Changes in fungal community composition of biofilms on limestone across a chronosequence in Campeche, Mexico

Abstract

Background and Aims:
The colonization of lithic substrates by fungal communities is determined by the properties of the substrate (bioreceptivity) and climatic and microclimatic conditions. However, the effect of the exposure time of the limestone surface to the environment on fungal communities has not been extensively investigated. In this study, we analyze the composition and structure of fungal communities occurring in biofilms on limestone walls of modern edifications constructed at different times in a subtropical environment in Campeche, Mexico.

Methods:
A chronosequence of walls built one, five and 10 years ago was considered. On each wall, three surface areas of 3 × 3 cm of the corresponding biofilm were scraped for subsequent analysis. Fungi were isolated by washing and particle filtration technique and were then inoculated in two contrasting culture media (oligotrophic and copiotrophic). The fungi were identified according to macro and microscopic characteristics.

Key results:
We found 73 genera and 202 species from 844 isolates. Our results showed that fungal communities differed in each biofilm. In the middle-aged biofilm a high number of isolates was found, but both species richness and diversity were low. In contrast, in the old biofilm species richness and diversity were high; Hyphomycete 1, Myrothecium roridum and Pestalotiopsis maculans were abundant. The dominant species in the middle-aged biofilm were Curvularia lunata, Curvularia pallescens, Fusarium oxysporum and Fusarium redolens, and in the young biofilm were Cladosporium cladosporioides, Curvularia clavata, Paraconiothyrium sp. and Phoma eupyrena.

Conclusions:
Our results suggest that the composition of the fungal community in each biofilm varies according to time of exposure to the environment. Furthermore, the fungal community was composed of a pool of uncommon species that might be autochthonous to limestone.

Full text

117: 59-77 Octubre 2016 Research article
59
Changes in fungal community composition of biolms on limestone
across a chronosequence in Campeche, Mexico
Cambios en la composición de la comunidad fúngica de biopelículas
sobre roca calcárea a través de una cronosecuencia en Campeche, México
Sergio Gómez-Cornelio1,4, Otto Ortega-Morales2, Alejandro Morón-Ríos1, Manuela Reyes-Estebanez2 and Susana de la
Rosa-García3
1 El Colegio de la Frontera Sur, Av. Ran-
cho polígono 2A, Parque Industrial
Lerma, 24500 Campeche, Mexico.
2 Universidad Autónoma de Campe-
che, Departamento de Microbiología
Ambiental y Biotecnología, Avenida
Agustín Melgar s/n, 24039 Campe-
che, Mexico.
3 Universidad Juárez Autónoma de Ta-
basco, División Académica de Cien-
cias Biológicas, Carretera Villahermo-
sa-Cárdenas km 0.5 s/n, entronque a
Bosques de Saloya, 86150 Villaher-
mosa, Tabasco, Mexico.
4 Author for correspondence:
sgomez@ecosur.edu.mx
To cite as:
Gómez-Cornelio, S., O. Ortega-Morales,
A. Morón-Ríos, M. Reyes-Estebanez
y S. de la Rosa-García. 2016. Changes
in fungal community composition of
biolms on limestone across a chrono-
sequence in Campeche, Mexico. Acta
Botanica Mexicana 117: 59-77.
Received: 28 de marzo de 2016.
Reviewed: 6 de julio de 2016.
Accepted: 2 de septiembre de 2016.
AbstrAct:
Background and Aims: The colonization of lithic substrates by fungal communities is determined by
the properties of the substrate (bioreceptivity) and climatic and microclimatic conditions. However, the
effect of the exposure time of the limestone surface to the environment on fungal communities has not
been extensively investigated. In this study, we analyze the composition and structure of fungal commu-
nities occurring in biolms on limestone walls of modern edications constructed at different times in a
subtropical environment in Campeche, Mexico.
Methods: A chronosequence of walls built one, ve and 10 years ago was considered. On each wall, three
surface areas of 3 × 3 cm of the corresponding biolm were scraped for subsequent analysis. Fungi were
isolated by washing and particle ltration technique and were then inoculated in two contrasting culture
media (oligotrophic and copiotrophic). The fungi were identied according to macro and microscopic
characteristics.
Key results: We found 73 genera and 202 species from 844 isolates. Our results showed that fungal
communities differed in each biolm. In the middle-aged biolm a high number of isolates was found,
but both species richness and diversity were low. In contrast, in the old biolm species richness and diver-
sity were high; Hyphomycete 1, Myrothecium roridum and Pestalotiopsis maculans were abundant. The
dominant species in the middle-aged biolm were Curvularia lunata, Curvularia pallescens, Fusarium
oxysporum and Fusarium redolens, and in the young biolm were Cladosporium cladosporioides, Cur-
vularia clavata, Paraconiothyrium sp. and Phoma eupyrena.
Conclusions: Our results suggest that the composition of the fungal community in each biolm varies
according to time of exposure to the environment. Furthermore, the fungal community was composed of
a pool of uncommon species that might be autochthonous to limestone.
Key words: dominant species, fungal colonization, fungal diversity, succession, trophic preference.
resumen:
Antecedentes y Objetivos: La colonización de los sustratos líticos por comunidades fúngicas está deter-
minada por las propiedades del sustrato (bioreceptividad) y las condiciones climáticas y microclimáticas.
Sin embargo, los efectos del tiempo de exposición de la supercie de la roca calcárea al ambiente sobre
la composición de las comunidades fúngicas no se ha investigado. En este estudio, analizamos la com-
posición y estructura de las comunidades fúngicas inmersas en biopelículas asociadas a roca calcárea,
en paredes de edicaciones modernas construidas a diferentes tiempos en un ambiente subtropical en
Campeche, México.
Métodos: Se consideró una cronosecuencia de paredes construidas a uno, cinco y 10 años. Sobre cada
pared, se rasparon tres supercies de 3 × 3 cm para cada biopelícula. Los hongos se aislaron por la técnica
de lavado y ltración de partículas, posteriormente se inocularon en dos medios de cultivo contrastantes
(un medio oligotróco y uno copiotróco). Los hongos se identicaron de acuerdo a sus características
macro y microscópicas.
Resultados clave: Encontramos 73 géneros y 202 especies de 844 aislados. Los resultados mostraron
que las comunidades fúngicas son diferentes en las tres biopelículas. En la biopelícula de desarrollo in-
termedio encontramos un alto número de aislados, pero tanto la riqueza como la diversidad fueron bajas.
En contraste, en la biopelícula avanzada, los valores de riqueza de especies y diversidad fueron altos, y
las especies abundantes fueron Hyphomycete 1, Myrothecium roridum y Pestalotiopsis maculans. Las
especies dominantes en la biopelícula intermedia fueron Curvularia lunata, Curvularia pallescens, Fu-
sarium oxysporum y Fusarium redolens, y en la biopelícula joven fueron Cladosporium cladosporioides,
Curvularia clavata, Paraconiothyrium sp. y Phoma eupyrena.
Conclusiones: Nuestros resultados sugieren que la composición de la comunidad fúngica en cada biope-
lícula cambia de acuerdo al tiempo de exposición de la roca calcárea al ambiente. Además, como parte
de la composición de la comunidad fúngica, encontramos un conjunto de especies poco comunes que
podrían ser autóctonas en la roca calcárea.
Palabras clave: colonización fúngica, diversidad fúngica, especies dominantes, preferencia tróca, sucesión.

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
60
IntroductIon
It is well known that rocks, either in natural geological
settings or as part of monuments, are common habitats
for a wide range of microorganisms (Scheerer et al., 2009;
Miller et al., 2012). The colonization of lithic substrates
by microbial communities is inuenced by the properties
of the substrate, such as porosity, surface roughness and
mineralogical composition -bioreceptivity- (see review
in Miller et al., 2012), in addition to climate and micro-
climatic conditions (Guillitte, 1995; Ortega-Morales et
al., 1999; Gaylarde and Gaylarde, 2005; Barberousse et
al., 2006). Furthermore, communities of microorganisms
growing on lithic substrates, including fungi, may res-
pond differentially to environmental conditions over time
based on their ecophysiological requirements (Scheerer et
al., 2009; Mihajlovski et al., 2014).
In the tropics and subtropics, rocks are capable of
being colonized by microorganisms due to high levels
of relative humidity and particular bioreceptivity of the
limestone (Kumar and Kumar, 1999; Gómez-Cornelio et
al., 2012). Gaylarde and Gaylarde (2005) found that the
macro- and micro-environments of different geographical
regions play an important role in the biomass and com-
position of the microorganism groups that compose bio-
lms. For example, microbial biomass in Latin America
is dominated by cyanobacteria and fungi, while in Europe
phototrophs, including algae and cyanobacteria, are the
most common organisms. Furthermore, the development
of biolms on rocks represents an important stage in the
primary succession of terrestrial ecosystems (Chertov et
al., 2004; Gorbushina, 2007). In this process, fungi that
form part of biolms physically and chemically deteri-
orate rock, and thus actively participate in the formation
of protosoil and minerals and also accelerate this pro-
cess, enabling subsequent colonization of the substrate
by mosses, lichens or plants (Gorbushina and Krumbein,
2000; Steringer, 2000; Gadd, 2007). Although molecu-
lar techniques are commonly used to study communities
in the eld of environmental microbiology, the traditional
techniques of isolation and identication of fungi are of
vital importance in order to phenotypically characterize
fungi and to determine their role on epilithic substrates
(Ruibal et al., 2005; Gleeson et al., 2010).
Fungal epilithic communities have been studied in
a wide range of environments and for several lithotypes
(Steringer and Krumbein, 1997; Steringer and Prill-
inger, 2001; Urzì et al., 2001; Gorbushina et al., 2002;
Ruibal et al., 2005; Ruibal et al., 2009; Tang and Lian,
2012). However, most research has not considered the
inuence of time on the colonization patterns of fungal
communities. One exception was the study of Lan et al.
(2010), in which fungal communities of young and old
biolms on sandstone were found substantially differ-
ent. Furthermore, the importance of lamentous fungi
as rock colonizers and their ecological role in environ-
ments are not well-understood, especially in tropical and
subtropical climates. More attention has been placed on
the microcolonial fungi, meristematic fungi and yeasts
of temperate climates (Steringer and Krumbein, 1997;
Gorbushina et al., 2002; Chertov et al., 2004; Gorbushina
et al., 2005; Ruibal et al., 2005; 2009; Steringer et al.,
2012). Therefore, in order to expand our current under-
standing of the fungal community associated with lime-
stone, we studied the culturable subset of fungi in bio-
lms exposed to similar environmental conditions and
substratum properties. A chronosequence was considered
by examining the biolms of three walls constructed one,
ve and 10 years ago.
mAterIAls And methods
Study area and climate variables
The coastal city of Campeche, Mexico has a subtropical
climate and an altitudinal range of 3-10 m. The studied
biolms were relatively categorized as young, middle-
aged and old, corresponding to walls that were construc-
ted with limestone rock fragments one, ve and 10 years
ago, respectively, according to historical documentation
(Fig. 1). The colonization of buildings by fungi may ini-
tiate shortly after construction but the formation of bio-
lm usually takes several years (Barberousse et al., 2006;
Gómez-Cornelio et al., 2012; Adamson et al., 2013). Hen-

117: 59-77 Octubre 2016
61
Limestone walls with comparable characteristics
of exposure to the surrounding environment were chosen.
All walls were composed of rock blocks and had similar
substratum properties (or bioreceptivity), vertical surfac-
es and homogeneous coverage of biolms. In addition,
all walls were oriented towards the north where low solar
irradiation and high relative humidity prevail in compar-
ison to facades oriented towards other directions (Ad-
amson et al., 2013; Ortega-Morales et al., 2013). Further
criteria for selecting the walls included the absence of sur-
rounding vegetation and low levels of human disturbance,
such as painting or washing. Sites with automobile traf-
c or post-construction remodeling works were avoided.
Samples were taken from above the height of one meter in
order to avoid confounding factors such as potential mi-
crobial colonization due to splashing water. Hence, time
elapsed since construction of the walls and establishment
of biolms was the main inuential variable considered in
the analysis of fungal community structure.
Biolm sampling and fungal isolation
In the dry season of January 2014, we sampled biolms
on the three selected limestone surfaces. Mean maximum
and minimum temperatures in January were 34 °C and
11.4 °C, respectively. Mean rainfall was 23 mm, and the
mean relative humidity was 83%. On each wall, we scra-
ped three surface areas of 3 × 3 cm to a maximum depth
of 3 mm, using a sterile scalpel. Scraping was performed
by the same person to avoid bias. The biomasses of the
biolms scraped from the wall were placed in sterile Petri
dishes and transferred to the laboratory for processing.
Fungi were isolated by washing and ltration
of particles technique (Bills et al., 2004). One gram of
each scraped biolm was placed in a washing apparatus
with micro-sieves with pores of 250, 125, 100 and 75
µm (MINI-SIEVE INSERT-ASTD, Bel-Art Products,
Pequannock, New Jersey, USA), and was consequently
washed and ltered for 10 min using bi-distilled water.
This technique reduces the isolation of propagules from
spores, favoring only the isolation of fungi attached to
rock particles (Bills et al., 2004; Arias-Mota and Here-
Figure 1: Biolm samples from rock fragments of limestone buildings
(time elapsed since construction): A. young biolm (one year); B.
middle-aged biolm (ve years); C. old biolm (10 years).
ce, the monthly climate data were obtained from the lo-
cal meteorological observatory in Campeche, in order to
calculate the annual means of climate variables as well as
the mean conditions corresponding to the number of years
since walls were constructed and exposed to the environ-
ment. The considered climatic variables were: minimum
and maximum temperature, minimum, mean and maxi-
mum relative humidity and mean rainfall (Table 1).

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
62
Table 1: Geographical location, color and climatic parameters of biolms developed on the surface of sampled limestone walls. (Values are
expressed as means ± 1SD).
Young biolm Middle-aged biolm Old biolm
Geographical location 19º49'30.7"N
90º32'51.2"W
19º49'30.5"N
90º33'16.3"W
19º49'29.4"N
90º33'14"W
Mean temperature (ºC) Minimum 16.5 ± 2.5 16.4 ± 4.7 16.7 ± 2.5
Maximum 37.1 ± 4.3 37.6 ± 2.6 37.2 ± 4.5
Mean relative humidity (%) Minimum 43.8 ± 10.7 39.9 ± 9.4 39.6 ± 9.4
Mean 78.6 ± 5.6 75 ± 5.8 74.5 ± 6.6
Maximum 97.9 ± 1.2 97.8 ± 1.1 97.4 ±1.4
Mean rainfall (mm) Rainy season 224.9 ± 11.3 192.6 ± 54 194.8 ± 29
Dry season 20.4 ± 19 19.8 ± 17 24.4 ± 20
Degree of colonization (visual inspection) Not observed Dark green Black
dia-Abarca, 2014) that may present active bioweathering
or serve a protective function on the surface of the lime-
stone.
Particles trapped on the 75 μm sieve were trans-
ferred to sterile lter paper and incubated for 24 h at 27
°C to remove excess water. In order to isolate the greatest
number of species, we used two culture media: a copi-
otrophic medium composed of 2% malt extract, 2% agar
and 0.2% CaCO3 (MEAC) and an oligotrophic medium of
0.2% CaCO3 and 2% agar (CCOA). Both media were ad-
justed to pH 7.7 and supplemented with chloramphenicol
(200 mg L-1) to inhibit the growth of bacteria. Media were
prepared with CaCO3, since it is the main component of
limestone (Burford et al., 2003). Under a stereomicro-
scope, 50 particles were transferred to 10 plates with
MEAC (5 particles per plate); this procedure was repeat-
ed for the CCOA medium. Plates were incubated at 27 °C
in darkness. After the fourth day, plates were inspected
daily for a period of four weeks. All fungal colonies that
emerged from the particles were puried in inclined tubes
with MEAC.
Morphological identication of fungi
The fungal isolates were identied according to macros-
copic characteristics, such as coloration, diameter, textu-
re, pigmentation, margin appearance, zonality and pro-
duction of exudates in the culture medium, in addition
to the morphological characteristics of their reproductive
and vegetative structures, including color, conidiogenesis,
spore type and size. Fungal isolates that sporulated were
identied using the taxonomic keys of Booth (1971), Ellis
(1971; 1976), Sutton (1980), Pitt (2000), Klich (2002),
Boerema et al. (2004), Domsch et al. (2007) and Seifert et
al. (2011). The identity of the species with more than eight
isolates was conrmed by performing genomic DNA ex-
traction and sequencing the ITS region (data not shown).
Fungal colonies that did not sporulate were inoc-
ulated into the following culture media: cornmeal agar,
oatmeal agar, potato-carrot agar, Czapek dox agar, potato
dextrose agar and V8 agar. Plates were then subjected to
cyclical periods of light/darkness (12/12 h) to promote
sporulation (Bills et al., 2004) and incubated at 27 °C.
Every fourth day for up to six weeks, plates were checked
for signs of reproductive structures. Isolates that not pro-
duced spores were separated into morphotaxa, according
to their macroscopic and microscopic morphology in the
different culture media. All fungal isolates were con-
served in malt extract broth supplemented with glycerol
(20% [vol/vol]) at -80 °C; agar plugs with mycelium were
conserved in sterile distilled water at room temperature.

117: 59-77 Octubre 2016
63
Data analysis
The fungal communities of the sampled biolms were
analyzed according to species richness, dened as the
number of different fungi species per biolm, in addition
to species abundance as the number of fungal isolates
per identied species. The colonization frequency of the
particles was determined as number of emerged fungal
species (one or two per particle) from particles divided
by the number of inoculated particles, multiplied by 100
in order to obtain the percentage of particles with adhered
mycelium (Bills et al., 2004). In order to determine the
substrates that have been reported for the fungi that were
identied at the species level, we used the literature pre-
viously employed in the identication of fungi and per-
formed a search in Summon system. Fungal diversity was
calculated with the Simpson´s (D’) and Shannon´s (H’)
diversity indices, in addition to the Shannon (J’) evenness
index, which were performed in the EstimateS 9.1 soft-
ware (Colwell, 2013). In order to determine the similarity
and composition of the fungal species found in the three
biolms, the Jaccard index was calculated, and a Venn
diagram was created.
results
Analysis of fungal composition and diversity
In the mycological analysis 844 isolates were recove-
red, distributed in 73 genera and 202 species (Table 2).
The identied species were grouped as follows: 149 As-
comycota, one Basidiomycota and 52 Mycelia sterilia.
Hyphomicetous asexual species of Ascomycota (108
species) dominated, while Coelomycetous species repre-
sented 21% of the isolates (38 species). The genera with
highest number of species (>4) and isolates (>17) in the
fungal community were Aspergillus P. Micheli ex Haller,
Cladosporium Link, Curvularia Boedijn, Fusarium Link,
Microsphaeropsis Höhn., Myrothecium Tode, Nodulispo-
rium Preuss, Paraconiothyrium Verkley and Phoma Sacc.
(Table 2).
We found the largest number of isolates (322) in
the middle-aged biolm, followed by the old and young
biolms with 268 and 254 isolates, respectively. Howev-
er, species richness and diversity were higher in the old
biolm and lower in the middle-aged biolm (Table 3).
The Shannon evenness index of the old biolm generated
a value close to 1, and in the middle-aged biolm, a value
of 0.47 (Table 3). The Jaccard’s similarity index showed
a low degree of similarity among the young, middle-aged
and old fungal communities inhabiting the biolms. The
resulting index values were similar for the comparisons of
young and middle-aged (0.18), young and old (0.16) and
middle-aged and old (0.15) biolms.
Overall, of the 202 species identied, 26% were
isolated from inoculated particles in both culture media
(52 species), while 36% (73 species) were found exclu-
sively in the oligotrophic medium (CCOA) and 38% in
the copiotrophic medium (MEAC). Additionally, in the
analysis of colonization frequency of particles from all
three biolms, we observed a high number of particles
with adhered mycelia (Table 3). The middle-aged biolm
showed the highest percentage (80%) of colonization;
however, most mycelium that emerged from these par-
ticles belonged to the species Curvularia lunata (Wak-
ker) Boedijn, Fusarium oxysporum Schltdl. and Fusarium
redolens Wollenw. (Table 2). The young and old biolms
presented a minor colonization frequency of particles (Ta-
ble 3).
In the old biolm, a high number of isolated species
(35%) was specic to only one of the media, and a slight-
ly lower proportion was present in both media (30 %).
In young and middle-aged biolms, 41% of species were
indistinctly isolated from both media (Table 2). Isolates
of Lasiodiplodia theobromae (Pat.) Griffon & Maubl.
and Nigrospora oryzae (Berk. & Broome) Petch were ob-
tained in the MEAC medium. A high percentage (>70%)
of species from the genera Aspergillus, Penicillium Link
and Trichoderma Pers. were also isolated. Meanwhile,
in the CCOA medium the lithic species Friedmanniomy-
ces simplex Selbmann, de Hoog, Mazzaglia, Friedmann
& Onofri (6 isolates) was found as well as Stachybotrys
Corda species and a large number of uncommon species
(Table 2).

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
64
Table 2: Epilithic fungal community in terms of abundance of species isolated from biolms on limestone at different stages of development and
color of their reproductive structures. (M: melanized and H: hyaline).
aCopiotrophic medium (MEAC).
bOligotrophic medium (CCOA).
*Mycelia sterilia with one or more isolates, which are added in the total.
Epilithic fungi Coloration Young biolm Middle-aged
biolm
Old biolm Total
Ascomycota
Sordaria micola (Roberge ex Desm.) Ces. & De Not. M 2 13a,b
Xylariales sp. 1 M1 1b
Xylariales sp. 2 M 1 1a
Xylariales sp. 3 M 1 1b
Coelomycetous asexual species of Ascomycota
Ascochyta carpathica (Allesch.) Keissl. M 2 2a
Clypeopycnis sp. M 2 136a,b
Coleophoma sp. M 7 7a,b
Colletotrichum crassipes (Speg.) Arx H1 1a
Colletotrichum dematium (Pers.) Grove M1 1a
Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. H1 1b
Coniothyrium multiporum (V.H. Pawar, P.N. Mathur &
Thirum.) Verkley & Gruyter
M1 1b
Cytospora polygoni-sieboldii Henn. M1 1a
Lasiodiplodia theobromae (Pat.) Griffon & Maubl. M 7 7a
Microsphaeropsis arundinis (S. Ahmad) B. Sutton M12 3a,b
Microsphaeropsis sp. 1 M1 1b
Microsphaeropsis sp. 2 M 2 2a,b
Neosetophoma samararum (Desm.) Gruyter, Aveskamp &
Verkley
M1 1a
Paraconiothyrium sp. M19 3 22a,b
Paraphoma chrysanthemicola (Hollós) Gruyter, Aveskamp
& Verkley
M1 1b
Paraphoma meti (Brunaud) Gruyter, Aveskamp & Verkley M 2 3 5a,b
Pestalotiopsis maculans (Corda) Nag Raj M5310 18a,b
Peyronellaea aurea (Gruyter, Noordel. & Boerema)
Aveskamp, Gruyter & Verkley
M 2 2a,b
Peyronellaea gardeniae (S. Chandra & Tandon) Aveskamp,
Gruyter & Verkley
M1 1b
Phlyctema lappae (P. Karst.) Sacc. M1 1a
Phoma adianticola (E. Young) Boerema M4 4a
Phoma crystallifera Gruyter, Noordel. & Boerema M1 1b
Phoma eupyrena Sacc. M 23 12 15 50a,b

117: 59-77 Octubre 2016
65
Table 2: Continuation.
Epilithic fungi Coloration Young biolm Middle-aged
biolm
Old biolm Total
Phoma herbarum Westend. M4 1 2 7a,b
Phoma heteroderae Sen Y. Chen, D.W. Dicks. & Kimbr. M 2 2a,b
Phoma leveillei Boerema & G.J. Bollen M1 1b
Phoma multirostrata (P.N. Mathur, S.K. Menon & Thirum.)
Dorenb. & Boerema
M1 1 2b
Phoma paspali P.R. Johnst. M5 5a,b
Phoma pratorum P.R. Johnst. & Boerema M1 1a
Phoma proteae Crous M1 1b
Phoma putamina Speg. M12 3a,b
Phoma sp. 1 M1 1a
Phoma sp. 2 M 2 2a,b
Phoma tropica R. Schneid. & Boerema M 2 2a,b
Phomopsis putator (Nitschke) Traverso M1 1a
Pleurophomopsis lignicola Petr. M1 1 2a
Pyrenochaetopsis pratorum (Berk. & M.A. Curtis) M.B.
Ellis
M1 1b
Westerdykella minutispora (P.N. Mathur) Gruyter, Aveskamp
& Verkley
M4 1 1 6a,b
Hyphomicetous asexual species of Ascomycota
Acremoniella velutina (Fuckel) Sacc. M1 1a
Acremonium brachypenium W. Gams H 2 2b
Acremonium fusidioides (Nicot) W. Gams H1 1b
Acremonium rutilum W. Gams H 2 2b
Acremonium sordidulum W. Gams & D. Hawksw. H1 1a
Agaricodochium sp. H1 1a
Alternaria longipes (Ellis & Everh.) E.W. Mason M1 1a
Alternaria tenuissima (Kunze) Wiltshire M1 1 2a,b
Arxiella terrestris Papendorf H1 1b
Aspergillus aculeatus Iizuka M 2 2a
Aspergillus alliaceus Thom & Church H1 1a
Aspergillus awamori Nakaz. M 2 2a
Aspergillus foetidus Thom & Raper M 2 2a
Aspergillus fumigatus Fresen. M 2 2a
Aspergillus japonicus Saito M1 1a
Aspergillus niger Tiegh. M 7 7a
Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud M1 1a
Badarisama sp. M1 1a

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
66
Baudoinia sp. M1 1b
Calcarisporium sp. M1 1a
Capnobotryella antalyensis Sert & Ster. M1 1a
Chaetasbolisia falcata V.A.M. Mill. & Bonar M1 1b
Chromelosporium sp. H1 1a
Cladosporium cladosporioides (Fresen.) G.A. de Vries M 22 10 8 40a,b
Cladosporium oxysporum Berk. & M.A. Curtis M10 8 18a,b
Cladosporium sphaerospermum Penz. M9211a,b
Cladosporium tenuissimum Cooke M 2 2 4a,b
Corynespora citricola M.B. Ellis M1 1a
Corynesporella pinarensis R.F. Castañeda M1 1b
Curvularia australiensis (Tsuda & Ueyama) Manamgoda, L.
Cai & K.D. Hyde
M1 1 5 7a,b
Curvularia brachyspora Boedijn M 2 2a
Curvularia clavata B.L. Jain M18 3122a,b
Curvularia fallax Boedijn M1 1b
Curvularia hawaiiensis (Bugnic. ex M.B. Ellis) Manamgoda,
L. Cai & K.D. Hyde
M1 1b
Curvularia lunata (Wakker) Boedijn M10 105 26 141a,b
Curvularia pallescens Boedijn M6 13 11 30a,b
Curvularia spicifera (Bainier) Boedijn M1 1a
Curvularia verruculosa Tandon & Bilgrami ex. M.B. Ellis M 7 1 8a,b
Curvularia sp. M 2 2a,b
Echinocatena sp. M1 1b
Exochalara longissima (Grove) W. Gams & Hol.-Jech. M 2 2a
Friedmanniomyces simplex Selbmann, de Hoog, Mazzaglia,
Friedmann & Onofri
M13 2 6b
Fusarium camptoceras Wollenw. & Reinking H1 1b
Fusarium equiseti (Corda) Sacc. H1 1a
Fusarium occiferum Corda H1 1 2a,b
Fusarium incarnatum (Desm.) Sacc. H1 1b
Fusarium oxysporum Schltdl. H8 40 4 52a,b
Fusarium redolens Wollenw. H14 41 9 64a,b
Fusarium sacchari (E.J. Butler & Haz Khan) W. Gams H1 1a
Fusarium solani (Mart.) Sacc. H 2 2 4a,b
Fusarium subglutinans (Wollenw. & Reinking) P.E. Nelson,
Toussoun & Marasas
H1 1b
Table 2: Continuation.
Epilithic fungi Coloration Young biolm Middle-aged
biolm
Old biolm Total

117: 59-77 Octubre 2016
67
Fusarium tabacinum (J.F.H. Beyma) W. Gams H1 1b
Fusarium ventricosum Appel & Wollenw. H1 1a
Gabarnaudia sp. H1 1b
Geotrichum candidum Link H 2 2a,b
Gilmaniella subornata Morinaga, Minoura & Udagawa M1 1a
Graphium penicillioides Corda M 2 2a,b
Hyphomycete 1 M11 11a,b
Microdochium dimerum (Penz.) Arx H12 3b
Microdochium nivale (Fr.) Samuels & I.C. Hallett H 2 2b
Monodictys uctuata (Tandon & Bilgrami) M.B. Ellis M1 1b
Monodictys paradoxa (Corda) S. Hughes M 3 3a,b
Myrothecium cinctum (Corda) Sacc. M 3 3a
Myrothecium roridum Tode M12 16 28a,b
Myrothecium sp. 1 M134a,b
Myrothecium sp. 2 M1 1a
Myrothecium sp. 3 M1 1a
Nalanthamala madreeya Subram. H1 1b
Nigrospora oryzae (Berk. & Broome) Petch M 3 1 1 5a
Nodulisporium acervatum (Massee) Deighton M1 1b
Nodulisporium ochraceum Preuss M1 1a
Nodulisporium puniceum (Cooke & Ellis) Deighton M 3 2 5a,b
Nodulisporium radians (Berk.) Deighton M1 1b
Nodulisporium sp. 1 M1 1b
Nodulisporium sp. 2 M1 1a
Nodulisporium sylviforme Deighton M1 4 5a,b
Nodulisporium thelenum (Sacc.) G. Sm. M1 1 2a,b
Ochroconis tshawytschae (Doty & D.W. Slater) Kiril. &
Al-Achmed
M1 1b
Penicillium citreonigrum Dierckx H1 1a
Penicillium dierckxii Biourge H1 1b
Penicillium islandicum Sopp H1 1a
Penicillium oxalicum Currie & Thom H1 1a
Periconia igniaria E.W. Mason & M.B. Ellis M1 1a
Periconiella mucunae M.B. Ellis M1 1a
Prathoda longissima (Deighton & MacGarvie) E.G.
Simmons
M1 1b
Table 2: Continuation.
Epilithic fungi Coloration Young biolm Middle-aged
biolm
Old biolm Total

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
68
Pseudohelicomyces albus Garnica & E. Valenz. H 2 2a,b
Pseudopithomyces chartarum (Berk. & M.A. Curtis) J.F. Li,
Ariyawansa & K.D. Hyde
M1 1a
Pseudoramichloridium brasilianum (Arzanlou & Crous)
Cheew. & Crous
M1 1a
Ramichloridium apiculatum (J.H. Mill., Giddens & A.A.
Foster) de Hoog
M 2 2b
Sarocladium kiliense (Grütz) Summerb. H1 1a
Sarocladium strictum (W. Gams) Summerb. H1 1b
Scolecobasidium constrictum E.V. Abbott M 3 4 1 8a,b
Sepedonium sp. H1 1b
Stachybotrys microspora (B.L. Mathur & Sankhla) S.C. Jong
& E.E. Davis
M1 1b
Stachybotrys nephrospora Hansf. M1 1b
Stachybotrys renispora P.C. Misra M1 1b
Tolypocladium sp. H1 1b
Torula herbarum (Pers.) Link M1 1a
Trichobotrys sp. M1 1a
Trichocladium sp. M1 1b
Trichoderma aggressivum Samuels & W. Gams H1 1 2a
Trichoderma harzianum Rifai H 2 13a,b
Trichoderma longibrachiatum Rifai H 2 2a
Trichoderma ovalisporum Samuels & Schroers H1 1a
Trichoderma strigosum Bissett H1 1a
Veronaea musae M.B. Ellis M1 1a
Verruconis verruculosa (R.Y. Roy, R.S. Dwivedi & R.R.
Mishra) Samerp. & de Hoog
M1 1a
Basidiomycota
Geotrichopsis sp. H1 1a
Mycelia sterilia
Mycelia sterilia (Morphotaxon 01) H1 1 1 3b
Mycelia sterilia (Morphotaxon 02) M 2 2a,b
Mycelia sterilia (Morphotaxon 03) H1 1 2b
Mycelia sterilia (Morphotaxon 04) M1 1 2b
Mycelia sterilia (Morphotaxon 05) M1 1 2a,b
Mycelia sterilia (Morphotaxon 06-10*) M1 5a
Mycelia sterilia (Morphotaxon 11-15*) M1 5b
Mycelia sterilia (Morphotaxon 16) H1 1b
Table 2: Continuation.
Epilithic fungi Coloration Young biolm Middle-aged
biolm
Old biolm Total

117: 59-77 Octubre 2016
69
Table 3: Abundance, richness, diversity and evenness of the fungal epilithic community colonizing biolms on limestone at different stages of
development.
State of biolms Abundance
(number of
isolates)
Richness (number
of species)
Simpson Diversity
index (D’)
Shannon Diversity
index (H’)
Shannon evenness
index (J’)
Particles
colonization (%)
Young 254 83 25.8 3.7 0.67 73
Middle-aged 322 59 6.8 2.7 0.47 86
Old 268 117 37.2 4.1 0.75 75
Of the 124 fungi identied at the species level,
many were associated with numerous substrates, based
on the literature review to determine with which sub-
strates identied fungal species had been previously as-
sociated (Fig. 2). Thirty-one species were identied as
cosmopolitan and belonged to the genera Cladosporium,
Curvularia, Fusarium and Penicillium, including sev-
eral common species, such as Aureobasidium pullulans
(de Bary & Löwenthal) G. Arnaud and Geotrichum can-
didum Link (Domsch et al., 2007). Most of the identied
species (81) have been reported in soil, mainly species of
Mycelia sterilia (Morphotaxon 17-18*) M 3 6a,b
Mycelia sterilia (Morphotaxon 19-20*) M 2 4a,b
Mycelia sterilia (Morphotaxon 21) M 2 2b
Mycelia sterilia (Morphotaxon 22-23*) M 12a
Mycelia sterilia (Morphotaxon 24-25*) H12b
Mycelia sterilia (Morphotaxon 26) M1 1b
Mycelia sterilia (Morphotaxon 27) H 3 3a,b
Mycelia sterilia (Morphotaxon 28) M 2 2b
Mycelia sterilia (Morphotaxon 29-34*) M1 6a
Mycelia sterilia (Morphotaxon 35-39*) H1 5a
Mycelia sterilia (Morphotaxon 40-50*) M111b
Mycelia sterilia (Morphotaxon 51-52*) H12b
Table 2: Continuation.
Epilithic fungi Coloration Young biolm Middle-aged
biolm
Old biolm Total
the genera Aspergillus, Microdochium Syd., Monodictys
S. Hughes, Myrothecium, Phoma, Scolecobasidium E.V.
Abbott and Trichoderma. Many species (78) were also
associated with plants, including the genera Alternaria,
Colletotrichum Corda, Microsphaeropsis, Monodictys,
Myrothecium, Phoma and Sarocladium W. Gams & D.
Hawksw. 44 of the identied species have been found
in litter, corresponding to the genera of Myrothecium,
Scolecobasidium and Stachybotrys. Forty species (genus
Monodictys) have been associated with air, and 39 spe-
cies with wood (genera Nodulisporium and Phlyctema

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
70
old biolms had the highest number of exclusive species
(82), among these species of the genera Aspergillus, La-
siodiplodia Ellis & Everh., Penicillium and Stachybotrys,
in addition to Xylariales spp. and Hyphomycete 1.
The Venn diagram shows 16 species in the core
group (three biolms) of the fungal community (Fig. 3
and Table 2). Meanwhile, Cladosporium cladosporioides
(Fresen.) G.A. de Vries, Curvularia clavata B.L. Jain,
Nigrospora oryzae, Phoma eupyrena Sacc., Phoma her-
barum Westend. and Westerdykella minutispora (P.N.
Mathur) Gruyter, Aveskamp & Verkley were abundant in
the young biolm. In the middle-aged biolm Curvularia
lunata, Curvularia pallescens Boedijn, Friedmanniomy-
ces simplex, Fusarium oxysporum, Fusarium redolens
and Scolecobasidium constrictum E.V. Abbott, and in the
old biolm Clypeopycnis sp., Curvularia australiensis
(Tsuda & Ueyama) Manamgoda, L. Cai & K.D. Hyde and
Pestalotiopsis maculans (Corda) Nag Raj were frequent
(Fig. 4 and Table 2). Twenty-ve species were present in
at least two biolms; the young and old biolms had the
highest number of shared species (12). Paraconiothyri-
um sp. was dominant and was isolated 19 times in the
young biolm, although its abundance diminished in the
Figure 2: Frequency of substrate types reported for the fungal species
isolated from biolms according to the literature.
Desm.). Finally, 40 and 31 species have been reported on
rocks and in water, respectively.
Species dominance
With respect to species abundance, 61% of all fungal spe-
cies were isolated only once, and 6% were isolated more
than 10 times. The remaining fungi (33%) were isolated
from 2 to 9 times. The encountered fungal community was
mainly dominated by fungi that contain melanin at some
or all of their reproductive stages (149 species). Only 53
species (26%) were found with hyaline structures without
pigments (Table 2). In all biolms, we found that species
composition has an approximate ratio of 4:1 of melanized
fungi to hyaline species.
In regard to the fungal communities, most co-exist-
ing groups of species may be associated with a particular
biolm, characterized by time of exposure of the substrate
(limestone) to the environment; this is shown in the Venn
diagram (Fig. 3). In the young biolm, we found 49 ex-
clusive species, including several from the genera Colle-
totrichum and Coleophoma Höhn.; the species Monodyc-
tis paradoxa (Corda) S. Hughes, Myrothecium cinctum
(Corda) Sacc., Phoma adianticola (E. Young) Boerema
and Phoma paspali P.R. Johnst. were also prominent. In
the middle-aged biolm, only 32 exclusive species were
found, although these were not frequent (> 2 isolates). The
Figure 3: Venn diagram indicating number of common and exclusive
fungal species to the studied biolms.

117: 59-77 Octubre 2016
71
middle-aged biolm. Cladosporium oxysporum Berk. &
M.A. Curtis, Cladosporium sphaerospermum Penz. and
Nodulisporium puniceum (Cooke & Ellis) Deighton were
found at a higher frequency in the young biolm in com-
parison to the old biolm. Curvularia verruculosa Tan-
don & Bilgrami ex. M.B. Ellis was also more frequent
in the middle-aged biolm than the old biolm. Mean-
while, the species Nodulisporium sylviforme Deighton
and Paraphoma meti (Brunaud) Gruyter, Aveskamp &
Verkley were more dominant in the old biolm than in the
young biolm. Finally, Myrothecium roridum Tode was
more frequent in the old biolm than in the middle-aged.
dIscussIon
The fungal communities of biolms occurring on limes-
tone walls, considering time elapsed since wall construc-
tion, presented different degrees of microbial coloniza-
tion. In the 5- and 10-year-old samples, colonization was
evident by the green and black biomass of the biolms
that were visibly observed (Fig. 1), which is consistent
with the ndings of Adamson et al. (2013). The phototro-
phs colonizing such substrates are mainly composed of -
lamentous cyanobacteria and cocoidal bacteria (Scheerer
et al., 2009); these have been found on Mayan buildings
in the Yucatan peninsula (Ortega-Morales et al., 1999;
2013). The metabolic products of these organisms pro-
vide nutritional support for the establishment of hetero-
trophic communities, including fungi, allowing for their
colonization (De la Torre et al., 1991).
The fungal composition was dominated by species
of the Ascomycota class, which are common in rock sub-
strates; these Hyphomicetous asexual species are able to
colonize rocks during the rst year of exposure (Ruibal
et al., 2008; Gleeson et al., 2010; Hallman et al., 2011;
Gómez-Cornelio et al., 2012). In contrast, Coelomycet-
ous species are known to colonize limestone in Mediter-
Figure 4: Fungal species with the highest number of isolates of the three biolms a different stages of development on limestone.

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
72
ranean regions (Wollenzien et al., 1995). The identica-
tion of only one taxon belonging to Basidiomycota may
indicate that this group of organisms is not common in
biolms developing on limestone, as conrmed by Tang
and Lian (2012), who used culture-independent methods;
however, species of this group may exist in sterile form.
The genera Aspergillus, Cladosporium, Curvularia, Fu-
sarium, Microsphaeropsis, Myrothecium, Nodulisporium,
Paraconiothyrium and Phoma are common in subtropical
environments and in this study (Table 2). For other re-
gions, some of the most common, dominant genera that
have been reported on epilithic substrates include Alter-
naria, Cladosporium, Fusarium, Penicillium, Phoma and
Trichoderma (Wollenzien et al., 1995; Kumar and Kumar,
1999; Gorbushina and Krumbein, 2000; Urzì et al., 2001;
Gorbushina et al., 2002). All these genera were also iden-
tied in this study, but only Cladosporium, Fusarium and
Phoma were common and dominant. These differences
may be dictated by environmental factors and the biore-
ceptivity of rocks.
Some isolates showed no reproductive structures
(8%) and were identied as Mycelia sterilia (25% of mor-
phospecies). However, few isolates belonged to these mor-
phospecies, indicating that they are rare in the community
(Table 2). The nding of sterile mycelia commonly occurs
in microbiological studies of other substrates (Arias-Mota
and Heredia-Abarca, 2014; Rocha et al., 2014; Khirilla et
al., 2015), including limestone substrates (Gómez-Corne-
lio et al., 2012). These fungi may be classied according
to morphotype based on their morphological characteris-
tics (Paulus et al., 2003). The production of less complex
structures and poorly elaborated reproduction systems
may represent an adaptation strategy in order to conserve
energy on certain substrates (Ruibal et al., 2005) due to
lack of nutrients or water.
In this study, greater species richness was found in
the old biolm. However, in another study on sandstone,
the highest number of species was reported for fresh bio-
lms in comparison to older biolms (Lan et al., 2010).
Meanwhile, in this study the Shannon evenness index
showed that the species identied from isolates were al-
most equitable in the old biolm. The lowest evenness
was obtained in the middle-aged biolm, probably due to
the presence of several dominant species, such as Curvu-
laria lunata and Fusarium redolens (Table 3). We found
higher diversity values in comparison to other studies on
fungal communities colonizing distinct substrates of plant
litter and endophytes (Collado et al., 2007; Reverchon et
al., 2010). This diversity may be due to the establishment
and accumulation of propagules on bare rock at a constant
and rapid speed. For example, an increase of 9 × 102 to
7.5 × 105 colony-forming units was documented in only
11 weeks (Gorbushina and Krumbein, 2000). Under ideal
environmental conditions, a high number of fungi could
colonize and grow on limestone.
These results, in addition to those of a previous
study that investigated the fungal communities on bare
limestone (Gómez-Cornelio et al., 2012), suggest that the
species richness of limestone in subtropical environments
is high in comparison to other rock surfaces, that have
been studied in Europe, for example, and in particular in
the Mediterranean (De la Torre et al., 1991; Steringer
and Prillinger, 2001; Gorbushina et al., 2002; Ruibal et
al., 2005; Hallman et al., 2011). Although the intrinsic
characteristics of rock, such as its mineral composition,
porosity and roughness, have been reported to inuence
the colonization of microbial communities (Guillitte,
1995; Burford et al., 2003; Lan et al., 2010), the study of
Tomaselli et al. (2000) did not nd a relationship among
existing organisms and the petrographic characteristics of
rock. In this study, the high values of species richness and
diversity may be attributed to favorable environmental
factors in the subtropics (Table 1) and time elapsed
since initial colonization (Gaylarde and Gaylarde, 2005;
Mihajlovski et al., 2014).
Furthermore, the Jaccard’s similarity index showed
low values of similarity among fungal communities cor-
responding to different ages. This is notable in consider-
ing that the middle-aged and old biolms were located
less than 1 km from each other. These biolms also had
a similar chromatic aspect; therefore, one might expect a
high degree of similarity. Meanwhile, the young biolm

117: 59-77 Octubre 2016
73
was located at an approximate distance of 10.5 kilometers
from the other biolms and had a visible although incipi-
ent colonization of microorganisms. Microclimatic differ-
ences present at each site may contribute towards the for-
mation of a unique and particular mycobiota (Mihajlovski
et al., 2014). Our results differed from those reported for
sandstone, in which no differences in eukaryotic compo-
sition were found between fresh and young biolms (Lan
et al., 2010). However, to the contrary, on a serpentine
substrate fungal communities were found to have low
similarity (Daghino et al., 2012). These ndings highlight
that the community composition and diversity of fungi
are not always determined by substrate bioreceptivity.
Based on the fungi that emerged from the analyzed
particles that were scraped from biolms on limestone,
we were able to evaluate the fungal network and nd a
high proportion of active mycelium. The use of an oli-
gotrophic culture medium (CCOA) allowed for the char-
acterization of the cultivable fungi, which represented a
complete, diverse and functional community (Ruibal et
al., 2005). Although many of these species require water
to grow, such as species of the Stachybotrys genus (Jain
et al., 2009), the products of the extracellular matrix and
the retention of water in rock pores could allow for the
growth of water-demanding species. A large variety of
specialized fungi were observed in this study; these spe-
cies could represent an autochthonous community specif-
ic to limestone, a substrate with limited nutrients. How-
ever, due to the intrinsic environmental conditions of the
tropics, nutrients may be transported by air and deposited
on the rock as dust (Kumar and Kumar, 1999; Gorbush-
ina and Krumbein, 2000; Ruibal et al., 2009), thereby
providing conditions for the colonization of specialized
fungal species in the production of exopolymers allow-
ing fungi to adhere to the surfaces; some fungi are also
capable of biomineralizing limestone. Furthermore, many
fungi are able to successfully establish on limestone since
they precipitate calcium; these represent the main sink of
toxic forms of calcium in the soil or other environments
(Steringer, 2000). Hence, fungi are essential members of
the microbial communities that develop in the biolms of
limestone.
The composition of fungal communities may also
be inuenced by surrounding substrates (Urzì et al., 2001;
Hallman et al., 2011). As previously mentioned, we identi-
ed the substrates previously reported in the literature for
the 124 fungi that were identied in our study at the spe-
cies level. A high proportion of fungi may have originated
from soil or plants, and to a lesser extent, from decompos-
ing litter and/or wood. Also, these fungal species could
come from other rocks or from spores suspended in air or
water (Fig. 2). However, the establishment and develop-
ment of fungi over a period of time may be determined by
the interactions of species with their environmental con-
ditions, such as relative humidity and temperature (Gor-
bushina and Krumbein, 2000; Gorbushina et al., 2005), in
addition to the bioreceptivity of the substrate. The variety
of fungi reported for other surrounding substrates con-
rms that a large quantity of propagules may potentially
reach and colonize rock surfaces. Therefore, according to
our ndings, limestone surfaces could act as a reservoir
of fungal species and function as a fungal source via the
dispersion of species under ideal conditions.
Most species isolated from biolms occurred only
once or twice, and few species showed dominance. The
isolation technique used in this study may promote the
growth of rare and uncommon species; these results do
concur with those found by other authors who used both
taxonomic and molecular identication techniques (Col-
lado et al., 2007; Ruibal et al., 2008; Gómez-Cornelio et
al., 2012). Additionally, the abundance of dominant spe-
cies of the three analyzed biolms was variable and may
be determined by time of exposure, similar to the varia-
tions observed in Lan et al. (2010) in the microorganism
community of old and fresh biolms.
The fungal community contained melanin at some
or all of their reproductive stages (74% of the species);
this concurs with the reports of other authors, in which
the dominant fungi isolated from monuments also con-
tained pigmentation (Steringer and Krumbein, 1997;
Gorbushina et al., 2002; Lan et al., 2010). Pigmentation
in fungi may have different functions or result from the

Gómez-Cornelio et al.: Fungal communities composition in biolms on limestone
74
environmental conditions experienced on the rock surface
(Scheerer et al., 2009; Hallman et al., 2011). Therefore,
melanized communities of fungi have been shown to oc-
cur at a high frequency on rock surfaces, although com-
position may differ from one community to another, as
mentioned in the results.
Moreover, fungal propagules are capable of quick-
ly colonizing rock surfaces. Gorbushina and Krumbein
(2000) have suggested that uctuations in environmen-
tal conditions, such as those that lead to decits in nutri-
ents and water on the rock surfaces, promote changes in
the diversity of the fungal community. In our results the
environmental conditions and the characteristics of the
limestone substrate of the biolms were similar; thus one
would expect to nd a relationship among the studied fun-
gal communities. However, it may be necessary to study
the roles of dominant species during succession of fungal
communities. For example, it has been shown that the hy-
phae of common fungal species biomineralize the surface
of the limestone (Burford et al., 2006) and thus lead to
subsequent changes. In this study the composition of the
fungal community is likely determined by time of expo-
sure to the environment and species interactions, which
may then facilitate or inhibit colonization by other spe-
cies, leading to changes in the composition of the fungal
chronosequence associated with limestone (Fryar, 2002).
During this process, the functional properties of fungi on
the limestone may also be potentially affected.
conclusIons
In this study, the fungal communities immersed in biolms
were different at each stage of development, dened by the
time of exposure of the limestone substrate to the environ-
ment. Although the mineral composition of rock substrates
has been found to have a certain degree of inuence on the
structure of fungal communities, in this study limestone
samples with similar characteristics of bioreceptivity, such
as rock color, roughness and porosity, were selected. Envi-
ronmental conditions were also similar across sites in the
city of Campeche, Mexico. Therefore, in addition to time
of exposure, the differentiation in the community structu-
re and diversity of fungi in this study may be determined
by the interactions among the species of each biolm; this
should be conrmed by subsequent studies. A particular
species composition was isolated in each biolm corres-
ponding to a different developmental stage, although a
common pool of hyaline and melanized fungi appear to
colonize rock with great success and may have specic
functions on the rock substrate. In future studies, biotic
factors, including interactions among bacterial, fungal and
algal species, should be studied in order to determine their
inuence on the structure of the fungal community.
Acknowledgements
We are grateful to Julio C. Rojas León and Hugo Pera-
les Rivera for their comments on an early version of the
manuscript. This research was supported by institutional
funding from El Colegio de la Frontera Sur and the Uni-
versidad Autónoma de Campeche. We extend our thanks
to the Comisión Nacional del Agua for the provision of
meteorological data and to the Consejo Nacional de Cien-
cia y Tecnología for the doctoral scholarship awarded to
S.G.C. We thank two anonymous reviewers and the edi-
tor of the manuscript for suggested improvements; also
thanks to Allison Marie Jermain for reviewing the English
version of the manuscript.
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