Acta Botánica Mexicana
ISSN: 0187-7151
actabotmex@inecol.mx
Instituto de Ecología, A.C.
México
Gómez-Cornelio, Sergio; Ortega-Morales, Otto; Morón-Ríos, Alejandro; Reyes-Estebanez,
Manuela; de la Rosa-García, Susana
Changes in fungal community composition of biofilms on limestone across a
chronosequence in Campeche, Mexico
Acta Botánica Mexicana, núm. 117, octubre, 2016, pp. 59-77
Instituto de Ecología, A.C.
Pátzcuaro, México
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117: 59-77
Octubre 2016
Research article
Changes in fungal community composition of biofilms 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
AbstrAct:
1 El Colegio de la Frontera Sur, Av. Rancho polígono 2A, Parque Industrial
Lerma, 24500 Campeche, Mexico.
2 Universidad Autónoma de Campeche, Departamento de Microbiología
Ambiental y Biotecnología, Avenida
Agustín Melgar s/n, 24039 Campeche, Mexico.
3 Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias Biológicas, Carretera Villahermosa-Cárdenas km 0.5 s/n, entronque a
Bosques de Saloya, 86150 Villahermosa, 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
biofilms on limestone across a chronosequence 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.
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.
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 determinada por las propiedades del sustrato (bioreceptividad) y las condiciones climáticas y microclimáticas.
Sin embargo, los efectos del tiempo de exposición de la superficie de la roca calcárea al ambiente sobre
la composición de las comunidades fúngicas no se ha investigado. En este estudio, analizamos la composición y estructura de las comunidades fúngicas inmersas en biopelículas asociadas a roca calcárea,
en paredes de edificaciones 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 superficies de 3 × 3 cm para cada biopelícula. Los hongos se aislaron por la técnica
de lavado y filtración de partículas, posteriormente se inocularon en dos medios de cultivo contrastantes
(un medio oligotrófico y uno copiotrófico). Los hongos se identificaron 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 intermedio 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, Fusarium 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 biopelícula 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 trófica, sucesión.
59
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
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 influenced 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 microclimatic 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 respond 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 composition of the microorganism groups that compose biofilms. 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 biofilms 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 biofilms physically and chemically deteriorate rock, and thus actively participate in the formation
of protosoil and minerals and also accelerate this process, enabling subsequent colonization of the substrate
by mosses, lichens or plants (Gorbushina and Krumbein,
2000; Sterflinger, 2000; Gadd, 2007). Although molecular techniques are commonly used to study communities
in the field of environmental microbiology, the traditional
techniques of isolation and identification of fungi are of
vital importance in order to phenotypically characterize
60
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
(Sterflinger and Krumbein, 1997; Sterflinger and Prillinger, 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
influence 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
biofilms on sandstone were found substantially different. Furthermore, the importance of filamentous fungi
as rock colonizers and their ecological role in environments 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 (Sterflinger and Krumbein, 1997;
Gorbushina et al., 2002; Chertov et al., 2004; Gorbushina
et al., 2005; Ruibal et al., 2005; 2009; Sterflinger et al.,
2012). Therefore, in order to expand our current understanding of the fungal community associated with limestone, we studied the culturable subset of fungi in biofilms exposed to similar environmental conditions and
substratum properties. A chronosequence was considered
by examining the biofilms of three walls constructed one,
five 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
biofilms were relatively categorized as young, middleaged and old, corresponding to walls that were constructed with limestone rock fragments one, five and 10 years
ago, respectively, according to historical documentation
(Fig. 1). The colonization of buildings by fungi may initiate shortly after construction but the formation of biofilm usually takes several years (Barberousse et al., 2006;
Gómez-Cornelio et al., 2012; Adamson et al., 2013). Hen-
117: 59-77
Octubre 2016
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 surfaces and homogeneous coverage of biofilms. In addition,
all walls were oriented towards the north where low solar
irradiation and high relative humidity prevail in comparison to facades oriented towards other directions (Adamson et al., 2013; Ortega-Morales et al., 2013). Further
criteria for selecting the walls included the absence of surrounding vegetation and low levels of human disturbance,
such as painting or washing. Sites with automobile traffic 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 microbial colonization due to splashing water. Hence, time
elapsed since construction of the walls and establishment
of biofilms was the main influential variable considered in
the analysis of fungal community structure.
Biofilm sampling and fungal isolation
Figure 1: Biofilm samples from rock fragments of limestone buildings
(time elapsed since construction): A. young biofilm (one year); B.
middle-aged biofilm (five years); C. old biofilm (10 years).
ce, the monthly climate data were obtained from the local 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 environment. The considered climatic variables were: minimum
and maximum temperature, minimum, mean and maximum relative humidity and mean rainfall (Table 1).
In the dry season of January 2014, we sampled biofilms
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 scraped 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
biofilms scraped from the wall were placed in sterile Petri
dishes and transferred to the laboratory for processing.
Fungi were isolated by washing and filtration
of particles technique (Bills et al., 2004). One gram of
each scraped biofilm 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 filtered 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-
61
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
Table 1: Geographical location, color and climatic parameters of biofilms developed on the surface of sampled limestone walls. (Values are
expressed as means ± 1SD).
Young biofilm
Middle-aged biofilm
Old biofilm
Minimum
19º49'30.7"N
90º32'51.2"W
16.5 ± 2.5
19º49'30.5"N
90º33'16.3"W
16.4 ± 4.7
19º49'29.4"N
90º33'14"W
16.7 ± 2.5
Maximum
37.1 ± 4.3
37.6 ± 2.6
37.2 ± 4.5
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
224.9 ± 11.3
192.6 ± 54
194.8 ± 29
20.4 ± 19
19.8 ± 17
24.4 ± 20
Not observed
Dark green
Black
Geographical location
Mean temperature (ºC)
Mean relative humidity (%)
Mean rainfall (mm)
Rainy season
Dry season
Degree of colonization (visual inspection)
dia-Abarca, 2014) that may present active bioweathering
or serve a protective function on the surface of the limestone.
Particles trapped on the 75 μm sieve were transferred to sterile filter 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 copiotrophic 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 adjusted 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 stereomicroscope, 50 particles were transferred to 10 plates with
MEAC (5 particles per plate); this procedure was repeated 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 purified in inclined tubes
with MEAC.
Morphological identification of fungi
The fungal isolates were identified according to macroscopic characteristics, such as coloration, diameter, textu-
62
re, pigmentation, margin appearance, zonality and production 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
identified 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 confirmed by performing genomic DNA extraction and sequencing the ITS region (data not shown).
Fungal colonies that did not sporulate were inoculated 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 produced spores were separated into morphotaxa, according
to their macroscopic and microscopic morphology in the
different culture media. All fungal isolates were conserved 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
Data analysis
The fungal communities of the sampled biofilms were
analyzed according to species richness, defined as the
number of different fungi species per biofilm, in addition
to species abundance as the number of fungal isolates
per identified 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
identified at the species level, we used the literature previously employed in the identification of fungi and performed 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 software (Colwell, 2013). In order to determine the similarity
and composition of the fungal species found in the three
biofilms, 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 recovered, distributed in 73 genera and 202 species (Table 2).
The identified species were grouped as follows: 149 Ascomycota, one Basidiomycota and 52 Mycelia sterilia.
Hyphomicetous asexual species of Ascomycota (108
species) dominated, while Coelomycetous species represented 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, Nodulisporium Preuss, Paraconiothyrium Verkley and Phoma Sacc.
(Table 2).
We found the largest number of isolates (322) in
the middle-aged biofilm, followed by the old and young
biofilms with 268 and 254 isolates, respectively. However, species richness and diversity were higher in the old
biofilm and lower in the middle-aged biofilm (Table 3).
The Shannon evenness index of the old biofilm generated
a value close to 1, and in the middle-aged biofilm, 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 biofilms. 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) biofilms.
Overall, of the 202 species identified, 26% were
isolated from inoculated particles in both culture media
(52 species), while 36% (73 species) were found exclusively in the oligotrophic medium (CCOA) and 38% in
the copiotrophic medium (MEAC). Additionally, in the
analysis of colonization frequency of particles from all
three biofilms, we observed a high number of particles
with adhered mycelia (Table 3). The middle-aged biofilm
showed the highest percentage (80%) of colonization;
however, most mycelium that emerged from these particles belonged to the species Curvularia lunata (Wakker) Boedijn, Fusarium oxysporum Schltdl. and Fusarium
redolens Wollenw. (Table 2). The young and old biofilms
presented a minor colonization frequency of particles (Table 3).
In the old biofilm, a high number of isolated species
(35%) was specific to only one of the media, and a slightly lower proportion was present in both media (30 %).
In young and middle-aged biofilms, 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 obtained 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 Friedmanniomyces 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).
63
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
Table 2: Epilithic fungal community in terms of abundance of species isolated from biofilms on limestone at different stages of development and
color of their reproductive structures. (M: melanized and H: hyaline).
a
Copiotrophic medium (MEAC).
b
Oligotrophic medium (CCOA).
*Mycelia sterilia with one or more isolates, which are added in the total.
Epilithic fungi
Coloration
Young biofilm
Middle-aged
biofilm
Old biofilm
Total
2
1
3a,b
Ascomycota
Sordaria fimicola (Roberge ex Desm.) Ces. & De Not.
M
Xylariales sp. 1
M
1
1b
Xylariales sp. 2
M
1
1a
Xylariales sp. 3
M
1
1b
Ascochyta carpathica (Allesch.) Keissl.
M
2
2a
Clypeopycnis sp.
M
2
3
6a,b
Coleophoma sp.
M
7
7a,b
Colletotrichum crassipes (Speg.) Arx
H
1
1a
Colletotrichum dematium (Pers.) Grove
M
1
1a
Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.
H
1
1b
Coniothyrium multiporum (V.H. Pawar, P.N. Mathur &
Thirum.) Verkley & Gruyter
Cytospora polygoni-sieboldii Henn.
M
M
1
1a
Lasiodiplodia theobromae (Pat.) Griffon & Maubl.
M
7
7a
Microsphaeropsis arundinis (S. Ahmad) B. Sutton
M
2
3a,b
Microsphaeropsis sp. 1
M
1
1b
Microsphaeropsis sp. 2
M
Neosetophoma samararum (Desm.) Gruyter, Aveskamp &
Verkley
Paraconiothyrium sp.
M
M
19
Paraphoma chrysanthemicola (Hollós) Gruyter, Aveskamp
& Verkley
Paraphoma fimeti (Brunaud) Gruyter, Aveskamp & Verkley
M
1
M
2
Pestalotiopsis maculans (Corda) Nag Raj
M
5
Peyronellaea aurea (Gruyter, Noordel. & Boerema)
Aveskamp, Gruyter & Verkley
Peyronellaea gardeniae (S. Chandra & Tandon) Aveskamp,
Gruyter & Verkley
Phlyctema lappae (P. Karst.) Sacc.
M
2
2a,b
M
1
1b
M
1
1a
Phoma adianticola (E. Young) Boerema
M
4
4a
Phoma crystallifera Gruyter, Noordel. & Boerema
M
Phoma eupyrena Sacc.
M
Coelomycetous asexual species of Ascomycota
64
1
1
1
1b
2a,b
2
1
23
1a
22a,b
3
1b
3
12
3
5a,b
10
18a,b
1
1b
15
50a,b
117: 59-77
Octubre 2016
Table 2: Continuation.
Coloration
Young biofilm
Phoma herbarum Westend.
M
4
Phoma heteroderae Sen Y. Chen, D.W. Dicks. & Kimbr.
M
2
Phoma leveillei Boerema & G.J. Bollen
M
Phoma multirostrata (P.N. Mathur, S.K. Menon & Thirum.)
Dorenb. & Boerema
Phoma paspali P.R. Johnst.
M
Phoma pratorum P.R. Johnst. & Boerema
M
Phoma proteae Crous
M
Phoma putamina Speg.
M
1
Phoma sp. 1
M
1
1a
Phoma sp. 2
M
2
2a,b
Phoma tropica R. Schneid. & Boerema
M
Phomopsis putator (Nitschke) Traverso
M
Pleurophomopsis lignicola Petr.
M
1
Pyrenochaetopsis pratorum (Berk. & M.A. Curtis) M.B.
Ellis
Westerdykella minutispora (P.N. Mathur) Gruyter, Aveskamp
& Verkley
Hyphomicetous asexual species of Ascomycota
M
1
M
4
Acremoniella velutina (Fuckel) Sacc.
M
1
Acremonium brachypenium W. Gams
H
Acremonium fusidioides (Nicot) W. Gams
H
1
1b
Acremonium rutilum W. Gams
H
2
2b
Acremonium sordidulum W. Gams & D. Hawksw.
H
1
1a
Agaricodochium sp.
H
Alternaria longipes (Ellis & Everh.) E.W. Mason
M
Alternaria tenuissima (Kunze) Wiltshire
M
Arxiella terrestris Papendorf
H
1
1b
Aspergillus aculeatus Iizuka
M
2
2a
Aspergillus alliaceus Thom & Church
H
1
1a
Aspergillus awamori Nakaz.
M
2
2a
Aspergillus foetidus Thom & Raper
M
2
2a
Aspergillus fumigatus Fresen.
M
2
2a
Aspergillus japonicus Saito
M
1
1a
Aspergillus niger Tiegh.
M
7
7a
Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud
M
1
1a
Badarisama sp.
M
Epilithic fungi
M
Middle-aged
biofilm
1
Old biofilm
Total
2
7a,b
2a,b
1
1
1b
1
2b
5
5a,b
1
1a
1
1b
2
3a,b
2a,b
2
1
1
2a
1b
1
1
6a,b
1a
2b
2
1
1a
1
1
1a
1
1
1a
2a,b
1a
65
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
Table 2: Continuation.
Young biofilm
Epilithic fungi
Coloration
Baudoinia sp.
M
Calcarisporium sp.
M
Capnobotryella antalyensis Sert & Sterfl.
M
Chaetasbolisia falcata V.A.M. Mill. & Bonar
M
Chromelosporium sp.
H
Cladosporium cladosporioides (Fresen.) G.A. de Vries
M
22
Cladosporium oxysporum Berk. & M.A. Curtis
M
Cladosporium sphaerospermum Penz.
Middle-aged
biofilm
Old biofilm
Total
1
1b
1
1a
1
1
1a
1b
1
1a
8
40a,b
10
8
18a,b
M
9
2
11a,b
Cladosporium tenuissimum Cooke
M
2
2
4a,b
Corynespora citricola M.B. Ellis
M
1
Corynesporella pinarensis R.F. Castañeda
M
Curvularia australiensis (Tsuda & Ueyama) Manamgoda, L.
Cai & K.D. Hyde
Curvularia brachyspora Boedijn
M
Curvularia clavata B.L. Jain
M
18
Curvularia fallax Boedijn
M
1
Curvularia hawaiiensis (Bugnic. ex M.B. Ellis) Manamgoda,
L. Cai & K.D. Hyde
Curvularia lunata (Wakker) Boedijn
M
M
10
105
26
141a,b
Curvularia pallescens Boedijn
M
6
13
11
30a,b
Curvularia spicifera (Bainier) Boedijn
M
1
Curvularia verruculosa Tandon & Bilgrami ex. M.B. Ellis
M
Curvularia sp.
1
M
10
1a
1
1
1b
5
7a,b
2a
2
3
1
22a,b
1b
1
1b
1a
1
8a,b
M
2
2a,b
Echinocatena sp.
M
1
1b
Exochalara longissima (Grove) W. Gams & Hol.-Jech.
M
2
2a
Friedmanniomyces simplex Selbmann, de Hoog, Mazzaglia,
Friedmann & Onofri
Fusarium camptoceras Wollenw. & Reinking
M
1
2
6b
H
1
1b
Fusarium equiseti (Corda) Sacc.
H
1
1a
Fusarium flocciferum Corda
H
1
Fusarium incarnatum (Desm.) Sacc.
H
Fusarium oxysporum Schltdl.
H
8
Fusarium redolens Wollenw.
H
14
Fusarium sacchari (E.J. Butler & Hafiz Khan) W. Gams
H
1
Fusarium solani (Mart.) Sacc.
H
2
Fusarium subglutinans (Wollenw. & Reinking) P.E. Nelson,
Toussoun & Marasas
H
1
66
7
3
1
2a,b
1
1b
40
4
52a,b
41
9
64a,b
1a
2
4a,b
1b
117: 59-77
Octubre 2016
Table 2: Continuation.
Epilithic fungi
Coloration
Young biofilm
Middle-aged
biofilm
1
Old biofilm
Total
1b
Fusarium tabacinum (J.F.H. Beyma) W. Gams
H
Fusarium ventricosum Appel & Wollenw.
H
Gabarnaudia sp.
H
1
1b
Geotrichum candidum Link
H
2
2a,b
Gilmaniella subornata Morinaga, Minoura & Udagawa
M
Graphium penicillioides Corda
M
2
2a,b
Hyphomycete 1
M
11
11a,b
Microdochium dimerum (Penz.) Arx
H
2
3b
Microdochium nivale (Fr.) Samuels & I.C. Hallett
H
2
2b
Monodictys fluctuata (Tandon & Bilgrami) M.B. Ellis
M
1
1b
Monodictys paradoxa (Corda) S. Hughes
M
3
3a,b
Myrothecium cinctum (Corda) Sacc.
M
3
3a
Myrothecium roridum Tode
M
Myrothecium sp. 1
M
Myrothecium sp. 2
1
1a
1
1
12
1
1a
16
28a,b
3
4a,b
M
1
1a
Myrothecium sp. 3
M
1
1a
Nalanthamala madreeya Subram.
H
1
1b
Nigrospora oryzae (Berk. & Broome) Petch
M
Nodulisporium acervatum (Massee) Deighton
M
Nodulisporium ochraceum Preuss
M
1
Nodulisporium puniceum (Cooke & Ellis) Deighton
M
3
Nodulisporium radians (Berk.) Deighton
M
Nodulisporium sp. 1
M
Nodulisporium sp. 2
M
Nodulisporium sylviforme Deighton
M
Nodulisporium thelenum (Sacc.) G. Sm.
M
Ochroconis tshawytschae (Doty & D.W. Slater) Kiril. &
Al-Achmed
Penicillium citreonigrum Dierckx
3
1
1
5a
1
1b
1a
2
5a,b
1
1b
1
1b
1
1a
1
4
5a,b
1
1
2a,b
M
1
1b
H
1
1a
Penicillium dierckxii Biourge
H
1
1b
Penicillium islandicum Sopp
H
1
1a
Penicillium oxalicum Currie & Thom
H
1
1a
Periconia igniaria E.W. Mason & M.B. Ellis
M
Periconiella mucunae M.B. Ellis
M
Prathoda longissima (Deighton & MacGarvie) E.G.
Simmons
M
1
1a
1
1
1a
1b
67
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
Table 2: Continuation.
Epilithic fungi
Coloration
Pseudohelicomyces albus Garnica & E. Valenz.
H
Pseudopithomyces chartarum (Berk. & M.A. Curtis) J.F. Li,
Ariyawansa & K.D. Hyde
Pseudoramichloridium brasilianum (Arzanlou & Crous)
Cheew. & Crous
Ramichloridium apiculatum (J.H. Mill., Giddens & A.A.
Foster) de Hoog
Sarocladium kiliense (Grütz) Summerb.
M
Young biofilm
Middle-aged
biofilm
Old biofilm
Total
2
2a,b
1
1a
M
1
1a
M
2
2b
H
1
1a
Sarocladium strictum (W. Gams) Summerb.
H
1
Scolecobasidium constrictum E.V. Abbott
M
3
Sepedonium sp.
Stachybotrys microspora (B.L. Mathur & Sankhla) S.C. Jong
& E.E. Davis
Stachybotrys nephrospora Hansf.
1b
4
1
8a,b
H
1
1b
M
1
1b
M
1
1b
Stachybotrys renispora P.C. Misra
M
1
1b
Tolypocladium sp.
H
Torula herbarum (Pers.) Link
M
Trichobotrys sp.
M
1
1a
Trichocladium sp.
M
1
1b
Trichoderma aggressivum Samuels & W. Gams
H
Trichoderma harzianum Rifai
H
Trichoderma longibrachiatum Rifai
1
1b
1
1
1a
1
2a
1
3a,b
H
2
2a
Trichoderma ovalisporum Samuels & Schroers
H
1
1a
Trichoderma strigosum Bissett
H
1
1a
Veronaea musae M.B. Ellis
M
1
1a
Verruconis verruculosa (R.Y. Roy, R.S. Dwivedi & R.R.
Mishra) Samerp. & de Hoog
Basidiomycota
M
1
1a
Geotrichopsis sp.
H
2
1
1a
1
3b
Mycelia sterilia
Mycelia sterilia (Morphotaxon 01)
H
1
Mycelia sterilia (Morphotaxon 02)
M
2
Mycelia sterilia (Morphotaxon 03)
H
1
Mycelia sterilia (Morphotaxon 04)
M
1
1
2b
Mycelia sterilia (Morphotaxon 05)
M
1
1
2a,b
Mycelia sterilia (Morphotaxon 06-10*)
M
1
5a
Mycelia sterilia (Morphotaxon 11-15*)
M
1
5b
Mycelia sterilia (Morphotaxon 16)
H
1
1b
68
1
2a,b
1
2b
117: 59-77
Octubre 2016
Table 2: Continuation.
Epilithic fungi
Coloration
Young biofilm
Old biofilm
Mycelia sterilia (Morphotaxon 17-18*)
M
Middle-aged
biofilm
3
Total
Mycelia sterilia (Morphotaxon 19-20*)
M
2
4a,b
Mycelia sterilia (Morphotaxon 21)
M
2
2b
Mycelia sterilia (Morphotaxon 22-23*)
M
1
2a
Mycelia sterilia (Morphotaxon 24-25*)
H
1
2b
Mycelia sterilia (Morphotaxon 26)
M
1
1b
Mycelia sterilia (Morphotaxon 27)
H
3
3a,b
Mycelia sterilia (Morphotaxon 28)
M
2
2b
Mycelia sterilia (Morphotaxon 29-34*)
M
1
6a
Mycelia sterilia (Morphotaxon 35-39*)
H
1
5a
Mycelia sterilia (Morphotaxon 40-50*)
M
1
11b
Mycelia sterilia (Morphotaxon 51-52*)
H
1
2b
6a,b
Table 3: Abundance, richness, diversity and evenness of the fungal epilithic community colonizing biofilms on limestone at different stages of
development.
State of biofilms
Young
Abundance
(number of
isolates)
254
Richness (number Simpson Diversity Shannon Diversity Shannon evenness
of species)
index (D’)
index (H’)
index (J’)
Particles
colonization (%)
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 identified at the species level,
many were associated with numerous substrates, based
on the literature review to determine with which substrates identified fungal species had been previously associated (Fig. 2). Thirty-one species were identified as
cosmopolitan and belonged to the genera Cladosporium,
Curvularia, Fusarium and Penicillium, including several common species, such as Aureobasidium pullulans
(de Bary & Löwenthal) G. Arnaud and Geotrichum candidum Link (Domsch et al., 2007). Most of the identified
species (81) have been reported in soil, mainly species of
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 identified 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 species with wood (genera Nodulisporium and Phlyctema
69
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
Figure 2: Frequency of substrate types reported for the fungal species
isolated from biofilms 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 species 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 biofilms, 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-existing groups of species may be associated with a particular
biofilm, characterized by time of exposure of the substrate
(limestone) to the environment; this is shown in the Venn
diagram (Fig. 3). In the young biofilm, we found 49 exclusive species, including several from the genera Colletotrichum and Coleophoma Höhn.; the species Monodyctis 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 biofilm, only 32 exclusive species were
found, although these were not frequent (> 2 isolates). The
70
Figure 3: Venn diagram indicating number of common and exclusive
fungal species to the studied biofilms.
old biofilms had the highest number of exclusive species
(82), among these species of the genera Aspergillus, Lasiodiplodia Ellis & Everh., Penicillium and Stachybotrys,
in addition to Xylariales spp. and Hyphomycete 1.
The Venn diagram shows 16 species in the core
group (three biofilms) 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 herbarum Westend. and Westerdykella minutispora (P.N.
Mathur) Gruyter, Aveskamp & Verkley were abundant in
the young biofilm. In the middle-aged biofilm Curvularia
lunata, Curvularia pallescens Boedijn, Friedmanniomyces simplex, Fusarium oxysporum, Fusarium redolens
and Scolecobasidium constrictum E.V. Abbott, and in the
old biofilm 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-five species were present in
at least two biofilms; the young and old biofilms had the
highest number of shared species (12). Paraconiothyrium sp. was dominant and was isolated 19 times in the
young biofilm, although its abundance diminished in the
117: 59-77
Octubre 2016
Figure 4: Fungal species with the highest number of isolates of the three biofilms a different stages of development on limestone.
middle-aged biofilm. Cladosporium oxysporum Berk. &
M.A. Curtis, Cladosporium sphaerospermum Penz. and
Nodulisporium puniceum (Cooke & Ellis) Deighton were
found at a higher frequency in the young biofilm in comparison to the old biofilm. Curvularia verruculosa Tandon & Bilgrami ex. M.B. Ellis was also more frequent
in the middle-aged biofilm than the old biofilm. Meanwhile, the species Nodulisporium sylviforme Deighton
and Paraphoma fimeti (Brunaud) Gruyter, Aveskamp &
Verkley were more dominant in the old biofilm than in the
young biofilm. Finally, Myrothecium roridum Tode was
more frequent in the old biofilm than in the middle-aged.
dIscussIon
The fungal communities of biofilms occurring on limestone walls, considering time elapsed since wall construction, presented different degrees of microbial colonization. In the 5- and 10-year-old samples, colonization was
evident by the green and black biomass of the biofilms
that were visibly observed (Fig. 1), which is consistent
with the findings of Adamson et al. (2013). The phototrophs colonizing such substrates are mainly composed of filamentous 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 provide nutritional support for the establishment of heterotrophic 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 substrates; these Hyphomicetous asexual species are able to
colonize rocks during the first year of exposure (Ruibal
et al., 2008; Gleeson et al., 2010; Hallman et al., 2011;
Gómez-Cornelio et al., 2012). In contrast, Coelomycetous species are known to colonize limestone in Mediter-
71
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
ranean regions (Wollenzien et al., 1995). The identification of only one taxon belonging to Basidiomycota may
indicate that this group of organisms is not common in
biofilms developing on limestone, as confirmed 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, Fusarium, Microsphaeropsis, Myrothecium, Nodulisporium,
Paraconiothyrium and Phoma are common in subtropical
environments and in this study (Table 2). For other regions, some of the most common, dominant genera that
have been reported on epilithic substrates include Alternaria, 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 identified in this study, but only Cladosporium, Fusarium and
Phoma were common and dominant. These differences
may be dictated by environmental factors and the bioreceptivity of rocks.
Some isolates showed no reproductive structures
(8%) and were identified as Mycelia sterilia (25% of morphospecies). However, few isolates belonged to these morphospecies, indicating that they are rare in the community
(Table 2). The finding 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-Cornelio et al., 2012). These fungi may be classified according
to morphotype based on their morphological characteristics (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 biofilm. However, in another study on sandstone,
the highest number of species was reported for fresh biofilms in comparison to older biofilms (Lan et al., 2010).
Meanwhile, in this study the Shannon evenness index
showed that the species identified from isolates were al-
72
most equitable in the old biofilm. The lowest evenness
was obtained in the middle-aged biofilm, probably due to
the presence of several dominant species, such as Curvularia 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; Sterflinger
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 influence
the colonization of microbial communities (Guillitte,
1995; Burford et al., 2003; Lan et al., 2010), the study of
Tomaselli et al. (2000) did not find 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 corresponding to different ages. This is notable in considering that the middle-aged and old biofilms were located
less than 1 km from each other. These biofilms also had
a similar chromatic aspect; therefore, one might expect a
high degree of similarity. Meanwhile, the young biofilm
117: 59-77
Octubre 2016
was located at an approximate distance of 10.5 kilometers
from the other biofilms and had a visible although incipient colonization of microorganisms. Microclimatic differences present at each site may contribute towards the formation of a unique and particular mycobiota (Mihajlovski
et al., 2014). Our results differed from those reported for
sandstone, in which no differences in eukaryotic composition were found between fresh and young biofilms (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 findings 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 biofilms on limestone,
we were able to evaluate the fungal network and find a
high proportion of active mycelium. The use of an oligotrophic culture medium (CCOA) allowed for the characterization 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 species could represent an autochthonous community specific to limestone, a substrate with limited nutrients. However, 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; Gorbushina and Krumbein, 2000; Ruibal et al., 2009), thereby
providing conditions for the colonization of specialized
fungal species in the production of exopolymers allowing 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
(Sterflinger, 2000). Hence, fungi are essential members of
the microbial communities that develop in the biofilms of
limestone.
The composition of fungal communities may also
be influenced by surrounding substrates (Urzì et al., 2001;
Hallman et al., 2011). As previously mentioned, we identified the substrates previously reported in the literature for
the 124 fungi that were identified in our study at the species level. A high proportion of fungi may have originated
from soil or plants, and to a lesser extent, from decomposing 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 development of fungi over a period of time may be determined by
the interactions of species with their environmental conditions, such as relative humidity and temperature (Gorbushina and Krumbein, 2000; Gorbushina et al., 2005), in
addition to the bioreceptivity of the substrate. The variety
of fungi reported for other surrounding substrates confirms that a large quantity of propagules may potentially
reach and colonize rock surfaces. Therefore, according to
our findings, 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 biofilms 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 identification techniques (Collado et al., 2007; Ruibal et al., 2008; Gómez-Cornelio et
al., 2012). Additionally, the abundance of dominant species of the three analyzed biofilms was variable and may
be determined by time of exposure, similar to the variations observed in Lan et al. (2010) in the microorganism
community of old and fresh biofilms.
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 contained pigmentation (Sterflinger and Krumbein, 1997;
Gorbushina et al., 2002; Lan et al., 2010). Pigmentation
in fungi may have different functions or result from the
73
Gómez-Cornelio et al.: Fungal communities composition in biofilms on limestone
environmental conditions experienced on the rock surface
(Scheerer et al., 2009; Hallman et al., 2011). Therefore,
melanized communities of fungi have been shown to occur at a high frequency on rock surfaces, although composition may differ from one community to another, as
mentioned in the results.
Moreover, fungal propagules are capable of quickly colonizing rock surfaces. Gorbushina and Krumbein
(2000) have suggested that fluctuations in environmental conditions, such as those that lead to deficits in nutrients 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 biofilms were similar; thus one
would expect to find a relationship among the studied fungal 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 hyphae 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 exposure to the environment and species interactions, which
may then facilitate or inhibit colonization by other species, 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.
re and diversity of fungi in this study may be determined
by the interactions among the species of each biofilm; this
should be confirmed by subsequent studies. A particular
species composition was isolated in each biofilm corresponding to a different developmental stage, although a
common pool of hyaline and melanized fungi appear to
colonize rock with great success and may have specific
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
influence on the structure of the fungal community.
Acknowledgements
We are grateful to Julio C. Rojas León and Hugo Perales 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 Universidad 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 Ciencia y Tecnología for the doctoral scholarship awarded to
S.G.C. We thank two anonymous reviewers and the editor of the manuscript for suggested improvements; also
thanks to Allison Marie Jermain for reviewing the English
version of the manuscript.
lIterAture cIted
Adamson, C., S., McCabe, P. A. Warke, D. McAllister and B.
J. Smith. 2013. The influence of aspect on the biological
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In this study, the fungal communities immersed in biofilms
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has been found to have a certain degree of influence 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. Environmental 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-
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