Characterization of Fungi from Hypersaline Environments of Solar Salterns Using Morphological and Molecular Techniques

mycological research 110 (2006) 962–970

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Characterization of fungi from hypersaline environments of solar salterns using morphological and molecular techniques

Sharon A. CANTRELLa,*, Lilliam CASILLAS-MARTI´NEZb, Marirosa MOLINAc aDepartment of Biology, School of Science and Technology, Universidad del Turabo, P. O. Box 3030, Gurabo, PR 00778, USA bBiology Department, University of Puerto Rico, CUH Station, Humacao, PR 00791, USA cEnvironmental Protection Agency, ORD, Athens, GA 30602 USA article info abstract

Article history: The Cabo Rojo Solar Salterns located on the southwest coast of Puerto Rico are composed of Received 22 December 2005 two main ecosystems (i.e., salt ponds and microbial mats). Even though these locations are Received in revised form characterized by high solar radiation (mean light intensity of 39 mol photons m2 d1)they 24 April 2006 harbour a diverse microscopic life. We used morphological and molecular techniques to Accepted 1 June 2006 identify a series of halotolerant fungi. A total of 183 isolates and 36 species were cultured Published online 10 August 2006 in this study. From the water from the salt ponds, 86 isolates of 26 species were cultured. Corresponding Editor: John Dighton The halotolerant fungi isolated from water were: Cladosporium cladosporioides, nine Aspergillus sp., five Penicillium sp. and the black yeast Hortaea werneckii. A distinctive isolate with a blue Keywords: mycelium was cultured from the salt ponds, representing a new species of Periconia based Evaporation ponds on morphology and rDNA analysis. Forty-four isolates from eight species were cultured Halophilic fungi from the sediments around the salt ponds. Most of the sediment isolates formed only sterile Halotolerant fungi mycelium, while several were Chaetomium globosum. A total of 53 isolates from 16 species Puerto Rico were isolated from the three layers of the microbial mats, of which Aspergillus niger was Tropical fungi the most frequent isolate. Phospholipid fatty acid profiles generated from the different layers of the microbial mats indicated that the uppermost layers of the mats contained fungal biomarker, 18:2w6. This fatty acid decreased with depth, the highest concentration was observed in the green upper layer and it disappeared in the black bottom anoxic layer. This correlates with the isolation of fungi using the serial dilution technique. This is the first study that documents the presence of fungi in microbial mats. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction domain can also be found living in extreme conditions, as long as there are enough energy sources available to sustain Living organisms can be found over a wide range of extreme their metabolism. Most eukaryotic extremophiles belong to conditions. Most of the organisms living in extreme environ- the algal and fungal categories; some examples are Cyanidium ments (i.e, extremophiles) belong to the prokaryotes, speciﬁ- caldarium, a red thermophilic alga, Chlamydomonas nivalis, cally to the domain Archaea. Extremophiles from the Eukarya a psycrophilic green alga, and Trichosporum cerebriae,an

* Corresponding author. E-mail address: [email protected]. 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.06.005 Fungi from Cabo Rojo Solar Salterns 963

acidophilic fungus. These groups also include Dunaniella sal- their characteristic lamellae and other bacteria especially ina, a halophilic green alga known to dominate in hypersaline spore formers. The most abundant organisms present in an- environments (Schleper et al. 1995). oxic black layers are spirochetes with their characteristic axial Hypersaline environments originate by the evaporation of filaments. These different communities of the mats are sub- sea water and are also called thalassohaline environments jected to the typical changes in pluvial precipitation of Carib- (Oren 2002). As water evaporates, gypsum and other minerals bean ecosystems (rainfall decreases from 177 mm in the rainy precipitate, eventually, sodium chloride (NaCl) precipitates season to 51 mm in the dry period). Increases in the salinity of and salinity increases above 300 psu (Oren 2002; Gunde- the locations causes specific changes in the community struc- Cimerman et al. 2000). An example of an extreme hypersaline ture of the different mat layers as reported after terminal RFLP environment is the crystallization ponds or solar salterns. (T-RFLP) analysis. We reported a decrease in the number of These ponds provide a diversity of environments where dif- peaks present in the electropherograms generated from ferent conditions of salinity, pH, temperature, light intensity, mats samples collected during the dry season. This result in- oxygen and nutrient concentrations are found, allowing the dicates the relationship between an increase in salinity and study of different microbial communities (Pedro´s-Alio´ 2004; a decrease in the diversity of the community structure. Pedro´s-Alio´ et al. 2000; Gunde-Cimerman et al. 2000). Most mi- The Cabo Rojo salterns are composed of two ecosystems, crobial diversity studies in salterns have focused on halophilic the salt ponds and the microbial mats. Physicochemical con- Archaea bacteria of the order Halobacteriales, which comprise ditions at this location were monitored during sampling to op- the main microbial component in these environments (Oren timize our growth media for the enrichment of a series of 2002). Other organisms such as algae, protozoa, eubacteria halophilic and halotolerant fungi. Specific layers of the micro- and even fungi are also found in the salterns, even though it bial mats were analysed for their content of the 18:2w6 fungal was thought that they could not survive under extreme salt biomarker. Fungal isolates were characterized based on mor- conditions (Gunde-Cimerman et al. 2004). phological and molecular traits. Fungi are ubiquitous in most ecosystems where they usu- ally colonize a diverse range of substrates. The highest diversity of fungi is found in tropical regions, mainly in tropical Materials and methods forests (Hawksworth 1991). In Puerto Rico, fungal studies have concentrated on forest and coastal ecosystems (Acevedo Site description 1987, 2001; Cantrell et al. 2004; Lodge et al. 2002, Nieves-Rivera 2005). However, many fungi can adapt to extreme environ- The study area is located in the Southwest coast of Puerto Rico mental conditions of water, temperature, pH and salinity (Grif- in Punta Los Morrillos in the municipality of Cabo Rojo (lat. 0 0 fith 1994). Most of the fungi that can be found in extreme 17 56 25 N, long. 67 11 W). The area is known as the Cabo environments belong to the imperfect stage of the Ascomycota, Rojo Solar Salterns (in Spanish Las Salinas de Cabo Rojo; which have been reported in mangroves, saline soils, marine Fig 1) and is characterized by a high solar radiation (46 mol 2 1 sediments, sea water, salt marshes, and sand dunes (Domsch photons m d ), low precipitation (51 mm) and salinity (up et al. 1993, Kohlmeyer & Volkmann-Kohlmeyer 1991, Hyde and to 600 psu) (Casillas-Martıńez et al. 2005). The salterns are Pointing et al. 2000, Newell 1996, Guiraud et al. 1995, Moubasher composed of an estuary surrounded by mangroves. Natural et al. 1990). Because fungi were only recently isolated from mats are fed from an inlet that also feeds a series of artificial hypersaline environments, their function in these extreme salt ponds with seawater. conditions is still unclear (Gunde-Cimerman et al. 2004). Most Pluvial precipitation in the salterns varies depending on of the studies of fungi in extreme salt conditions have been the time of the year, a dry period with a mean precipitation performed in the northern latitudes, in the region of Russia of 51 mm runs from April to November. A short rainy season and the Dead Sea (Gunde-Cimerman et al. 2004, Kis-Papo takes place from December to March in which life flourishes et al. 2001, 2003, Buchalo et al. 1998). Butinar et al. (2005a,b) again at the salterns as pluvial precipitation rises to 177 mm. described fungi isolated from several natural and man-made Sampling in this study took place during a transient period hypersaline environments including those found in France, within the dry period (from May through July, 2003–2004) Namibia, Portugal, Slovenia, Spain, and Dominican Republic. that was characterized by higher-than-usual precipitation. Montalvo-Rodrı´guez et al. (1998) described Halogeometricum Consequently, salinity levels in the ponds fluctuated and the borinquense, a new genus of halophilic Archaea isolated from mats were always submerged with the incoming water from the solar salterns in Puerto Rico. Dıáz-Mun˜ oz & Montalvo- the inlet (in contrast to the usual dry period in which the Rodrı´guez (2005) reported for the first time Hortaea werneckii mats are completely dry). from Puerto Rico. Casillas-Martıńez et al. (2005) describe the microbial mat associated with the surrounding environment Physicochemical analysis of the Cabo Rojo Solar Salterns. The microbial mats located in the Cabo Rojo salterns are approximately 50–70 mm in Physicochemical conditions during sampling were deter- depth with three distinct layers (green, pink and black). As ob- mined using portable instrumentation as previously described served by transmission electron micrographs the top green by Casillas-Martıńez et al. 2005. Briefly, the pH and tem- layer is dominated by cyanobacteria, diatoms and benthic perature of salt pond water and overlying water of the mats dinoflagelates, which are abundant during the rainy periods. was measured using a combined pH and temperature The pink zone underneath this top layer harbours an abun- meter (Hanna Instruments, Woonsuckel, RI, HI 9024C). The dance of unidentified filamentous anoxyphototrophs with salinity readings were taken with a handheld refractometer 964 S. A. Cantrell et al.

Man-made salt ponds (SP) NSF NSF

Natural salt flats (NSF) SP

Inlet and mat

Fig 1 – Detail map showing Los Morrillos point in the southwest coast of Puerto Rico where the Cabo Rojo Solar Salterns are located. Two main ecosystems comprise the salterns salt ponds and microbial mats. Top right photo shows the constructed salt ponds (SP) and the natural salt ﬂat (NSF) within which microbial mats will develop (January 2006). Bottom right photo shows the inlet within which microbial mats have developed (June 2004).

(Westover, Mill Creek, WA, RHS-10-AT) and the photosynthet- MEA and a spore suspension was made in the inoculating ﬂuid ically active radiation was determined using a quantum sen- FF-IF. The spore suspension was inoculated in FF microplates sor (LiCor, Lincoln, NE, 190SA). that are speciﬁc for fungi and incubated at 25 C for 96 h.

Isolation and characterization of fungi Halotolerance test

A sediment core (10 2.5 cm) and water samples (500 ml) were The halotolerance test was performed following a modified collected during June 2003 and 2004. Microbial mat samples procedure described in Moubasher et al. (1990). The culture were collected during June 2004. Four aliquots of 10 ml of medium used in this study was MEA amended with the fol- each water sample were filtered through a 0.45 mm membrane. lowing NaCl concentrations: 10, 15, 20 and 25 %. Isolates Filters were placed onto two different media: marine agar were inoculated in the centre of the plates and incubated at o (MA) prepared using water from the same sample at two con- 27 C for 10 d. After this period, the diameter of the colony centrations (10 %, 90 %) and malt extract agar (MEA). Isolation was measured. of fungi from sediments and microbial mats was performed using the serial dilution technique in combination with two selective media prepared in the laboratory: MA prepared with sea water and MEA. Four 2 2 inch squares of microbial mats were collected and divided into three distinctive layers: green (first layer), pink (second layer), and black (third layer; Fig 2). Isolations were made from a composite of each layer. In addition, fungi were isolated from dry microbial mats. The plates were incubated at 30 oC for one month. Pure cultures were isolated in MEA and MA. Fungi were identified using standard taxonomic references. Some isolates were characterized using a Biolog MicroSta- tion Plate Reader and MicroLog 3 (Hayward, CA) to determine Fig 2 – Layers in a microbial mat collected in June 2004 at the the different carbohydrates metabolized. Fungi were grown in Cabo Rojo Solar Salterns in Cabo Rojo, Puerto Rico. Fungi from Cabo Rojo Solar Salterns 965

DNA extraction and phylogenetic analysis 700 600 Genomic DNA of selected pure cultures was extracted using 500 CTAB following the protocol of Graham et al. (1994). DNA was 400 stored in a 20 oC freezer. The ITS1–5.8 S ITS region was ampli- fied using a combination of primers ITS1 and ITS4 (White et al. 300 200 1990) with the Qiagen PCR Core Kit (Qiagen, Valencia, CA). PCR Salinity (psu) amplifications were conducted using the standard protocol 100 suggested by the manufacturer using 2–4 ml of DNA template. 0 Thermal cycling parameters for amplification consisted of one Inlet New Pond Pond E Pond A Matt Channels initial cycle of denaturing at 95 oC for 2 min, annealing at 55 oC Fig 3 – Average salinity measurements in the different for 30 s and extension at 72 oC for 1 min. This cycle was fol- water samples. lowed by 38 cycles with denaturing at 95 oC for 30 s, annealing at 55 oC for 30 s and extension at 72 oC for 1 min. A final cycle was added consisting of an extension time of 10 min. Sequencing in both directions was performed on an ABI include: the inlet that feeds the ponds, the water column on 310 automated sequencer (ABI, Foster city, CA) using primers top of the microbial mats and three salt ponds (A, E and ITS1 and ITS4. A few amplicons were cloned with the Topo New Pond). Even though all locations were subjected to the TA cloning system (Invitron, Carlsbad, CA). Sequences were typical seasonal changes of the Caribbean, the summer compiled using Auto Assembler (ABI) software. Sequence sim- months are considered a transient period as the dry period ilarities were obtained using the BLAST tool from NCBI. Align- is interrupted with occasional rainy events. Mean rainfall dur- ments and phylogenetic trees were reconstructed in Clustal X ing sampling was 68 mm and the daylight intensity was version 1.81 and the NJ distance method with Jukes-Cantor 39 mol photons m2 d1. The mean salinity in the locations correction (Thompson et al., 1997). BS confidence levels were sampled ranged 250–600 psu in the salt ponds and 50–80 psu defined from 1K iterations of tree reconstruction. in the water column of the mats and the inlet (Fig 3). As all Phospholipid fatty acid (PLFA) extraction and analysis locations exceeded salinity of 35 psu they were considered hypersaline. Other parameters such as temperature values Mat and sediment samples collected for lipid analysis were (ranging from 26–32 C) and pH (7.3 to 8.2.) also differ depend- frozen within a few hours of collection and stored at 20 C ing on the sample location. until freeze-dried. Phospholipid fatty acids were extracted, methylated and purified from 2 g of the freeze-dried samples Salt pond water samples following the procedure described by Burke et al. (2003). The extracted fatty acid methyl esters (FAMEs) were analysed The total number of isolates was calculated by the addition of with an Agilent 6890 Series gas chromatography (GC) system the number of isolates in each aliquot. A total of 86 isolates (Agilent; Palo Alto, CA) equipped with a flame ionization detec- from 26 species were obtained, most of them are halotolerant tor (FID) and a 50-m DB-5 capillary column (film thickness (Table 1, Fig 4). These isolates were obtained primarily from m 0.33 m, internal diameter 0.2 mm; Agilent; Palo Alto, CA). Indi- MEA plates. Only four specimens were isolated from MA pre- vidual compounds were quantified by FID response relative to pared with 10 % pond water, no isolates grew on MA þ 90 % an internal standard (20:0 ethyl ester) added prior to GC analy- seawater. In 2004, it was not possible to obtain water samples sis. Identifications were assigned based on relative retention from pond E and New Pond because they were in the process times compared to a prepared standard mixture (Sigma, St of salt extraction. The most frequent species in water samples Louis, MO; and Matreya, Pleasant Gap, PA) that was run with was Cladosporium cladosporioides, which accounted for 27 % of each batch of samples. Subsequent analysis by GC–mass spec- the isolates. Several species of Aspergillus and Penicillium trometry (MS) was used to verify compound identification and were identified, such as A. candidus, A. caespitosus, A. flavus, to identify FAMEs not in the standard mixture. The same A. flavipes, A. melleus, A. nidulans, A. ochraceus, A. penicillioides, temperature program and similar 30-m DB-5 (film thickness A. unguis, P. citrinum, P. chrysogenum, P. oxalicum and P. varia- m 0.25 m, internal diameter 0.25 mm) as used in the GC–FID in- bile. The black yeast, Hortaea werneckii was also isolated from strument were used in an HP 5890 Series II GC interfaced to one of the water samples. This yeast has been isolated an HP 5972 mass selective detector. The oven temperature pro- from soil and hypersaline environments in temperate gram was programmed to hold at 70 C for 2 min, ramp from 70 regions, where it seems to be more abundant (Butinar et al. 1 to 160 Cat40Cmin , then ramp from 160 to 280 Cat 2005a, Gunde-Cimerman, personal communication). In addi- 1 5 Cmin , with a final isothermal period of 20 min. tion, Dıáz-Mun˜ oz & Montalvo-Rodrı´guez (2005) have isolated H. werneckii from the Cabo Rojo salt ponds. Other dematia- Results ceous fungi isolated in this study were Curvularia lunata, and Nigrospora sphaerica. Physicochemical parameters The number of total isolates and the number of isolates that belong to the dematiaceous fungi from water samples We determined the physicochemical parameters that prevail taken in 2003 were compared with the salinity of the water. in four different locations within the Cabo Rojo salterns that The results indicate that there is a high correlation between 966 S. A. Cantrell et al.

30 Table 1 – Species, number of isolates and salt tolerance of each species obtained from each environment sampled 25

Species Salt Water Sediment Microbial 20 tolerancea mat 15 Aspergillus caespitosus HH 1 10 Aspergillus candidusb HH 1

Aspergillus carneus HH 6 Num. of Isolates 5 Aspergillus flavipes WH 1 0 Aspergillus flavus MH 1 1 Inlet New Pond Pond E Pond A Matt Sea Water Aspergillus NT 1 Channels heteromorphus Sample Aspergillus japonicus NT 1 Aspergillus melleusb MH 1 Fig 4 – Total number of isolates obtained in each water Aspergillus nidulans HH 2 1 sample. Number of isolates represents the total in 40 ml of b Aspergillus niger WH 20 water (2003, 2004). Aspergillus ochraceusb MH 3 Aspergillus penicillioides MH 2 Aspergillus sp. MH 5 9 3 Aspergillus unguis WH 1 Chaetomium globosumb WH 5 2 This new species is characterized by producing a blue pig- Cladosporium MH 24 1 2 b mented mycelium on MEA and PDA, and spherical brown cladosporioides Cladosporium WH 2 ornamented spores produced in a brown conidiophore. The oxysporum new species was characterized using Biolog and can metabo- Cladosporium WH 1 1 lize several carbohydrates such as arabinose, fucose, maltitol, b sphaerospermum mannitol, methyl-D-Glucoside, palatinose, d-psicose, raffi- Curvularia lunata WH 6 nose, rhamnose, salicin, threhalose and quinic acid. In liquid Dreschlera sp. WH 1 media, this species grew differently depending on the me- Graphium like WH15 % 1 Hortaea werneckiib HH 1 dium and the percentage of dextrose. In malt extract (MEB) Humicola sp. MH 1 and Sabouraud dextrose broth (SDB), the species produced Sterile mycelium NT 16 2 a blue-pigmented mycelium (Fig 6). Plates containing MEA ‘‘Dematiaceae’’ and Sabourand Dextrose Agar (SDA) also turned blue. In con- Sterile mycelium HH&MH 11 10 4 trast, in marine and Sabouraud broths, the mycelium is white ‘‘Moniliaceae’’ and there is no pigment produced in the medium. The pig- Myrothecium roridumb MH 2 ment was extracted in acetone and acetonitrile and was ana- Nigrospora sphaerica HH 1 1 Penicillium citrinumb MH 1 lysed using hplc. The results show three peaks between 600– Penicillium HH 1 630 nm, suggesting that the blue colour is produced by three chrysogenumb isomers of the same pigment (data not shown). Penicillium oxalicum WH 1 Penicillium sp. HH 12 1 6 Penicillium variabile NT 1 1 Sediment samples Periconia sp.nov. WH15 % 1 Phialophora sp. NT 1 A total of 44 isolates from eight species were obtained from Scopulariopsis sp. NT 1 sediment samples (Table 1, Fig 8). All the species were iso- Sporothrix sp. NT 1 lated on MEA; none on MA. Most of the sediment isolates Total number 27 8 16 of species Total number 86 44 53 of isolates 30

a See text for explanation. HH, highly halotolerant; MH, moder- 25 ately halotolerant; WH, weakly halotolerant. 20 b Reported from other hypersaline waters from temperate and tropical regions. 15

Num. of Isolates 5 the total number of fungal isolates present in the ponds and 0 salinity. Pond A produced both the highest number of isolates Inlet New Pond Pond E Pond A Matt Channels and the highest salinity, while the inlet and mat channel had the least number of isolates and the lowest salinity (Figs 3–5). Fig 5 – Total number of fungi and dematiaceous fungi in A distinctive isolate was obtained from one water sample water samples taken in 2003. Number of isolates represents and based on cultural, morphological and phylogenetic char- the total in 40 ml of water. (Total isolates, number of de- acteristics; it belongs to a new species of Periconia (Figs 6–7). matiaceous isolates.) Fungi from Cabo Rojo Solar Salterns 967

Fig 6 – Periconia sp. nov, ined. isolated from water from one of pond. Culture grown in MEA.

were Sterile mycelium belonging to the ‘‘Moniliaceae’’ (23 %) and Microbial mat samples ‘‘Dematiaceae’’ (36 %) but several isolates belonged to Aspergil- A total of 53 isolates from 16 species were obtained from the lus sp., A. japonicus, Chaetomium globosum, Cladosporium clado- different layers of the microbial mats (Table 1, Fig 9). All the sporioides, Penicillium sp., and P. variabile. species were isolated on MEA; none on MA. In contrast to the water samples, 83 % of the isolates belong to the moniliaceous fungi. Aspergillus niger was the most common species, 0.02 accounting for 38 % of the isolates. Chaetomium globosum, as Pyricularia variabilis AY265333 100 100 Periconia macrospinosa AJ246159 well as Cladosporium cladosporioides, C. sphaeropermum and 100 Periconia macrospinosa AJ246158 Nigrospora sphaerica were isolated from the dry microbial 74 Periconia sp. nov. DQ336713 mat. Most fungal isolates obtained from the live and active mi- 100 Helminthosporium solani AF145703 Helminthosporium velutinum AF145704 crobial mat belonged to Aspergillus and Penicillium. The num- Penicillium commune AF455544 ber of isolates decreased from the first (green) to the third 65 Penicillium commune AF455436 Penicillium chrysogenum AF455490 (black) layer, which agrees with the results obtained by PLFA 72 Penicillium chrysogenum DQ336710 analysis. 77 Penicillium chrysogenum AY373902 Aspergillus restrictus AY373864 95 Halotolerance test 99 Aspergillus penicillioides AB006432 100 Aspergillus penicillioides DQ336711 The majority of the species isolated in this study can grow in Aspergillus penicillioides AY373862 Aspergillus ostianus AY373858 0–25 % NaCl, however, none of them can be classified as halo- 99 Aspergillus elegans AF459736 philic because they all can grow in NaCl-free media. Based on Aspergillus elegans AF128860 68 their growth rate in the presence of 25 % NaCl, species were Aspergillus ochraceus DQ336712 Aspergillus ochraceus AY373856 classified as (see Table 1): (1) highly halotolerant (species 86 Aspergillus ochraceus AY373855 with a growth rate >3 cm over 10 d in 25 % NaCl): Penicillium Claviceps purpurea U57669 sp. (SACCR #10 and #50AA), P. chrysogenum, Aspergillus caespito- 99 91 Chaetomium sp. AY560520 100 Chaetomium globosum AY429054 sus, A. candidus, A. carneus, A. nidulans, Nigrospora sphaerica and 100 Chaetomium globosum DQ336707 moniliaceous Sterile mycelium (SACCR #62AA). In MEB Hortaea 91 Chaetomium globosum AY429051 Chaetomium globosum AY429050 Hortaea werneckii AB087201 25 99 Hortaea werneckii AY820141 Hortaea werneckii AB087199 Hortaea werneckii AY128704 20 99 71 Hortaea werneckii DQ336709 Trimmatostroma salinum AJ238676 15 Phaeotheca triangularis AJ244256 88 Cladosporium sphaerospermum DQ336706 10 98 Cladosporium sphaerospermum AJ244228 Cladosporium cladosporioides AF393689

99 Num. of Isolates Cladosporium oxysporum AF393720 5 Cladosporium tenuissimum CTE300331 Cladosporium cladosporioides AF455535 0 Cladosporium oxysporum DQ336708 12345 Sample Fig 7 – Phylogenetic tree that resulted from the alignment of the ITS1–5.8 S–ITS2 region of the ribosomal DNA. Species Fig 8 – Total number of isolates in each sediment sample. in bold represent isolates from this study. Letters and num- Number of isolates represents the total in two replicates bers after species represent GenBank accession numbers. of a 1:1000 dilution. 968 S. A. Cantrell et al.

20 diatoms First-Green Layer 18 actinomycetes 16 anaerobic bact 14 fungi 12 G - bacteria 10 G + bacteria 8 0 5 10 15 20 25

Num. of Isolates 6

4 diatoms Second-Peach Layer 2 actinomycetes 0 anaerobic bact Dry Mat 1st Layer 2nd Layer 3rd Layer fungi Fig 9 – Total number of organisms isolated from each G - bacteria layer of the microbial mat. Number of isolates represents the total in two replicates of a 1:1000 dilution. G + bacteria 0 5 10 15 20 25

diatoms Third-Black Layer werneckii can grow up to 25 % NaCl. (2) Moderately halotolerant actinomycetes (species with a growth rate between 2–3 cm over 10 d in 25 % NaCl): Aspergillus sp. (SACCR #48AA), A. flavus, A. melleus, A. anaerobic bact ochraceus, A. penicillioides, Cladosporium cladosporioides, Humi- fungi cola sp., moniliaceous mycelia sterilia (SACCR #61AA), Myrothe- G - bacteria cium roridum and P. citrinum. (3) Weakly halotolerant species G + bacteria can be divided into two categories, those that present a growth 0 5 10 15 20 25 rate <2 cm over 10 d in 25 % NaCl and those that can grow in % PFLA Composition up to 15 % NaCl. The following species are in the first category: A. flavipes, A. niger, C. oxysporum, C. sphaerospermum, Chaeto- Fig 10 – Percent composition of PLFAs extracted from the mium globosum, Curvularia lunata, Dreschlera sp. (SACCR # distinct layers of a microbial mat collected at the Cabo Rojo 121), and P. oxalicum. Periconia sp., and Graphium-like fall in solar salterns in Puerto Rico. Wet season (gray color bar), dry the second category. season (black color bar).

Phylogenetic relationships similar, the relative composition of individual PLFAs suggests drastic changes in the structure of the microbial community Several isolates were hard to identify and for their identifica- across layers. The top layer (green) contained the highest per- tion nucleotide sequences from the ITS1–5.8 S ITS2 region of cent composition of PLFAs commonly found in G- bacteria the ribosomal DNA were used. We selected representatives of (16:1w7), diatoms (20:5w3) and fungi (18:2w6), while the bot- all the different genera obtained in this study. In Fig 7, the phy- tom layer (black) had a higher composition of the markers logenetic relationship of these isolates is presented. Isolates for anaerobic bacteria (cy17:0, cy19:0; Fig 10). Both diatoms that were confirmed using nucleotide sequences are Aspergillus and fungi biomarkers were absent in this bottom layer. Sea- ochraceus (SACCR #97), Chaetomium globosum (SACCR #13M1), sonal differences (dry versus wet periods) were mostly noticed Cladosporium oxysporum (SACCR #103), and Hortaea werneckii in the composition of the intermediate, pink layer. Fungi, (SACCR #16). Also, phylogenetic analysis confirmed that the G- and Gþ bacteria (i15:0, i16:0) were all found at a higher per- isolate thought to belong to the genus Periconia (SACCR #64) cent composition during the dry season in this layer, while was indeed a new species. Isolates that were identified to the anaerobic bacteria were more abundant during the wet sea- species level and supported by morphology are A. penicillioides son. In contrast, the fungi biomarker was more abundant (SACCR #43b), C. sphaerospermum (SACCR #25) and Penicillium during the wet season in the top layer and anaerobic bacteria chrysogenum (SACCR #2). The above halotolerant isolates from were more abundant during the dry season in both the top and the Cabo Rojo Solar Saltern relate phylogenetically with bottom layers. Overall, the fungi biomarker accounted for less non-halophilic or non-halotolerant species. More isolates and than 5 % of the total PLFA composition. species obtained in this study will be sequenced.

PLFA analysis Discussion

PLFA analysis was used to assess the structure of the micro- In this study, we identiﬁed species of fungi present in an bial communities inhabiting the different mat layers. Al- extreme environment believed to be too harsh for fungi to though the types of fatty acids identiﬁed in each layer were thrive. Our results support recent reports presented by others Fungi from Cabo Rojo Solar Salterns 969

and challenge the idea that such organisms could not survive present study, 36 species, 15 genera and several isolates of under the extreme conditions present in solar salterns. mycelia sterilia were obtained, representing less than 1 % of Gunde-Cimerman et al. (2004) reported that fungi belonging the total number of species described in the world and tropical to the Dematiaceae can populate salterns with extreme NaCl regions. From these species, 75 % are halotolerant fungi and concentrations. In addition, Meristemic black yeasts and Cla- might be growing and reproducing in this extreme dosporium sp. were among the most common species identi- environment. fied along with various species from the genera Aspergillus The relationship between the concentration of nitrogen, and Penicillium. Before publication of these studies, it was the number of fungal isolates and salinity was established thought that the occurrence of fungi in hypersaline environ- by Butinar et al. (2005b). They observed two peaks of nitrogen, ments was due to a random event caused by airborne inocu- one at salinities of 8–10 (0.3–1.7 mg l-1 nitrogen) and the other lums; therefore, the fungi had no specific ecological function. at salinities of 21–25 (4–13.5 mg l-1 nitrogen). These two peaks However, recent evidence indicates that some species are correspond to the highest number of colony forming units. capable of growing and reproducing (Gunde-Cimerman et al., Some of the nutrients in the solar saltern come from bacteria, 2004). algae and the brine shrimp that bloom in the ponds right Thirty-one percent of the 36 species of isolates in this study, before salt starts to precipitate above a salinity of 300 including Aspergillus candidus, A. melleus, A. niger, A. ochraceus, (Pedro´ s-Alio´ s 2004). As the salinity increases above 450 psu Chaetomium globosum, Cladosporum cladosporioides, C. sphaero- many organisms start to die and the ecosystem might be spermum, Hortaea werneckii, Myrothecium roridum, Penicillium devoid of life. Fungi will live on the excreted nutrients, and citrinum and P. chrysogenum, have been reported in other hyper- the dead organic matter that is produced from the die-off of saline waters from temperate to tropical regions (Butinar et al. the algae and brine shrimp. Fungal colonies then develop on 2005a,b; Gunde-Cimerman et al. 2004; Kis-Papo, et al. 2001, the surface of the dead algae and shrimp, forming aggregates. 2003). Dematiaceous fungi account for 34 % of the total number The outer cells of the aggregate are exposed to high salt con- of isolates identified in the Cabo Rojo solar saltern and their centrations, and help protect the centre of the colony abundance seems directly proportional to the salt concentra- (Gunde-Cimerman et al. 2004). In our study, results indicate tion. The abundance increased from 17 % at a salinity of that the number of isolates increases with salinity. This 50 psu to 70 % at a salinity of 600 psu, which agrees with results peak in numbers of fungal isolates coincides with the highest reported by Butinar et al. (2005b) (Figs 3, 5) At lower salinities, measured concentration of nutrients and organic matter in dematiaceous or melanized fungi occur occasionally and are the saltern, suggesting that a similar sequence of events as de- replaced by moniliaceous fungi, where Aspergillus and Penicil- scribed by Gunde-Cimerman et al. (2004) could be taking place lium were the dominant species. In addition, the presence at the Cabo Rojo salterns. Also, the number of dematiaceous and abundance of fungi in the microbial mats is a function of fungi increases with salinity which is an expected shift of the oxic level in each layer, with fungi present at the top layers the microbial community due to its adaptation advantage. and disappearing as the amount of anaerobic biomarkers in- These fungal species with dark cell walls in the spores and creased, as indicated by the PLFA data. In the first layer 72 % mycelium can tolerate dehydration, and solar radiation better of the isolates belong to moniliaceous fungi. The second and than the moniliaceous fungi whose cells are devoid of pig- third layer was dominated by A. niger (90 % and 75 %, respec- ments. The dark pigment is melanin, and at high salt concen- tively). The dominance in the first layer of moniliaceous fungi trations melanin granules will be densely packed protecting that produce hyaline mycelium and spores might be related to the fungal cell (Gunde-Cimerman et al. 2004). Another adapta- the presence of photosynthetic organism that might be provid- tion to this hypersaline environment consists of an alteration ing protection. Conversely, A. niger is a moniliaceous species in the composition of phospholipid fatty acids in the fungal characterized by brown spores and its presence in the second cell membrane. At high salt concentrations the membrane and third layer of the mat can be related to the ability of this needs to be more fluid to overcome the higher osmotic pres- fungus to grow in the absence of oxygen and the production sure. This fluidity is achieved by increasing the unsaturated of pigmented spores that are more resistant than hyaline PLFA:sterol ratio (Gunde-Cimerman et al. 2004). spores to extreme conditions (Domsch et al. 1993). In general, the results obtained in the present study agree It is particularly interesting that 60 % of the sediment with similar studies on fungi from hypersaline environments. isolates belong to Mycelia Sterilia, of which dematiaceous A total of 36 species were identified including a new species of isolates are dominant and Chaetomium globosum accounted Periconia. Most of the isolated species are fungi that are com- for 11 % of the isolates. C. globosum was reported from the mon in other non-saline environments. None of the identified Dead Sea and vegetative mycelia, and spores stored in species are truly halophilic because they present extensive undiluted Dead Sea water were viable after 12 weeks (Kis- growth on non-salt media. Most of the species obtained in Papo et al. 2003). The C. globosum isolates obtained in this this study (75 %) can tolerate up to 25 % salt concentration study were from the sediments and dry mat, and can grow and a few species can grow up to 15 %. This is the first study up to 2 cm in diameter in 10 d in MEA amended with 25 % that documents the presence of fungi in microbial mats pro- NaCl. Interestingly, this species was not found in live viding new information on the occurrence and distribution microbial mat. of fungi in these habitats. Future work will concentrate on The exact number of total species that can thrive in hyper- the possible role of fungi in the biogeochemical cycling of saline environments is unknown, and it is possible that the nutrients in water, sediments and microbial mats in these number varies as a function of latitude and surrounding habi- hypersaline environments. In addition, we will implement tat (Gunde-Cimmerman et al. 2004; Pedro´ s-Alio´ s 2004). In the modern molecular techniques such as T-RFLP and large-scale 970 S. A. Cantrell et al.

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