMA and BBM, respectively (Fig. 3). At this stage bility of successful identification for the broadest range of some of the colonies also showed coloration due to pigment fungi (Schoch et al. 2012). PCR amplification, DNA sequencing synthesis. The proportion of colonies able to synthesize these 354 mycoscience 56 (2015) 351e358

Fig. 1 e Subglobular, bipolar and uniseptate germinated ascospore from Anaptychia ciliaris. A: Individual and group spores ejected onto BBM, some of them also germinated and poorly developed after one month in this inorganic media. B: Details of capsule ornamentations (c, capsule; s, e Anaptychia ciliaris septum). C: Arrows show breaking points of spore capsule Fig. 2 Developmental stages of spore with germ tubes. Bars: A 200 mm; B, C 10 mm. germination (s, septum). A: Compacted growth with globular cells observed on 1% G-BBM media. B: Poor growth with elongated cells observed on BBM media. Bars: 100 mm.

pigments was different for different media (Fig. 4). After 100 days of growing in new media, there was also signiﬁcant dif- with abundant white aerial hyphae on the surfaces. Compact ¼ ¼ < ferences [H5 61.8; n 78; p 0.001 maintaining similar growth was observed without holes on the inside (Fig. 5D). In pattern to 50 days of incubation (Fig. 3)]. After 300 days of in- general, the colonies had numerous folds (Fig. 5). Finally, cubation, the pattern changed and the highest growth rates development on BBM and 1% G-BBM were very poor and the ¼ ¼ < was observed at 3% G-LBM (H5 67.6; n 78; p 0.001). The growth was not signiﬁcant (Fig. 3). DNA amount from pure cultures maintained in 3% G-LBM during six months was approximately 60 mg DNA per g of fresh weight after media was removed. Both color and morphological appearance of the cultured 4. Discussion mycobiont after 300 days varied with the type of media it had grown on (Fig. 5). When the mycobiont grew on 2% G-BBM Spores from asci were propelled upward and frequently, they media, the colonies was gelatinous, brown (YR; 5/6 Munsell were attached to the media as single spores (Fig. 1). This kind color system), and three-dimensional growth, having devel- of dispersion of single spores has already been observed oped a hole in the center of each colony (Fig. 5B). In 3% G-LBM in other species (Sangvichien et al. 2011) and also in A. ciliaris media, well-developed colonies were dull brown (YR; 2/2), also (Honegger and Zippler 2007), though it is very common in with three-dimensional growth and hole in the center Parmeliaceae and Physciaceae that ascospores from an ascus (Fig. 5A). In CMA media, brown-colored (YR; 3/4) cultures remaining attached as a small packets of spores on media showed white aerial hyphae with red tips (R; 8/9) and compact (Molina and Crespo 2000; Sangvichien et al. 2011; Molina et al. growth without holes in the structure (Fig. 5C). In 0.2G-MY 2013). All the apothecia ejected spores and the number of media, the colonies showed a reddish brown color (YR; 3/4), spores produced and discharged was very abundant, always mycoscience 56 (2015) 351e358 355

increasing the apothecia's genetic variation. Furthermore, lichens have a very slow growth rate, and can have apothecia of several ages on each lichen thallus. This allows for apothecia with different maturity rate and genetic constitution to persist through time at close range within one lichen individual. The spores took 6e7 days to germinate, a timeframe relatively common for lichen ascospore germination (Yoshimura et al. 1989). The germination rate was higher in inorganic media without glucose than with glucose enrichment, but differences were not statistically significant. The inhibition by glucose of germination has previously been observed in other studies (Molina et al. 1997; Molina and Crespo 2000; Molina et al. 2013) and related with control mechanism between bionts (Molina et al. 1997). In contrast, Denison (2003) emphasized the need to incorporate adsorbents and sugars, or polyols on basal, inorganic media to achieve germination. Fig. 3 e Growth rates of Anaptychia ciliaris spores in During the early stages of hyphae development (between different enriched media: CMA (C), 3% G-LBM (B), 0.2% G- 30 and 50 days) the mycobiont growth rate was slower, more MY (+), 2% G-BBM (þ), 1% G-BBM (:), and BBM (◊). Lower filamentous and superficial in the inorganic medium (BBM) case letter denote significantly different mean values than in the same medium enriched with glucose (1G-BBM). (p < 0.05; multiple comparisons, normal z-values). This shows that a carbon source supplement, without addi- tional additives such as vitamins, growth hormones, etc., improves the mycobiont development and biomass yield in the early development stages (Fig. 2). This differential more than a hundred per apothecia. It is worth to stress this behavior dependent on the presence or absence of energy because frequently apothecia from lichen don't eject spores supply has been described previously in other lichen species in vitro (Crittenden et al. 1995; Sangvichien et al. 2011). These (Molina and Crespo 2000; Behera et al. 2009). Fungal filamen- results suggest that all apothecia had reached sexual maturity tous growth has been associated with lichenized and parasitic and that the season for collection and in vitro sporulation fungi that need extra energy intake to complete their devel- conditions were appropriate. Sangvichien et al. (2011) re- opment (Molina and Fontaniella 2008). Glucose has often been ported that the discharge spores pattern correlates with the selected to in vitro experiment as a carbon source for its low seasonal maturity of ascoma. The environmental conditions economical cost and the fact that it is labile and easily like temperature, relative humidity, light etc., also can influ- metabolized. It is also common to use other carbon sources ence on discharge rate. such as fructose, sucrose, etc. in experiments (Behera and The production of meiospores from apothecia was not Makhija 2001; Brunauer et al. 2007). In nature, the mycobiont homogeneous and variation was observed even though the in the lichen symbiosis obtains the carbon source from green diameter of the apothecia was previously determined to algae usually in the form of polyols, such as ribitol, erythritol, 5 mm, approximately. This is not surprising considering the and sorbitol (Palmqvist et al. 2008). However, Behera and heterothallic life cycle in A. ciliaris (Honneger and Zippler Makhija (2001) did not find large differences in growth using 2007), where potentially cross fertilization and genetic mannitol instead of glucose or fructose. recombination among different genotypes can occur within CMA was found to be the best medium for development one individual thallus or between thalli (Tehler 1982). In het- during the first days after multisporic cultures were trans- erothallic lichens, the apothecia emerge from dikaryotic hy- ferred to growth media (Fig. 3). The CMA medium contains phae where nuclei could be genetically different, hence corn meal infusion as a carbon source and vitamins. Denison (2003) also used it successfully for the germination of several Lobaria species. After the inflection point observed at 100 days, the best media found for growth were 3G-LBM and 2G-BBM. The former has been successfully used for in vitro cultures of a large number of lichenized fungi (Crittenden et al. 1995). These culture conditions allow getting enough DNA amount to perform the whole genome sequencing of this fungi species, according to the data handled by McDonald et al. (2013). Anaptychia ciliaris is capable of synthesizing pigments after 50 days of growing in carbon-enriched media (Fig. 4) but not in inorganic media. This suggests that the availability of sugars and sugar alcohols in the nutrient medium could be a precondition to start pathways for pigment production (as proposed by Stocker-Worg€ otter€ 2007). Key morphogenetic Fig. 4 e Percentage of colored colonies after 50 days in differences were also observed between the aposymbiotic cell several organic enrichment media. aggregates of A. ciliaris growing in different media after 300 356 mycoscience 56 (2015) 351e358

Fig. 5 e Ontogenetic development of Anaptychia ciliaris on four enriched media. A: 3% G-LBM. B: 2% G-BBM. C: CMA. D: 0.2 % G-MY. Bars: 5 mm.

days (Fig. 5). These support the idea that the nutrients and 3% G-LBM and 2% G-BBM media resulted in three-dimensional additives in media can at least partly inﬂuence the growth in the cultures, while CMA and 0.2% G-MY promoted morphology and metabolic capacity of the aposymbiotic col- deep downward growth of most of the fungal biomass into the onies (Yamamoto et al. 1987a, b; Yoshimura et al. 1989; Wang media. These results conﬁrm that the presence of certain et al. 2009). Variations in the synthetic capacity of secondary nutrients and their balanced relationship could also be an compounds depending on the culture media have been widely important factor in morphogenesis (Stocker-Worg€ otter€ and described in the literature (Stocker-Worg€ oter€ and Elix 2006; Elix 2006). The proliferation of aerial hyphae in some col- Brunauer et al. 2007). However, other abiotic variables (white onies (Fig. 5D) could be related increased phenolic compounds light intensity, UV exposure, temperature, water stress, production on a drier substrate (Culberson and Armaleo 1992). desiccation on culture, etc.) and biotic variables (such as Aerial hyphae can be interpreted as an early phase of cortical presence or absence of compatible algal symbionts) have also hyphae, which could later form a dense cortex after a period of been shown to contribute to lichen growth (Yamamoto et al. desiccation (Fazio et al. 2009; Stocker-Worg€ otter€ 2010). 1994; Stocker-Worg€ otter€ 2007; Fazio et al. 2009; Stocker- In this study we have successfully grown axenic culture of Worg€ otter€ 2010). The three-dimensional growth patterns of A. ciliaris mycobiont in four enrichment culture media. The mycobiont in vitro conditions could be genetically pro- following process is recommended as the optimal culturing grammed (Stocker-Worg€ otter€ 2010), but the genes that are conditions of this species: 1) sporulation and germination on supposed to be involved in regulating morphogenesis in inorganic media (BBM), 2) after germination, transfer to CMA lichen fungi have not been studied yet (Stocker-Worg€ otter€ (organic media) for early stages of development (about 50 2010). Three-dimensional architecture in colonies can also days), and 3) later inoculation in 3% G-LBM to increase increase due to environmental factors such as presence of biomass production. So far, we do not know the chemical compatible algae, and the incorporation of ribitol and phyto- nature of the pigments observed in aposymbiotic cultures, hormones (Wang et al. 2009) or stress conditions (Stocker- however, certainly determining if they are phenolic com- Worg€ otter€ 2007). In our A. ciliaris mycobiont experiment, the pounds or not could be of great interest. mycoscience 56 (2015) 351e358 357

Experimental Mycology 16: 52e63; http://dx.doi.org/10.1016/ Disclosure 0147-5975(92)90041-O. Deason DR, Bold HC, 1960. Phycological studies. I. Exploratory studies The authors declare no conflict of interest. All the experi- of Texas soil algae, 6022. University Texas Publications 1e70. ments undertaken in this study comply with the current laws Denison WC, 2003. Apothecia and ascospores of Lobaria oregana Lobaria pulmonaria Mycologia e of Japan. and investigated. 95: 513 518. Divakar PK, Amo de Paz G, del Prado R, Esslinger TL, Crespo A, 2007. Upper cortex anatomy corroborates phylogenetic hypothesis in species of Physconia (Ascomycota, e Acknowledgments Lecanoromycetes). Mycological Research 111: 1311 1320; http:// dx.doi.org/10.1016/j.mycres.2007.08.009. Favelo-Longo SE, Piervittori R, 2012. Cultivation of isidia and We acknowledge Ministerio de Ciencia e Innovacion (CGL2008- transplantation of adult thalli of Xanthoparmelia tinctina in an 01600, CGL2010-21646) for providing the funds for this abandoned asbestos mine. Lichenologist 44: 840e844; http:// research. This project was also supported by a research grant dx.doi.org/10.1017/S0024282912000485. from Rey Juan Carlos University to MCM (Estancias Breves de Fazio AT, Bertoni MD, Adler MT, Ruiz LB, Rosso ML, Muggia L, € € Investigacion ). The authors thank the following colleagues, Hager A, Stocker-Worgotter E, Maier MS, 2009. Culture studies on the mycobiont isolated from Parmotrema reticulatum who provided supplies or other input for this project: Ning (Taylor) Choisy: metabolite production under different Zhang, James White, Marshall Bergen, Joan Bennett, and conditions. Mycological Progress 8: 359e365; http://dx.doi.org/ Monica Torres (Rutgers University), Constantino Ruibal (Uni- 10.1007/s11557-009-0609-1. versidad Complutense de Madrid), Gregorio Aragon and Gagunashvili AN, Davı´dsson SP, Jonsson ZO, Andresson OS, 2009. Debora Banuelos~ (Rey Juan Carlos University). Special thanks Cloning and heterologous transcription of a polyketide to Lena Struwe (Rutgers University) for her interesting con- synthase gene from the lichen Solorina crocea. Mycological Research e tributions to this manuscript and easy accessibility. MCM 113: 354 363; http://dx.doi.org/10.1016/ j.mycres.2008.11.011. greatly thank Rutgers University for her time there as a Honegger R, Zippler U, 2007. Mating systems in representatives of Visiting Scientist. Parmeliaceae, Ramalinaceae and Physciaceae (Leconoromycetes, lichen-forming ascomycetes). Mycological e references Research 111: 424 432; http://dx.doi.org/10.1016/ j.mycres.2007.02.005. 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