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The endophytic mycobiota of Arabidopsis thaliana
Elena García &Ángela Alonso &Gonzalo Platas &
Soledad Sacristán
Received: 1 November 2012 /Accepted: 10 December 2012
#Mushroom Research Foundation 2012
Abstract Fungal endophytes are receiving increasing atten-
tion as resources to improve crop production and ecosystem
management. However, the biology and ecological signifi-
cance of these symbionts remains poorly understood, due to
a lack of model systems for more efficient research. In this
work, we have analyzed the culturable endophytic myco-
biota associated, in the wild, with leaves and siliques of the
model plant A. thaliana. We have studied the effect of biotic
and abiotic factors in the frequency of fungal endophytes in
plant specimens, and in the species composition of the
endophytic community. Our results indicate that the fre-
quency of Arabidopsis plants hosting endophytes depends
on the time of the year and the phenological stage of the
plant, and that the probability of endophyte colonization
increases as the life cycle of the plant progresses. The
diversity of the endophytic assemblages of natural A. thali-
ana populations was high, and precipitation and temperature
were the two main factors determining the diversity and
species composition of the communities. We propose A.
thaliana and its endophytes as a model system for an inte-
gral approach to the principles governing the endophytic
lifestyle, taking advantage of the molecular tools and the
abundant knowledge accessible from the host plant.
Keywords Endophyte .Mycobiota .Arabidopsis .Wild
populations .Ecology
Introduction
Fungal endophytes live within plant tissues without causing
symptoms of disease (Hyde and Soytong 2008; Stone 2006;
Wilson 1995). The existence, ubiquity, abundance and high
diversity of fungal endophytes have been long recognized,
and the ability of many endophytes to protect plants from
herbivores by producing toxins, improve plant tolerance to
abiotic stress or confer resistance to plant pathogens is well
known. Indeed, endophytes offer promising solutions to
improve crop production and ecosystems management
(Aly et al. 2011a,b; Debbab et al. 2011,2012; Li et al.
2012; Saunders et al. 2010; Suryanarayanan et al. 2012).
However, the ecological significance of these symbionts,
including their contribution to plant fitness and adaptation,
remains poorly characterized (Rodriguez et al. 2009;
Saikkonen et al. 2010; Sieber 2007). There is also a large
gap in the basic knowledge about the way in which endo-
phytes interact with the host and the mechanisms responsi-
ble for the beneficial attributes of colonization (Porras
Alfaro and Bayman 2011; Purahong and Hyde 2011;
Schulz and Boyle 2005). The lack of data about the
Electronic supplementary material The online version of this article
(doi:10.1007/s13225-012-0219-0) contains supplementary material,
which is available to authorized users.
E. García :S. Sacristán (*)
Centro de Biotecnología y Genómica de Plantas
(UPM-INIA) and E.T.S.I. Agrónomos, Universidad Politécnica de
Madrid, Campus de Montegancedo, Autopista M40 (Km. 38),
28223 Pozuelo de Alarcón Madrid, Spain
e-mail: soledad.sacristan@upm.es
Á. Alonso
Departamento de Biotecnología, E.T.S.I. Agrónomos, Universidad
Politécnica de Madrid, Avda. Complutense s/n,
28040 Madrid, Spain
G. Platas
Fundación Centro de Excelencia en Investigación de
Medicamentos Innovadores en Andalucía MEDINA, Parque
Tecnológico de las Ciencias de la Salud de Granada, Avda del
Conocimiento 3,
18100 Armilla, Granada, Spain
Present Address:
E. García
Instituto de Recursos Naturales y Agrobiología,
Consejo Superior de Investigaciones Científicas,
Avenida Reina Mercedes 10,
41012 Sevilla, Spain
Fungal Diversity
DOI 10.1007/s13225-012-0219-0
endophytic functional interactions may be due to the scar-
city of model systems set into a point for molecular biology
approaches (Arnold 2007; Hamilton et al. 2012).
Arabidopsis thaliana is, by far, the best studied model
among all known species of flowering plants (Koornneef
and Meinke 2010). This Brassicacea lacks the agronomic
significance of other related species such as rapeseed or mus-
tard, but it offers important advantages for basic research in
genetics and molecular biology: it has a short generation time,
a small size that limits the requirement for growth facilities,
and prolificseed productionthrough self-pollination; it can be
genetically transformed very efficiently and it has a small
genome, which has been sequenced more than a decade ago
(AGI 2000). Hence, there is a large amount of available
information about its functional genetics and genomics and a
variety of tools for deepen in the knowledge of plant biology
(Ehrhardt and Frommer 2012;Keurentjesetal.2011). Even
so, A. thaliana is not just a laboratory model plant: the knowl-
edge generated in the laboratory is being applied to the study
of its wild populations, so it has become a model, as well, for
the study of plant ecology, adaptation and evolution
(Bergelson and Roux 2010). Arabidopsis thaliana is an annu-
al forb that occurs worldwide throughout temperate regions,
after expansion from its native geographical range in Eurasia
and North Africa (Hoffmann 2002). It tends to colonize dis-
turbed, low-competition habitats, so it can be found in a high
diverse range of environments, including anthropic and wild
habitats (Pico et al. 2008). Although A. thaliana is one of the
best studied hosts for a diverse range of plant pathogens
(Nishimura and Dangl 2010) the study of its interactions at
community level has started only recently (Anderson and
Mitchell-Olds 2011) and research in interactions other than
highly pathogenic is now just emerging. The study of the
bacterial communities associated with A. thaliana is currently
gaining importance (Knief et al. 2010; Kniskern et al. 2007;
Micallef et al. 2009), including the recent metagenomic anal-
yses of the composition of the bacterial microbiomes of A.
thaliana roots (Bulgarelli et al. 2012;Lundbergetal.2012).
As for fungi, there are examples of endophytes isolated from
different hosts that are able to colonize A. thaliana roots and
shoots (Piriformospora indica, Peskan Berghofer et al. (2004)
and Acremonium alternatum, Jaschke et al.(2010)). Very
recently, it has been shown that some endophytic isolates from
wild Arabidopsis plants are able to colonize the plant in axenic
conditions (Junker et al. 2012).
The purpose of this work was to acquire basic information
about the occurrence, composition and diversity of fungal
endophytes associated with A. thaliana, evaluating the contri-
bution of biotic and abiotic factors on endophyte occurrence
and diversity. To achieve that, we have isolated and identified
the culturable endophytic mycobiota of leaves and siliques of
A. thaliana in wild populations in Central Iberian Peninsula,
which is considered one of the centers of genetic diversity of
this species (Pico et al. 2008). We have studied the effect of
location, date of sampling, plant phenological stage and organ
in the frequency of fungal endophytes in plant specimens and
in the species composition of the endophytic fungal commu-
nity. The diversity of the fungal endophytic assemblages of
natural occurring A. thaliana populations was high compared
to those found in surveys of other hosts in temperate regions
(Arnold and Lutzoni 2007; Sanchez Marquez et al. 2012).
Among the variables analyzed, precipitation and temperature
were the two main factors determining the diversity and spe-
cies composition of the fungal endophytic communities. This
is, to our knowledge, the first study of the endophytic myco-
biota of A. thaliana at different ecological environments,
showing that this community can be used as a model system
to improve our knowledge about the ecological function of
endophytes and their contribution to plant adaptation. The
availability of endophytic isolates naturally infecting A. thali-
ana offers a great opportunity for molecular approaches to the
study of the endophytic functional interactions with the host.
Materials and methods
Field surveys
Sampled populations (Table 1) are described in Pico et al.
(2008) and Pagan et al. (2010), except of Rascafría popula-
tion, which was not previously described. They are situated in
acidic soils from different geological substrata and with flo-
ristic formations that differ among zones: Mediterranean ev-
ergreen oaks (Quercus rotundifolia Lam and Q. coccinea L)
dominate in Polán, Menasalbas and Marjaliza . The ecological
site in Rascafría is a transition from deciduous melojo oak
(Quecus pyrenaica Willd) forest to Scots pine (Pinus sylvest-
ris L.) forest, vegetation in Ciruelos de Coca is dominated by
Pinus pinaster Aiton, and Las Rozas is an antropic xeric
grassland associated to Q. rotundifolia. Field surveys were
carried out during year 2008. Ciruelos de Coca, Las Rozas,
Polán, Menasalbas and Marjaliza populations were surveyed
during the growth period of A. thaliana,fromFebruaryto
June, and also in October and November, in order to find a
possible second germination after the autumn rains. Rascafría
was surveyed during May, June, October and November.
In each survey, 5 to 30 plants were randomly collected in
at least three sites 2 to 20 m distant depending on population
size. At each sampled site, plant density was estimated by
counting the number of A. thaliana plants in a 0.5 ×0.5 m
quadrat. The phenological stage of all the sampled plants
was recorded considering three categories (Boyes et al.
2001): vegetative growth as rosette (stages 1.04–3.9), repro-
ductive growth with plants producing the inflorescence
stems, flowers and fruits (stages 5–6.90) and shattering
(plants in which at least one silique had shattered, stages
Fungal Diversity
8 and later). The diameter of the rosette and the inflores-
cence stem length of the sampled plants that were at the end
of the cycle (shattering stage) were measured, and these
plants were classified in three classes depending on the
number of siliques (less than 5, more than 5 and more than
10). The collected plants lacked obvious disease symptoms
such as chlorosis, leaf spots, or other types of pathogen-
induced lesions. Whole plants were removed from the field
and transported to the laboratory, kept at 4 °C, and pro-
cessed within 48 h. Plants at early vegetative stage were
transplanted and kept in a growth chamber at 22 °C, 16
photoperiod, until flowering, to confirm their identification
as A. thaliana.
Climatic data were obtained from the Spanish Metereology
Agency. Data from weather stations closer than 20 km from
each location, and with a difference in elevation of less than
60 m with Arabidopsis populations, were used.
Endophyte isolation
From each plant, three different organs (rosette leaves,
stem leaves –ie. leaves from the inflorescence stem-, and
siliques), were sampled when present. Two fragments of
approximately 10 mm
2
of each sampled organ were sur-
face disinfected by submerging them in 20 % household
bleach (1 % active chlorine) and gently shaking during
5 min. After rinsing two times in sterile water, one frag-
ment was placed in a sterile moist chamber and the other
in a potato dextrose agar plate. Plates were kept at room
temperature (20–24 °C). Fragments were inspected peri-
odically and the fungal mycelium growing from them was
isolated in potato dextrose agar (PDA) plates containing
200 mg/L of chloramphenicol. The effectiveness of the
surface sterilization was controlled by making imprints of
disinfected leaf fragments on PDA plates (Schulz et al.
1993). The isolation frequency was calculated as the num-
ber of plants from which endophytes were isolated in
relation to the analyzed plants.
Endophyte identification
Endophytes were preliminary classified by the homology of
their ITS1-5.8S rRNA-ITS2 sequence region with the
sequences in the fungal EMBL/GenBank database
(Table 5). The identification of the isolate was limited to
the genus, unless the sequences of type species were found
among the better blast hits, and these unequivocally indicat-
ed the same species (Ko Ko et al. 2011). The molecular
identification was confirmed with the morphology when the
fungal reproductive structures were present. Fungal DNA
was extracted from small mycelial fragments scraped from
the surface of culture plates using a commercial kit
(RedExtract-N-Amp Plant PCR, Sigma Aldrich). The
ITS1-5.8S rRNA-ITS2 region was PCR amplified as de-
scribed in Sanchez Marquez et al. (2007), using the primers
ITS4 and ITS5 (White et al. 1990). PCR amplicons were
purified by column (Quiagen), and sequenced in an ABI
Prism sequencer (MACROGEN). Sequencing reaction chro-
matograms were visualized with Chromas 1.45 software
(Technelysium, Australia). The DNA sequences obtained
in sequencing reactions were assembled with Genestudio
2.1.1.5. (Genestudio, Inc., Suwanee, GA, USA). The
sequences obtained were compared with the fungal sequen-
ces in the EMBL/GenBank database using the nBLASTx
algorithm. Sequences were aligned using the program
ClustalX (Thompson et al. 1997) and a dendrogram was
made with MEGA 3.1 using the neighbour-joining method
with Kimura 2-parameter distances (Kumar et al. 2004). The
percentage of nucleotide similarity was determined for the
groups of sequences within the same branch of the dendro-
gram. Because for most fungal species the range of intra-
specific variation in ITS sequences is unknown (Taylor et al.
2000), sequences with a nucleotide identity higher than
97 % were arbitrarily considered to belong to the same
species as it has been done in other studies (Higgins et al.
2007; Neubert et al. 2006; Sanchez Marquez et al. 2007).
The sequences of this study were deposited in the GenBank
Table 1 Geographical, ecological and climatic description of A. thaliana populations
Population
a
Latitude/
Longitude
Elevation
(m a.s.l)
b
Genus of dominant
tree
T
max abs
c
T
max
c
T
m
c
T
min
c
T
min abs
c
P
c
Ciruelos de Coca 41º12′N/ 4º32′W 789 Pinus sp. 37.7 20.0 13.0 6.0 −9.3 372
Rascafría 40º53′N/ 3º53′W 1280 Quercus sp. / Pine sp. 33.3 18.1 10.7 3.4 −12.6 697
Las Rozas 40º30′N/ 3º53′W 724 Quercus sp. 37.0 20.3 14.6 8.9 −5.0 464
Polán 39º49′N/ 4º15′W 513 Quercus sp. 39.9 23.4 16.9 10.4 −5.0 391
Menasalbas 39º39′N/ 4º20′W 735 Quercus sp. 39.5 21.9 15.7 9.4 −5.5 493
Marjaliza 39º34′N/ 3º55′W 983 Quercus sp. 37.1 20.0 14.8 9.7 −8.0 668
a
Populations Ciruelos de Coca, Las Rozas, Polán, Menasalbas and Marjaliza are described in Pagan et al. 2010 and Pico et al. 2008.
b
meters above
sea level.
c
T
max abs
: annual absolute maximum temperature (°C); T
max
: annual mean of maximum temperatures (°C); T
m
: annual mean temperature
(°C); T
min
: annual mean of minimum temperatures (°C); T
min abs
: annual absolute minimum temperature (°C); P: annual mean precipitation (mm)
Fungal Diversity
database under the accession numbers: JX982366 - JX982485
(Table 5). Full alignment of the sequences has been submitted
to the TreeBASE under the accession S13513 (http://purl.org/
phylo/treebase/phylows/study/TB2:S13513).
Phylogenetic Bayesian analysis based on the Markov
Chain Monte Carlo (MCMC) approach was run in the pro-
gram MrBayes 3.01 (Ronquist and Huelsenbeck 2003). To
improve mixing of the chain, four incrementally heated simul-
taneous Monte Carlo Markov chains were run over 2,000,000
generations. MrModeltest 2.2 (Posada and Buckley 2004)was
used to perform hierarchical likelihood ratio tests to calculate
the Akaike Information Criterion (AIC) and hierarchical like-
lihood ratio tests (hLRTs) values of the nucleotide substitution
models. The model selected by hLRT and AIC was the GTR+
I+G model. This model allowed for six classes of substitution
types, a portion of invariant alignment positions, and mean
substitution rates varying across the remaining positions
according to a gamma distribution. Priors used for the
MCMC process were a Dirichlet distribution for substitution
rates and nucleotide frequencies and a uniform prior for the
rate parameter of the gamma distribution (Huelsenbeck et al.
2008). The MCMC analysis used a sampling frequency of
100. The resulting consensus tree was a majority rule consen-
sus tree including compatible groups of lower frequencies.
Tree graphical output was performed using ITOL on line
software http://itol.embl.de/
Analysis of composition and diversity of endophytic
assemblages
Shannon Index of Diversity was calculated as H′=−∑p
i
lnp
i
,
(Shannon and Weaver 1949) where p
i
is the proportional
abundance of each species in each sampled plant, i.
Evenness is the ratio between observed diversity and max-
imum diversity (Zak and Willig 2004) and was calculated as
e=H′/lnS, where Sis the species richness or the number of
species observed. Diversity of the endophytic communities
of plants at different populations, plants at different pheno-
logical stages and samples from different plant organs was
measured using Fisher’sα, which is robust for comparisons
among samples of different sizes (Magurran 2004). Fisher’s
αis the alpha parameter of a fitted logarithmic series distri-
bution (Magurran 2004) and was computed using the pro-
gram EstimateS 8.2 (Colwell 2005). Species accumulation
curves were calculated with data from all the species or only
the plural species identified from each plant using the ana-
lytical formulas of Colwell et al. 2004 provided by the
program EstimateS 8.2 (Colwell 2005). Beta diversity, or
the amount of change in species composition between
assemblages, was estimated as the average proportion of
species in each assemblage that are not found at other
assemblages (Sanchez Marquez et al. 2010). This number
was calculated by dividing the average number of shared
species per assemblage by the average number of species
per assemblage, and subtracting this number from one.
Jaccard (J) species presence/absence based similarity index
(Zak and Willig 2004) were computed using the program
EstimateS 8.2 (Colwell 2005).
Statistical analysis
Correlation between data was analyzed by calculating the
Pearson correlation coefficient. For that, plant density was
logarithmically transformed in order to obtain the normal
distribution of the data. Frequencies were compared in con-
tingency tables where the probability of rejecting the null
hypothesis of equal frequencies was derived from the sim-
ulation of 1,000 tables in which marginal totals were fixed
according to the data (Model III). These statistical analyses
are described in Sokal and Rohlf (1995) and were performed
using the statistical package STATGRAPHICS Centurion
XVI.16.0.08 (Stat Point Technologies, Inc). Canonical
Correspondance Analysis (CCA) was carried out in order
to test the effect of different variables on fungal species
occurrence (Ter Braak and Verdonschot 1995). Only sam-
ples with endophytic species with total abundance of 3 or
higher were included. The forward selection method was
used in order to rank environmental variables in their im-
portance for determining species composition (Escoufier
and Roberts 1979). The statistical significance of the effect
of each variable was tested by a Monte Carlo permutation
test with 500 permutations (Ter Braak 1992). These analyses
and the constrained ordination were performed using default
settings and untransformed species data with the program
CANOCO 4.5 (Ter Braak and Smilauer 2002).
Results
Environment and phenology of A. thaliana wild populations
Sampled populations are located in an approximately 300 km
N-S transect in the Central Plateau of Spain (Fig. 1), at
different elevations, with floristic formations associated to
Quercus sp. or Pinus sp. (Table 1). Latitude and elevation
affected the climatic variables temperature and precipitation
(Table 1). Elevation of each location was positively correlated
with the annual mean precipitation (r=0.87; P= 0.01) and
negatively correlated with annual maximum, minimum or
mean temperatures (r=−0.86 to −0.92; P<0.04).Hence,more
elevated populations had more precipitation and lower tem-
peratures. Latitude was negatively correlated with minimum
and mean temperatures, so temperatures decreased to the
North (r=−0.77 and −0.82, respectively; P<0.04). Plant size
at the end of the cycle (rosette diameter and stem length) and
plant productivity (number of siliques per plant) also
Fungal Diversity
depended on the latitude (Table 2), being the plants of
Northern populations larger than the plants in the South (r=
0.91, 0.94 and 0.87, respectively, P<0.03). A negative corre-
lation was also found between minimum and mean temper-
atures and the variables associated with the size and
productivity of the plants (r=−0.85 to −0.94, P<0.03).
Both growth period and life cycle of Arabidopsis plants
varied at the different locations (Table 3). The longest cycle
was found in the plants of Menasalbas, and the shortest
cycle was found in Marjaliza. In Ciruelos de Coca, Las
Rozas, Polán and Menasalbas, plants were already present
in mid February, while in Marjaliza no plants were found
until April. Plants at different phenological stages could be
found in the same sampling site depending on the popula-
tion and month of sampling. In Ciruelos de Coca and Las
Rozas, some plants had entered the reproductive stage al-
ready in February and the cycle finished in May, when most
of the plants were at shattering. In Polán and Menasalbas,
phenology was delayed with regard to Ciruelos de Coca and
Las Rozas, with most of the plants still green in May.
Rascafría was only sampled from May on, when all plants
had entered the reproductive stage, being most of them at
shattering in June. No plants were found in October at any
site, but in mid- November we found plants at early vege-
tative stage at Ciruelos de Coca, Las Rozas, Polán and
Menasalbas.
Isolation frequency of endophytes
We analyzed 208 Arabidopsis plants for the presence of
endophytic isolates, obtaining a total number of 530 sam-
ples (Table 4). Endophytes were isolated from 94 plants,
which means an isolation frequency (ie. proportion of plants
from which endophytes were isolated) of 45 %. We have
Fig. 1 Geographical location of A. thaliana populations in Central Spain
Table 2 Mean and standard error of plant density, rosette diameter, stem length and number of siliques in each population at the end of the cycle
Population Plant density
a
Rossette diameter (cm
2
)
b
Stem length
b
(cm) Number of siliques
b,c
Ciruelos de Coca 23.0± 9.1 1.8±0.2 27.1± 3.9 2.0
Rascafría 24.0± 8.7 1.9±0.2 25.2± 3.8 2.0
Las Rozas 256.0± 71.4 1.6±0.1 11.4± 1.1 1.0
Polán 18.7 ±10.9 1.5± 0.2 8.3± 1.7 1.0
Menasalbas 8.0±0.0 1.1±0.1 8.2±2.2 1.0
Marjaliza 8.0±4.0 1.0 ±0.0 7.0 ±0.0 1.0
a
Number of plants per square meter. Data correspond to the months of May or June, depending on the phenology of the population.
b
Data
correspond to plants at shattering.
c
Median of the classes in which plants were classified depending on the number of siliques: less than 5 (1), more
than 5 (2) and more than 10 (3)
Fungal Diversity
analyzed if there were statistically significant differences in
the isolation frequencies between the different data sets by
means of contingency tables that take into account the
differences in sampling sizes. The isolation frequency was
significantly different depending on the month of sampling
(Table 5,P<10
−3
). It increased significantly from March to
April (P=0.01) and from April to May (P<10
−3
), reaching
its maximum in June and decreasing significantly from June
to November (P<10
−3
). This pattern coincides with the
progress of Arabidopsis life cycle in the sampled popula-
tions, which attained the end of the cycle in May and June
(Table 3). The increase of the isolation frequency was main-
ly due to the plants at reproductive and shattering stages.
Hence, when analyzing the temporal variation of the isola-
tion frequency in plants of the same phenological stage, no
significant differences were found at vegetative stage (P=
0.24, Table 4). However, at reproductive and shattering
stages, this frequency increased from March or April to
June, when it attained its maximum (P<10
−3
and P=
0.002, respectively, Table 4). In order to further analyze if
the isolation frequency depended on the phenological stage
of the plant independently of the month of sampling, we
compared the isolation frequency of plants sampled at dif-
ferent phenological stages in the same month. Significant
differences in the isolation frequency were found in
February and May. In these months, most of the isolates
Table 3 Temporal variation of
phenological stage in A. thaliana
populations measured as per-
centage of plants at each pheno-
logical stage in the different
months
a
Vegetative: growth as rosette
(stages 1.04–3.9); Reproductive:
growth with plants producing
the inflorescence stems, flowers
and fruits (stages 5–6.90);
Shattering: plants in which at
least one silique had shattered
(stages 8 and later Boyes et al.
2001).
b
No A. thaliana plants
present.
c
Rascafría population
was sampled from May on
Population Month Nª of sampled
sites
Phenological stage
a
Vegetative Reproductive Shattering
Ciruelos de Coca February 1 100 % 0 % 0 %
March 3 49 % 51 % 0 %
April 5 0 % 44 % 56 %
May 4 0 % 0 % 100 %
June —
b
November 1 100 % 0 % 0 %
Rascafría
c
May 5 0 % 85 % 15 %
June 5 0 % 23 % 77 %
November —
b
Las Rozas February 1 25 % 75 % 0 %
March 4 0 % 100 % 0 %
April 5 2 % 44 % 54 %
May 4 1 % 19 % 80 %
June —
b
November 2 100 % 0 % 0 %
Polán February 1 50 % 50 % 0 %
March —
b
April —
b
May 3 0 % 74 % 26 %
June —
b
November 2 100 % 0 % 0 %
Menasalbas February 1 100 % 0 % 0 %
March —
b
April 4 3 % 61 % 36 %
May 5 0 % 58 % 42 %
June 1 0 % 0 % 100 %
November 3 100 % 0 % 0 %
Marjaliza February —
b
March —
b
April 1 50 % 50 % 0 %
May 3 33 % 58 % 8 %
June —
b
November —
b
Fungal Diversity
were obtained from plants at reproductive or shattering stages
(P=0.02,Table4). Regarding the plant population, significant
differences in the isolation frequency were also found in the
populations sampled in February and in May (P=0.03andP=
0.004 respectively, Table 4). In February, isolates were only
obtained from plants from the populations Polán and Las
Rozas, which were the most advanced in their cycle at that
time of the year, with plants at reproductivestage (Table 3). In
May, endophytes were obtained less frequently in Marjaliza,
which was the most retarded population, being the only one
with plants at vegetative stage at that time of the year. Hence,
the isolation frequency depends on both the month of
Table 4 Isolation frequency (%
of plants from which endophytes
were isolated) each month in
each population, according to
the phenological stage of the
sampled plant. The number of
analyzed plants is shown be-
tween parentheses
a
Vegetative: growth as rosette
(stages 1.04–3.9); Reproductive:
growth with plants producing
the inflorescence stems, flowers
and fruits (stages 5–6.90);
Shattering: plants in which at
least one silique had shattered
(stages 8 and later Boyes et al.
2001).
b
No A. thaliana plants
present.
c
Rascafría population
was sampled from May on
Month Population Total Phenological stage
a
Vegetative Reproductive Shattering
February Ciruelos de Coca 0 % (7) 0 % (7)
Las Rozas 75 % (4) 50 % (2) 100 % (2)
Polán 50 % (2) 0 % (1) 100 % (1)
Menasalbas 0 % (1) 0 % (1)
Marjaliza –
b
Total 29 % (14) 9 % (11) 100 % (3)
March Ciruelos de Coca 13 % (23) 0 % (11) 25 % (12)
Las Rozas 20 % (10) 20 % (10)
Polán –
b
Menasalbas –
b
Marjaliza –
b
Total 15 % (33) 10 % (21) 25 % (12)
April Ciruelos de Coca 61 % (18) 71 % (7) 55 % (11)
Las Rozas 24 % (21) 0 % (1) 17 % (6) 29 % (14)
Polán –
b
Menasalbas 45 % (11) 43 % (7) 50 % (4)
Marjaliza 67 % (3) 67 % (3)
Total 43 % (53) 50 % (4) 45 % (20) 41 % (29)
May Ciruelos de Coca 77 % (13) 77 % (13)
Rascafría
c
89 % (9) 88 % (8) 100 % (1)
Las Rozas 44 % (9) 0 % (1) 100 % (2) 33 % (6)
Polán 100 % (7) 100 % (5) 100 % (2)
Menasalbas 100 % (10) 100 % (7) 100 % (3)
Marjaliza 25 % (4) 0 % (1) 33 % (3)
Total 77 % (52) 0 % (2) 88 % (25) 72 % (25)
June Ciruelos de Coca –
b
Rascafría
c
88 % (17) 100 % (2) 87 % (15)
Las Rozas –
b
Polán –
b
Menasalbas 100 % (4) 100 % (4)
Marjaliza –
b
Total 86 % (21) 100 % (2) 89 % (19)
November Ciruelos de Coca 0 % (1) 0 % (1)
Rascafría
c
–
b
Las Rozas 19 % (16) 19 % (16)
Polán 0 % (8) 0 % (8)
Menasalbas 0 % (10) 0 % (10)
Marjaliza –
b
Total 9 % (35) 9 % (35)
GRAND TOTAL 45 % (208) 11 % (73) 65 % (62) 64 % (73)
Fungal Diversity
sampling and the phenological stage of the plant. No correla-
tions were found between the isolation frequency and the
geographic and climatic conditions of the populations
(Table 1) and neither with the variables related with the
productivity of the population (plant density, rosette and stem
size and number of siliques, Table 2).
In order to analyze if there were differences in the pres-
ence of endophytes in the different plant organs, we com-
pared the frequencies of samples with endophytes from
rosette leaves, stem leaves and siliques taken from the same
plant. Forty six plants were sampled both in the rosette and
stem leaves, with no significant differences in the isolation
frequency from each organ (endophytes were isolated from
38 rosette samples and 29 stem samples out of 81 samples,
respectively). Twenty eight plants were sampled in the sil-
iques and either rosette or stem leaves. In this case, the
isolation frequency was significantly lower in siliques than
in rosette or stem leaves from the same plant (endophytes
were isolated from 25 silique samples out of 56 and from 50
rosette or stem samples out of 72 samples, respectively,
P=0.004).
Diversity and composition of endophyte assemblages
We sequenced the ITS1-5.8S rRNA-ITS2 region of 120 ran-
domly chosen isolates, classifying the isolates based on se-
quence similarity (Table 5). We obtained a total of 48 species,
which belonged to 38 genera in 17 orders and 6 classes
(Table 6and Online Resource 1). Most of the isolates (117)
were Ascomycota,two isolates belonged to Basidiomycota
and one isolate belonged to subphylum Mucoromycotina
(Zygomycota). Seventy three isolates (61 % of the total)
belonged to the Class Dothideomycetes, which was the most
abundant with 4 orders, 17 genera and 20 species represented.
The most abundant order was Pleosporales,with 13 genera, 14
species and 54 isolates represented. The most diverse genus
was Colletotrichum, from which 5 different species were
found that accounted for 11 isolates. Eleven species lacking
taxonomically informative morphological structures could not
be identified to genus and even to order rank because their
sequences were less than 95 % similar to any identified
accession from the EMBL/GenBank fungus database. Only
eight species had five or more isolates (Alternaria sp.,
Cladosporium sp. 1, Colletotrichum sp. 5, Lewia sp.,
Embellisia sp. 1, Hipocrea lixii, Phoma sp.and Ulocladium
sp.), accounting for 50 % of the isolates. In contrast with these
dominant species, 58 % of the species were represented by just
one isolate (ie. singletons). The species accumulation curve
was non-asymptotic (Fig. 2). In contrast, we obtained a curve
approaching an asymptote when only plural species consisting
of two or more isolates were considered.
Total diversity was measured using Fisher’sα(32.77)
and Shannon diversity index (H′=3.48), with an evenness
e=0.73. Fisher’sαin each population (Table 7) was posi-
tively correlated with the annual precipitation (r=0.89;
P=0.04). As for sampled organ and vegetative stage of the
plant, Fisher’sαwas higher in rosette leaves than in stem
leaves and siliques, and it was lower in plants at vegetative
stage than at reproductive or shattering stages (Table 7). The
average number of species found in each population was
12.50 and the average number of shared species between
populations was 2.87, what results in a beta diversity index
(β
div
) of 0.77. The same index was used to compare organs
in the same plant (β
div
=0.82), plants in the same sampling
site (β
div
=0.92) and sites in the same sampling date within
each population (β
div
=0.92). These results indicate that it is
less probable to find different fungal species in different
organs of the same plant than in different plants from the
same population, independently of their position.
Assemblages from rosette and stem leaves were the most
similar (JI=0.32) and assemblages from siliques were the
most differentiated, with JI=0.28 and JI =0.24 compared to
stem and rosette leaves, respectively. Hence, the species
composition was more similar between closer organs. The
eight dominant species were shared between the three types
of organs, with the exception of H. lixii, and Ulocladium sp.,
which were not found in siliques. Jaccard similarity indexes
of the endophyte assemblages and geographical distances
between Arabidopsis populations are shown in Table 8.
There was no correlation between the similarity and the
spatial proximity of the populations. In order to determine
which variables were significant predictors of the dominant
fungal species in each population, forward selection CCA of
the geographical (elevation), ecological (genus of the dom-
inant tree) and climatic variables (temperature and precipi-
tation, Table 1) was performed. Only mean annual
precipitation and temperature were significant predictors
(P=0.004 and P=0.03, respectively, Fig. 3). Among the
different temperature measures, absolute maximum temper-
ature was the best adjusted to the data. Hence, the species
composition of fungal assemblages in each population
depended both on the precipitation and maximum tempera-
ture of the locations.
Discussion
The present work analyses the diversity of the endophytic
mycobiota of A. thaliana, the plant model par excellence, in
the wild. Much of the available knowledge about plant-
pathogen interactions comes from model systems with A.
thaliana as host (Nishimura and Dangl 2010). More recent-
ly, the ecological and evolutive aspects of plant-pathogen
interactions are also being studied in wild populations of A.
thaliana (Anderson and Mitchell-Olds 2011; Pagan et al.
2010; Salvaudon et al. 2008). However, the study of other
Fungal Diversity
Table 5 GenBank accession numbers of the ITS sequences and taxo-
nomic determination of 120 randomly chosen isolates. The taxonomic
determination was based on the homology with the sequences in the
fungal EMBL/GenBank database. The identification of the isolate was
limited to the genus, unless the sequences of the type species were
found among the better blast hits and these unequivocally indicated the
same species. The molecular identification was confirmed with the
morphology when the fungal reproductive structures were present
Isolate Accession
number
Taxonomic
determination
BEST hit
a
Identity
b
Reference Reproductive
structures
081 JX982366 Undetermined 1 HE653990. Pleosporales sp. 98 % Feldman et al. (2012) Not present
08147 JX982367 Undetermined 1 HE653990. Pleosporales sp. 98 % Feldman et al. (2012) Not present
08148 JX982368 Undetermined 1 HE653990. Pleosporales sp. 98 % Feldman et al. (2012) Not present
08160 JX982369 Undetermined 2 DQ979515. Fungal
endophyte isolate
94 % Higgins et al. (2007) Not present
0878 JX982370 Phoma sp. HQ630999. Phoma sp. 99 % Shrestha et al. (2011) Present
0886 JX982371 Phoma sp. FJ985695. Phoma sp. 99 % Sen et al. (2009) Present
08106 JX982372 Phoma sp. EU343173. Phoma exigua
var. exigua CBS 118.94
99 % Simon and Weiss (2008) Present
08137 JX982373 Phoma sp. HM755951. Phoma macrostoma 99 % Caesar et al. (2012) Present
08141 JX982374 Phoma sp. HM755951. Phoma macrostoma 99 % Caesar et al. (2012) Present
08145 JX982375 Phoma sp. HM755951. Phoma macrostoma 99 % Caesar et al. (2012) Present
08103 JX982376 Undetermined 3 FJ553169. Uncultured
Pleosporales clone
99 % Hartmann et al. (2009) Not present
0847 JX982377 Leptosphaeria sp. M96383. Leptosphaeria maculans 99 % Morales et al. (1993) Not present
083 JX982378 Lewia sp. FN539064. Lewia infectoria 99 % Partfitt et al. (2010) Not present
0811 JX982379 Lewia sp.FN539064. Lewia infectoria 99 % Partfitt et al. (2010) Not present
0815 JX982380 Lewia sp.FN539059. Uncultured Lewia 99 % Partfitt et al. (2010) Not present
08140 JX982381 Lewia sp.FN539059. Uncultured Lewia 99 % Partfitt et al. (2010) Not present
0813 JX982382 Lewia sp.FN539059. Uncultured Lewia 99 % Partfitt et al. (2010) Not present
0810 JX982383 Lewia sp.FN539059. Uncultured Lewia 99 % Partfitt et al. (2010) Not present
0821 JX982384 Lewia sp.FN539059. Uncultured Lewia 99 % Partfitt et al. (2010) Not present
084 JX982385 Embellisia sp. 1 JN383490. Embellisia eureka 98 % Lawrence et al. (2012) Not present
0867 JX982386 Embellisia sp. 1 JN383490. Embellisia eureka 99 % Lawrence et al. (2012) Not present
08105 JX982387 Embellisia sp. 1 JN383490. Embellisia eureka 99 % Lawrence et al. (2012) Not present
0850 JX982388 Embellisia sp. 1 JN383490. Embellisia eureka 98 % Lawrence et al. (2012) Not present
08122 JX982389 Embellisia sp. 1 JN383490. Embellisia eureka 99 % Lawrence et al. (2012) Not present
0812 JX982390 Embellisia sp. 1 JN383490. Embellisia eureka 100 % Lawrence et al. (2012) Not present
08127 JX982391 Embellisia sp. 1 JN383490. Embellisia eureka 99 % Lawrence et al. (2012) Not present
08128 JX982392 Embellisia sp. 1 JN383490. Embellisia eureka 100 % Lawrence et al. (2012) Not present
08108 JX982393 Embellisia sp. 1 JN383490. Embellisia eureka 99 % Lawrence et al. (2012) Not present
0826 JX982394 Embellisia sp. 1 JN383490. Embellisia eureka 99 % Lawrence et al. (2012) Not present
08107 JX982395 Embellisia sp. 1 JN383490. Embellisia eureka 100 % Lawrence et al. (2012) Not present
0822 JX982396 Embellisia sp. 2 JN383492. Embellisia lolii 99 % Lawrence et al. (2012) Not present
0820 JX982397 Alternaria sp. FJ176475. Alternaria sp. 99 % Qi et al. (2009) Present
08185 JX982398 Alternaria sp. FJ176475. Alternaria sp. 99 % Qi et al. (2009) Present
0838 JX982399 Alternaria sp. GQ121322. Alternaria alternata 100 % Shanmugam et al. (2011) Present
0860 JX982400 Alternaria sp. GQ121322. Alternaria alternata 99 % Shanmugam et al. (2011) Present
08135 JX982401 Alternaria sp.FJ176475. Alternaria sp. 99 % Qi et al. (2009) Present
0832 JX982402 Alternaria sp. GQ121322. Alternaria alternata 100 % Shanmugam et al. (2011) Present
0856 JX982403 Alternaria sp. GQ121322. Alternaria alternata 99 % Shanmugam et al. (2011) Present
08166 JX982404 Alternaria sp. GQ121322. Alternaria alternata 99 % Shanmugam et al. (2011) Present
0896 JX982405 Alternaria sp. GQ121322. Alternaria alternata 100 % Shanmugam et al. (2011) Present
0859 JX982406 Alternaria sp. GQ121322. Alternaria alternata 99 % Shanmugam et al. (2011) Present
08153 JX982407 Alternaria sp. GQ121322. Alternaria alternata 99 % Shanmugam et al. (2011) Present
0841 JX982408 Ulocladium sp. FN868451. Ulocladium sp. 99 % Botella and Diez (2011) Present
0881 JX982409 Ulocladium sp. FN868451. Ulocladium sp. 99 % Botella and Diez (2011) Present
Fungal Diversity
Table 5 (continued)
Isolate Accession
number
Taxonomic
determination
BEST hit
a
Identity
b
Reference Reproductive
structures
0885 JX982410 Ulocladium sp. FN868451. Ulocladium sp. 99 % Botella and Diez (2011) Present
0855 JX982411 Ulocladium sp. FN868451. Ulocladium sp. 99 % Botella and Diez (2011) Present
08150 JX982412 Ulocladium sp. FN868451. Ulocladium sp. 99 % Botella and Diez (2011) Present
0899 JX982413 Stemphylium sp. GU062207. Stemphylium sp. 99 % Arhipova et al. (2011) Present
08101 JX982414 Stemphylium sp. GU062207. Stemphylium sp. 99 % Arhipova et al. (2011) Present
08100 JX982415 Stemphylium sp. AF203450. Stemphylium solani 99 % Mehta et al. (2002) Present
0823 JX982416 Drechslera sp. JN712472. Pyrenophora
leucospermi CPC 1298
98 % Crous et al. (2011) Not present
0824 JX982417 Drechslera sp. JN712474. Pyrenophora
leucospermi CPC 1298
98 % Crous et al. (2011) Not present
08115 JX982418 Cyclothyrium sp. FJ025227 Cyclothyrium sp.
B14-3242.
99 % Sun and Guo (2008) Not present
08165 JX982419 Cyclothyrium sp. FJ025227 Cyclothyrium sp.
B14-3242.
98 % Sun and Guo (2008) Not present
0853 JX982420 Preussia sp. JN225887. Preussia sp.
ICMP:18938
99 % Johnston et al. (2012) Not present
08110 JX982421 Undetermined 4 AY568066. Ascomycete sp. 95 % Cunnington 2004 Not present
088 JX982422 Undetermined 5 FJ237141. Uncultured
fungus clone.
94 % Kuhnert et al. (2012) Not present
087 JX982423 Aureobasidium sp. FN868454. Aureobasidium
pululans
100 % Botella and Diez (2011) Present
089 JX982424 Aureobasidium sp. FN868454. Aureobasidium
pululans
100 % Botella and Diez (2011) Present
0857 JX982425 Aureobasidium sp. FN868454. Aureobasidium pululans 100 % Botella and Diez (2011) Present
0848 JX982426 Cladosporium
cladosporioides
AY251074. Cladosporiun
cladosporioides STE-V 3683
99 % Braun et al. (2003) Present
08136 JX982427 Cladosporium
cladosporioides
JF499834. Cladosporium
cladosporioides CPC18230
100 % Crous and Groenewald (2011) Present
08114 JX982428 Cladosporium
cladosporioides
GU566222. Cladosporium
cladosporioides
99 % Bukovska et al. (2010) Present
08180 JX982429 Cladosporium
cladosporioides
JF499834. Cladosporium
cladosporioides CPC18230
99 % Crous and Groenewald (2011) Present
08117 JX982430 Cladosporium sp.
1
JN906977. Cladosporium
allii CBS 101.81
99 % Bensch et al. (2012) Present
08156 JX982431 Cladosporium sp.
1
HQ540688. Davidiellaceae 99 % Larkin et al. (2012) Present
08121 JX982432 Cladosporium sp.
1
JN906977. Cladosporium
allii CBS 101.81
99 % Bensch et al. (2012) Present
08119 JX982433 Cladosporium sp.
1
JN906977. Cladosporium
allii CBS 101.81
99 % Bensch et al. (2012) Present
08130 JX982434 Cladosporium sp.
1
JN906977. Cladosporium
allii CBS 101.81
99 % Bensch et al. (2012) Present
08144 JX982435 Cladosporium sp.
1
JN906977. Cladosporium
allii CBS 101.81
99 % Bensch et al. (2012) Present
08183 JX982436 Cladosporium sp.
1
EU167591. Davidiella macrospora
CBS 138.40
99 % Simon et al. (2009) Present
08155 JX982437 Cladosporium sp.
1
JN906977. Cladosporium
allii CBS 101.81
99 % Bensch et al. (2012) Present
0854 JX982438 Cladosporium sp.
2
EU167574. Cladosporium
sp. CBS 280.49
99 % Simon et al. (2009) Present
0827 JX982439 Podospora sp. HQ829072. Podospora sp. 99 % Baynes et al. (2012) Present
0828 JX982440 Podospora sp. HQ829072. Podospora sp. 99 % Baynes et al. (2012) Present
08200 JX982441 Podospora sp. HQ829072. Podospora sp. 99 % Baynes et al. (2012) Present
08174 JX982442 Sarocladium
strictum
JX094774 Sarocladium
strictum ATCC 10141
99 % Mycology, American Type Culture
Collection
Present
0845 JX982443 Sarocladium
strictum
JX094774 Sarocladium
strictum ATCC 10141
99 % Mycology, American Type Culture
Collection
Present
Fungal Diversity
Table 5 (continued)
Isolate Accession
number
Taxonomic
determination
BEST hit
a
Identity
b
Reference Reproductive
structures
086 JX982444 Hypocrea lixii AF443919 Hypocrea lixii
G.J.S. 92–100
98 % Chaverri et al. (2003) Present
0835 JX982445 Hypocrea lixii AF443919 Hypocrea lixii
G.J.S. 92–100
98 % Chaverri et al. (2003) Present
0891 JX982446 Hypocrea lixii AF443919 Hypocrea lixii
G.J.S. 92–100
98 % Chaverri et al. (2003) Present
0858 JX982447 Hypocrea lixii AF443926. Hypocrea lixii
G.J.S. 90–254
99 % Chaverri et al. (2003) Present
0897 JX982448 Hypocrea lixii AF443926. Hypocrea lixii
G.J.S. 90–254
99 % Chaverri et al. (2003) Present
08173 JX982449 Hypocrea lixii AF443926. Hypocrea lixii
G.J.S. 90–254
99 % Chaverri et al. (2003) Present
0817 JX982450 Trichoderma sp. EU280100. Trichoderma ghanense 99 % Hoyos-Carvajal et al. (2009) Present
08131 JX982451 Undetermined 6 FJ524321. Uncultured
endophytic fungus clone
99 % Yuan et al. (2010) Not present
0819 JX982452 Plectosphaerella
cucumerina
JQ647434. Plectosphaerella
cucumerina CABI:IMI 312228
98 % Cannon et al. 2012a Present
0831 JX982453 Plectosphaerella
cucumerina
JQ647434. Plectosphaerella
cucumerina CABI:IMI 312228
98 % Cannon et al. 2012a Present
08177 JX982454 Plectosphaerella
cucumerina
JQ647434. Plectosphaerella
cucumerina CABI:IMI 312228
98 % Cannon et al. 2012a Present
0825 JX982455 Plectosphaerella
sp
L36640. Plectosphaerella
cucumerina
97 % O’Donnell and Gray (1995) Present
08169 JX982456 Undetermined 7 GU062315. Plectosphaerella sp. 94 % Arhipova et al. (2011) Not present
0888 JX982457 Sordaria sp. AY681188. Sordaria fimicola
CBS 508.50
100 % Cai et al. (2006) Not present
08161 JX982458 Apodus deciduus AY681199. Apodus deciduus
CBS 506.70
99 % Cai et al. (2006) Not present
08123 JX982459 Undetermined 8 EU686153. Fungal endophyte 99 % Davis and Shaw (2008) Not present
0842 JX982460 Colletotrichum sp.
5
EU070911. Colletotrichum
destructivum
99 % Sun and Zhang (2009) Present
0865 JX982461 Colletotrichum sp.
5
EU070911. Colletotrichum
destructivum
99 % Sun and Zhang (2009) Present
08198 JX982462 Colletotrichum sp.
5
EU070911. Colletotrichum
destructivum
99 % Sun and Zhang (2009) Present
08116 JX982463 Colletotrichum sp.
5
EU070911. Colletotrichum
destructivum
99 % Sun and Zhang (2009) Present
08124 JX982464 Colletotrichum sp.
5
EU070911. Colletotrichum
destructivum
99 % Sun and Zhang (2009) Present
08102 JX982465 Colletotrichum sp.
1
AJ301976. Colletotrichum
truncatum
99 % Nirenberg et al. (2002) Present
08125 JX982466 Colletotrichum sp.
1
AJ301976. Colletotrichum
truncatum
99 % Nirenberg et al. (2002) Present
0861 JX982467 Colletotrichum sp.
4
AJ301954. Colletotrichum
dematium
99 % Nirenberg et al. (2002) Present
0866 JX982468 Colletotrichum sp.
4
DQ286154. Colletotrichum dematium 98 % Farr et al. (2006) Present
08195 JX982469 Colletotrichum sp.
2
JQ005765. Colletotrichum linicola 95 % O’Connell et al. (2012) Present
0869 JX982470 Colletotrichum sp.
3
GU227803. Colletotrichum
tofieldiae IMI 288810
97 % Damm et al. (2009) Present
08182 JX982471 Biscogniauxia
mediterranea
JF295128. Biscognauxia
mediterránea CPC:18216
99 % Mirabolfathy et al. (2011) Not present
0849 JX982472 Penicillium sp. 1 HQ649932. Penicillium sp. 98 % Macia-Vicente et al. (2012) Present
0864 JX982473 Penicillium sp. 2 DQ681334. Penicillium granulatum 99 % Redondo et al. (2009) Present
08143 JX982474 Penicillium sp. 3 FN548146. Penicillium sp. 99 % Unterseher and Schnittler (2010) Present
0829 JX982475 Geomyces sp. JF439476. Geomyces sp. 99 % Han et al. (2011) Not present
08134 JX982476 Geomyces sp. JX171175. Geomyces sp. 99 % Slemmons et al. (2012) Not present
Fungal Diversity
symbiotic associations, such as mutualistic and neutral, rep-
resents a small part of the extensive knowledge generated by
this plant species, and the lack of data about fungal symbi-
otic communities is striking, with only few published exam-
ples of endophytes able to colonize roots and shoots of
Arabidopsis plants (Jaschke et al. 2010; Junker et al. 2012;
Peskan Berghofer et al. 2004; Weiss et al. 2011). In this
work, we have compared the occurrence and diversity of
endophytic fungi of A. thaliana populations situated at
different ecological environments, taking into account the
effect of location, date of sampling, plant phenological stage
and organ in the frequency of fungal endophytes and in the
species composition of the endophytic fungal community.
We have surveyed six different locations at Central
Spain, with various geographical and ecological attributes,
during the growing season of A. thaliana. Our results agree
with former demographical analyses in Spain, showing that
Arabidopsis populations consist of one or two cohorts of
plants that either germinate in the spring or germinate in the
autumn and overwinter as rosettes, flowering in the late
winter or beginning of the spring (Pagan et al. 2010). We
have obtained isolates during the whole growing season of
A. thaliana, from plants that were at different phenological
stages at the same time of the year. This situation has
allowed us to show that the proportion of plants with endo-
phytic isolates depended on the time of the year, but also on
the phenological stage of the plant. Hence, there were sig-
nificant differences in the isolation frequency in February
and May, when plants at reproductive and shattering stages
had endophytes more frequently than plants at vegetative
stage. Also, significant differences between locations were
only found in February and May, when differences between
populations in the developmental stage of the plants were
patent: the isolation frequency in the populations that still
had plants at vegetative stage was lower. Seasonal changes
in endophytes incidence have been shown in other studies
(Mishra et al. 2012;Terhonenetal.2011; Wearn et al.
2012), and also changes associated to the phenological stage
of the host (Cook et al. 2012; Tadych et al. 2012), although
both factors are difficult to disassociate and have not been
deeply studied. Our results also show evidence of plant age
as a factor that increases the probability of hosting endo-
phytes. At vegetative stage, the isolation frequency did not
depend on the time of the year, while at reproductive and
shattering stages, this frequency increased from March or
April to June, when it attained its maximum. Plants at
vegetative stage may mainly be due to new germinations
independently of the time of the year and, hence, may have
always a similar age. However, plants at reproductive and
shattering stages are older than plants at vegetative stage and
their age increases from March to June. Further evidence of
the effect of the age in the isolation frequency was the
difference found between younger and older organs: the
isolation frequency in siliques was significantly lower than
in rosette or stem leaves of the same plant. Other works also
relate the isolation frequency to the age of the plant or the
plant organ (Arnold and Herre 2003; Tadych et al. 2012;
Terhonen et al. 2011). This could be caused because the
colonization of the plant by the initial inoculum expands
with time, or because the time of exposure to endophyte
inoculum increases with age (Guo and Wang 2008; Herre et
al. 2007; Osono 2008; Sanchez Marquez et al. 2012; Sieber
2007). The last hypothesis would be corroborated by a
higher diversity in species composition in older plants or
organs, being our data consistent with this hypothesis: al-
though not highly significant, the Fisher’sαdiversity was
Table 5 (continued)
Isolate Accession
number
Taxonomic
determination
BEST hit
a
Identity
b
Reference Reproductive
structures
08167 JX982477 Geomyces sp. JX415263. Geomyces sp. 99 % Muller et al. (2012) Not present
0844 JX982478 Undetermined 9 JX171175. Geomyces sp. 81 % Slemmons et al. (2012) Not present
08118 JX982479 Undetermined 10 HQ611334. Uncultured
fungus clone
98 % Lindner et al. (2011) Not present
08113 JX982480 Undetermined 11 JN032492. Uncultured
fungus clone
90 % Lindahl et al. (2007) Not present
0837 JX982481 Mycoarthris sp. JX270444 Mycoarthris sp. 99 % Lorch et al. 2012 Not present
08154 JX982482 Lophodermium sp. AM921705.
Lophodermium sp.
97 % Sanchez Marquez
et al. (2007)
Not present
0874 JX982483 Mortierella sp. HQ608143. Mortierella sp. 99 % Rodrigues et al. (2011) Not present
08138 JX982484 Leucosporidium
sp.
FN908919. Leucosporidium
drumii CBS 11562
97 % Yurkov et al. (2012) Not present
08158 JX982485 Leucosporidium
sp.
FN908919. Leucosporidium
drumii CBS 11562
96 % Yurkov et al. (2012) Not present
a
Best published hit in the fungal EMBL/GenBank database.
b
Nucleotide maximum identity with the best published hit in the fungal EMBL/
GenBank database
Fungal Diversity
higher in rosette (older) leaves than in stem leaves and
siliques, and it was lower in plants at vegetative stage than
at reproductive or shattering stages.
The isolation frequency of endophytes found in our work is
comparable to other host species in similar latitudes (Arnold
and Lutzoni 2007). The fungal species diversity found in
Arabidospis assemblages is also similar, ifnot higher, to foliar
assemblages of other hosts in temperate and tropical regions
(Feldman et al. 2012; Rivera-Orduna et al. 2011;
Thongsandee et al. 2012; Wearn et al. 2012)and,inparticular,
it is in consonance with the highdiversity found in other hosts
in Spain (Gonzalez and Tello 2011; Sanchez Marquez et al.
2012). Species abundance distribution showed the typical
pattern of microbial communities, with a long right tail formed
by the species represented by just one isolate (Fuhrman 2009).
Species accumulation curve was non-asymptotic, which is
typical from species rich ecosystems (Arnold and Lutzoni
2007; Suryanarayanan et al. 2003) but, when only plural
species were considered, the species accumulation curve
attained an asymptote. This result indicates that most of the
dominant species have been identified, and more intensive
samplings will mostly find species that rarely occur (Arnold
and Lutzoni 2007; Sanchez Marquez et al. 2012). Many of the
species isolated in this work showed high nucleotide identities
with other endophytic isolates of the EMBL/GenBank in Blast
searches. However caution must be used when referring to
these names as in many cases they were not the sequences
from the type strains and thus could be wrongly named in
GenBank (Ko Ko et al. 2011). The vast majority of the isolates
obtained pertained to Ascomycota phylum, and the most
abundant and diverse orders were Pleosporales and
Table 6 Phyllum, class, order, species name and abundance of the
different fungal endophytic species in A. thaliana populations
PHYLUM/
Class/Order
Species
code
a
Species Abundance
ASCOMYCOTA
Dothideomycetes
Dothideales AUR Aureobasidium sp. 3
Capnodiales CL_CL Cladosporium
cladosporioides
4
CL_1 Cladosporium sp. 1 8
Cladosporium sp. 2 1
Pleosporales ALT Alternaria sp. 11
Drechslera sp. 2
EMB Embellisia sp 1 11
Embellisia sp. 2 1
Leptosphaeria sp. 1
LEW Lewia sp.7
PHO Phoma sp. 6
Preussia sp. 1
STE Stemphylium sp. 3
ULO Ulocladium sp. 5
UND Undetermined 1 3
Undetermined 2 1
Undetermined 3 1
Undetermined 4 1
Mytosporic
Dothideomycetes
Cyclothyrium sp. 2
Undetermined 5 1
Eurotiomycetes
Eurotiales Penicillium sp. 1 1
Penicillium sp. 2 1
Penicillium sp. 3 1
Leotiomycetes
Helotiales Mycoarthris sp. 1
Undetermined 10 1
Undetermined 11 1
Rhytismatales Lophodermium sp. 1
Leotyomycetes
incertae sedis
GEO Geomyces sp. 3
Sordariomycetes
Glomerellales Colletotrichum sp. 1 2
Colletotrichum sp. 2 1
Colletotrichum sp. 3 1
Colletotrichum sp. 4 2
COLL Colletotrichum sp. 5 5
Hypocreales Sarocladium strictum 2
HYP Hypocrea lixii 6
Trichoderma sp. 1
Phyllacorales PLEC Plectosphaerella
cucumerina
3
Plectosphaerella sp. 1
Undetermined 7 1
Sordariales POD Podospospora sp. 3
Table 6 (continued)
PHYLUM/
Class/Order
Species
code
a
Species Abundance
Sordaria sp. 1
Apodus deciduus 1
Xylariales Biscognauxia
mediterranea
1
Undetermined Undetermined 6 1
Undetermined 8 1
BASIDIOMYCOTA
Mycobotryomycetes
Sporidiobolales Leucosporidium sp. 2
Early diversifying
fungal lineages
(ZYGOMYCOTA)
Mucoromycotina
(subphylum)
Mortierellales Mortierella sp. 1
a
A code has been assigned for species with abundance equal or higher
than 3. These species have been included in the CCA s (see Fig. 3)
Fungal Diversity
Capnodiales, as in most of the foliar endophytic assemblages
described to date (Arnold 2007; Botella and Diez 2011;
Feldman et al. 2012;Rodriguezetal.2009; Sanchez
Marquez et al. 2012). However, the abundance of the order
Glomerellales is not that usual in other described assemblages.
This abundance was due to the occurrence of five different
species of the genus Colletotrichum, which was the most
diverse genus found in our work. Species of this genus are
frequent endophytic fungal colonizers of tropical plants (Lu et
al. 2004; Rakotoniriana et al. 2008; Rojas et al. 2010), and are
notable for their ability for crossing the border between
pathogenesis and endophytism, displaying one or another
lifestyle depending on the host (Freeman et al. 2001; Redman
et al. 2001). The most abundant species found in our work
within this genus (Colletotrichum sp. 5) belongs to the C.
destructivum clade, as described by Cannon et al. (2012b).
This clade contains species such as C. destructivum and C.
higginsianum that are pathogenic on A. thaliana (O’Connell et
al. 2004; Sun and Zhang 2009). The ITS sequence is not
differential enough within the C.destructivum clade to act as
a species-level marker in isolation (Hyde et al. 2009; Cannon et
al. 2012b), but multilocus phylogenies of strains provisionally
accepted as representative of C. higginsianum and C. destruc-
tivum indicate that these two species are distinct entities
(Cannon et al. 2012b;O’Connelletal.2012). C. higginsianum
has recently been sequenced, forming one of the reference
model pathosystems for hemibiotrophic fungal pathogens
(O’Connell et al. 2012). Our work is the first reference and
isolation of strains within the C. destructivum clade naturally
infecting A. thaliana, being these asymptomatic.
The first order factor accounting for species richness of
endophytes in A. thaliana was the variation in composition
between plants in the same population, with a beta diversity
index (β
div
) of 0.92. The beta diversity index represents the
probability that two samples from two different assemblages
are different and, thus, measures the amount of change in
species composition between them (Magurran 2004). It was
more probable to find different fungal species in different
plants of the same population than in different organs of the
same plant and, thus, the species variation within plants was
lower than among plants. The species variation within pop-
ulations was not spatially structured, since the β
div
between
plants in the same sampling site was equal to the β
div
between
sites, suggesting a random distribution of the different endo-
phytic species. Neither the diversity between populations was
due to isolation by distance, since JI similarity indexes were
not correlated with the geographical proximity of the
0
10
20
30
40
50
60
70
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91
a
b
Fig. 2 Species accumulation curves showing the relationship between
the number of samples analyzed and the number of endophytic species
found. When all fungal species identified were considered (a) the curve
was non asymptotic, but when only species represented by more than
one isolate were considered (b), an asymptotic curve resulted.
Continuous lines: sample-based rarefaction curves. Dashed lines:
95 % confidence intervals. Points: empirical data
Tab le 7 Number of isolates sequenced, nº of species, Fisher’sα
values and standard deviation (SD) of endophytic assemblages accord-
ing to Arabidopsis population, vegetative stage of the sampled plants
and organ sampled
Assemblage Nº of isolates
sequenced
Nº of species Fisher’sα±SD
Population
Ciruelos de Coca 14 11 3.14±1.94
a
Rascafría 32 22 9.85 ±2.4
c
Las Rozas 19 18 4.71± 2.4
ab
Polán 7 7 6.18±3.81
ab
Menasalbas 18 13 6.89 ±2.46
b
Marjaliza 4 4 –
#
Plant organ
Rosette leaves 47 30 14.28± 3.07
b
Stem leaves 27 20 9.79±2.77
a
Siliques 20 21 11.84± 3.82
ab
Phenological stage
Vegetative 8 8 6.83 ±5.16
a
Reproductive 37 27 13.95± 3.57
b
Shattering 49 33 13.85± 2.66
b
a, b, c
Different letters mean significant differences at 95 % confidence.
#
Not calculated because of low amount of data
Fungal Diversity
populations. Hence, other factors may account for the occur-
rence of the dominant species in the different populations. We
have examined variables related with the geography, ecology
and climate of the locations as factors determining the species
composition of the endophytic community. Among all the
variables analyzed, precipitation and temperature were the
significant predictors of the dominant fungal species in each
population. Both variables are determined by the elevation of
the locations, with close locations at different elevations hav-
ing differences in precipitation and temperature, what may
explain the differences in species composition found in close
populations. Thus, our work determines the climate as the
main factor in shaping the foliar endophytic composition in
thesamehostspecies,evenatregionalscale,inconsonance
with other works (Carroll and Carroll 1978; Suryanarayanan
et al. 2002; Zimmerman and Vitousek 2012). We have also
found a significant correlation between the precipitation and
the diversity of the endophytic assemblages. Humid condi-
tions are, in general, more favorable for fungal sporulation and
infection (Wilson 2000), and this is consistent with an increase
in endophyte incidence and diversity with precipitation
(Mishra et al. 2012; Tejesvi et al. 2005). The correlation
between precipitation and diversity can also be related to a
better dispersal of endophytes by rain (Carroll 1988;
Rodriguez et al. 2009; Saikkonen 2007).
Both temperature and precipitation are important factors in
fungal biology and, hence, may determine the adaptation of
fungal species to the environment (Zak et al. 2011). The inter-
action with the host is a third factor that may take part in the
adaptation of the endophytic fungi to the environment and may
also be affected by climatic factors (Compant and Sessitsch
2010). It has been shown, for example, that temperature has an
important role in the modulation of the immune responses in A.
thaliana (Alcazar and Parker 2011) and, thus, differences in
temperature may affect the composition of the interacting fun-
gal community (Traw and Bergelson 2010). Other genetic
factors of the plant may also be involved in the composition
of the assemblages in the different populations (Bulgarelli et al.
2012;Lundbergetal.2012;Micallefetal.2009). Hence,
further work is necessary in order to determine the effect of
host and environmental factors, or the interactions between
both, in the endophytic assemblages (Hoffman and Arnold
2008). A. thaliana grows in very diverse ecosystems all over
the world, both as native and as introduced species (Hoffmann
2002). Thus, Arabidopsis can serve as a standard host for
comparing the endophytic communities at global scale in order
to improve our understanding of the factors that shape the
endophytic assemblages and their contribution to the adaptation
of plants to the environment. The genotyping of Arabidopsis
plants of natural populations is nowadays very straight forward
at a reasonable cost (Pico et al. 2008) and it could be included in
standardized culture free-methods connecting host genetic and
endophytic variation at different scales. A. thaliana,withits
rapid cycle, adaptation ability, high diversity in genotypes and
mutants availability, also offers a great tool for experiments
aimed to split up environmental and genetic factors, such as
common garden experiments and genetic analysis using endo-
phytic isolates. In our study, we have obtained more than 120
endophytic isolates of 48 species. Some of these species are
unknown, others are frequent components of endophytic
assemblages and others are related with very well studied
pathogens of A. thaliana,suchasC. higginsianum or
Plectosphaerella cucumerina (O’Connelletal.2012;Ramos
et al. 2013). These isolates can be used as model systems for
molecular approaches for the study of the endophytic functional
Table 8 Jaccard similarity in-
dexes (JI, in bold) between the
fungal endophytic assemblages
A. thaliana populations and dis-
tance (km) between the locations
where the populations are
situated
Ciruelos de Coca Rascafría Las Rozas Polán Menasalbas Marjaliza
Ciruelos de Coca 69 101 164 179 193
Rascafría 0.14 46 127 145 150
Las Rozas 0.09 0.13 82 102 104
Polán 0.27 0.07 0.17 20 32
Menasalbas 0.14 0.13 0.19 0.11 31
Marjaliza 0.00 0.18 0.10 0.00 0.13
Fig. 3 First and second axes of the CCA based species-conditional
biplot showing the ordination of species with abundance equal or
higher than 3 in relation with the variables annual mean precipitation
(P) and annual absolute temperature (Tmaxabs). The diagram displays
100 % of the variance in the species-variables relations. F-ratio=2.29,
P=0.002. Eighenvalues (λ): axis 1, λ= 0.523; axis 2, λ=0.310. The
complete name of fungal species is shown in Table 6
Fungal Diversity
interactions with the host (Junker et al. 2012). Hence, the
endophytic mycobiota associated to the natural populations of
A. thaliana offers a whole range of possibilities to increase our
knowledge about the endophytic lifestyle, in order to optimize
the promising applications of fungal endophytes.
Acknowledgments We thank Drs. Carlos Alonso-Blanco and
Fernando García-Arenal for showing us the localization of the wild
populations of A. thaliana. We also thank Drs. Fernando García-Arenal,
and Mª Ángeles Ayllón, and an anonymous reviewer, for critical review
and suggestions for improving the manuscript. Mª Ángeles Portal pro-
vided excellent technical assistance. Meteorological data have been gent-
ly provided by the Spanish Metereology Agency (AEMET). This work
was funded by grants CAM CCG07-UPM/GEN-1899 of DGUI of
Comunidad de Madrid and UPM and AGL2008-00818 of Ministerio de
Educación y Ciencia of the Spanish Government to Soledad Sacristán.
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