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Environmental Microbiology Reports (2014)
Functional congruence of rhizosphere microbial
communities associated to leguminous tree from
Brazilian semiarid region
Rodrigo Gouvêa Taketani,*
Vanessa Nessner Kavamura, Rodrigo Mendes and
Itamar Soares Melo
Laboratory of Environmental Microbiology, Embrapa
Environment, Brazilian Agricultural Research
Corporation-EMBRAPA, Jaguariuna, São Paulo, Brazil.
Summary
Semiarid environments are characterized by the
uneven spread of rain throughout the year. This leads
to the establishment of a biota that can go through
long periods without rain. In order to understand
the dynamics of rhizosphere microbial communities
across these contrasting seasons in Caatinga, we
used the Ion Torrent platform to sequence the
metagenome of the rhizosphere of a native legumi-
nous plant (Mimosa tenuiflora). The annotation indi-
cated that most abundant groups detected were the
Actinobacteria and Proteobacteria, and the dominant
functional groups were carbohydrate and protein
metabolisms, and that in the wet season, the commu-
nities carried carbohydrate and amino acid meta-
bolisms. The major differences observed between
seasons were higher abundance of genes related to
carbohydrate and amino acid metabolisms in the
rainy season, indicating that the populations present
might be better adapted to a higher abundance of
organic matter. Besides, no clear separation of
samples was detected based on their taxonomic com-
position whereas the functional composition indi-
cates that samples from the rain season are more
related. Altogether, our results indicate that there is a
large functional stability in these communities mostly
due to the selection of features that aid the biota to
endure the dry season and blossom during rain.
Introduction
Semiarid environments are scattered around the globe
and are characterized by the uneven spatial and seasonal
Received 12 February, 2014; revised 4 June, 2014; accepted 7 June
2014. *For correspondence. E-mail rgtaketani@yahoo.com.br; Tel.
+55-19-33112665; Fax +55-10-33112640.
© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd
doi:10.1111/1758-2229.12187
distribution of rain (Pointing and Belnap, 2012). Hence, its
inhabitants have the ability to withstand the contrast
between dry and rainy seasons (Kavamura et al., 2013a).
The Caatinga biome is a semiarid environment located in
Brazilian Northeast covering approximately 12% of its
territory (Trovão et al., 2007). The precipitation distribution
is predominantly concentrated in a period of 3–4 months
over the year, resulting in two contrasting seasons, i.e. dry
and wet; eventually, the dry season can last for more than
1 year (Trovão et al., 2007). The hydric stress caused by
the lack of rainfall associated with a significant increase in
temperature and ultraviolet exposure for surface dwellers
(Kavamura et al., 2013a) in combination with human
predatory activity makes the Caatinga prone to desertifi-
cation (Silva et al., 2004).
While many studies have addressed above-ground
seasonal changes, effects on the below-ground micro-
flora have being neglected. In this context, we hypoth-
esized that differences in climatic conditions between
seasons would select for some key features in the
rhizosphere. In order to test this hypothesis, we evaluate
the rhizosphere microbiome of one of the most com-
mon leguminous tree found in the Caatinga, Mimosa
tenuiflora. Leguminous trees can represent 70% of the
number of plant individuals in the caatinga landscape
(Queiroz, 2006). This high prevalence can be attributed
to the low amount of nitrogen often observed in these
soils (Queiroz, 2006). Among these plants, M. tenuiflora
is one of the most abundant and widespread leguminous
trees in this biome (Queiroz, 2006). It is also intensively
used as firewood and in constructions by the local
farmers (Queiroz, 2006). Hence, we have chosen this
species as our model because of its distribution, ecologi-
cal and social roles.
The interaction between the plant, the soil and its micro-
bial inhabitants shapes what we call the rhizosphere envi-
ronment (Bais et al., 2006). Many studies have shown the
participation of each of these as drivers of selection in this
environment (Fierer and Jackson, 2006; Martiny et al.,
2006; Ramette and Tiedje, 2007). In some cases, this
interaction between plant and rhizospheric community
even allows the plant to withstand abiotic or biotic
stresses, such as desiccation (Kavamura et al., 2013a) or
diseases (Philippot et al., 2013). In turn, the rhizosphere
2 R. G. Taketani, V. N. Kavamura, R. Mendes and I. S. Melo
environment created by the plant is a nutrient hotspot that
allows for a high abundance of microorganisms (Bais
et al., 2006).
Recent advances in high throughput sequencing tech-
nology have provided the ability to sequence metagenomic
samples at much lower costs. This, together with the
availability of computational tools to analyse these data,
has led many researchers to invest in direct metagenomic
sequencing as a tool to investigate the effects of several
factors over microbial communities (Venter et al., 2004;
Jones, 2009; Gilbert et al., 2010; Fierer et al., 2012). This
approach allows the description of the components of a
community without any selective procedure (besides DNA
extraction) as opposed to amplicon sequencing which is,
more often than not, specific to certain phylogenetic or
functional groups, displaying some limitations (Petrosino
et al., 2009). Besides that, the direct sequencing of a
metagenome provides information about the genes and
metabolic capabilities of a community (Hugenholtz and
Tyson, 2008); this allows researchers to estimate how a
factor affects the community from two different and com-
plementary perspectives. Here, we used metagenomic
techniques to analyse M. tenuiflora rhizosphere samples
collected during the dry and rainy seasons in three different
sites of Caatinga biome to gain further insight into meta-
bolic and taxonomic shifts that occur in this plant-
associated microbial communities across seasons.
Materials and methods
Ethics statement
This project was conducted with the authorization of
the Institute of Environment and Renewable Natural
Resources process number 02001.004527/2011-90 and it
did not involve endangered or protected species.
Sampling, DNA extraction quality and yield
Samples from the M. tenuiflora rhizosphere were col-
lected in three different sites of typical Caatinga vegeta-
tion. Coordinates were: site 1 – 09° 13′ 24.8″ S, 41° 05′
11.4″ W; site 2 – 08° 50′ 01.6″ S, 42° 33′ 13.3″ W; site 3
– 06° 42′ 44.2″ S, 38° 15′ 08.2″ W. Samples were col-
lected during the spring (October 2010) and winter (May
2011) which corresponds to the peak of the dry and wet
seasons respectively. Samples were taken from three dif-
ferent plants of similar sizes (height and trunk width) at
each site and were mixed in equal amounts. Samples
were kept in plastic bags and stored at room temperature
until processing. Physical and chemical analyses were
performed in each sample and were published previously
(Lançoni et al., 2013). According to this study, sites 1 and
2 did not present many significant differences in the ana-
© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
lysed characteristics, while site 3 did, which makes site 1
and 2 much more similar between them than with site 3.
These data were used as environmental data on the mul-
tivariate statistics analysis.
Total community DNA from soil and rhizosphere of
M. tenuiflora samples were extracted using Power Soil
DNA Isolation Kit (MoBio Laboratories, EUA) according to
the manufacturer’s protocol. Total community DNA was
checked in a NanoDrop spectrophotometer for quantity
and quality (A260/280 was ~ 1.8) and in a Qubit® 2.0
fluorometer using Qubit® dsDNA BR Assay Kit to confirm
DNA quantity. Six to eight independent DNA extractions
were pooled to obtain 1 µg of metagenomic DNA, which
was used to construct a library. In total, six libraries were
obtained for sequencing, three from the dry season and
three from the rainy season.
Metagenomic DNA sequencing using Ion Torrent
Personal Genome Machine (PGM) platform
The total community DNA obtained as described above
was enzymatically fragmented and ligated into the appro-
priate adapters using the Ion Xpress Plus Fragment
Library Kit following the manufacturer’s protocol. The
obtained fragments were then bound into the ion spheres
and amplified by emulsion polymerase chain reaction
using the Ion Xpress Template Kit. The enriched beads
were then sequenced in an Ion Torrent (PGM) using 314
chips and Ion PGM sequencing kit. Raw data were ana-
lysed on a torrent server for base calling. Sequence files
(sff) were exported and analysed as described later.
Sequence analysis and statistical treatment
The sff were uploaded to the MG-RAST server (Meyer
et al., 2008) for automatic annotation using the default
settings and are stored under the accession num-
bers: 4487345.3, 4487344.3, 4487567.3, 4487653.3,
4487639.3 and 448787638.3. All phylogenetic analysis
presented here are the result of the Best Hit Classification
against the M5NR database using an E-value cut-off of
10−5, minimum identity of 60% and a minimum alignment
of 50 bp (Delmont et al., 2011). The functional annotation
was performed by Hierarchical Classification against the
Subsystems database using an E-value cut-off of 10−5,
minimum identity of 60% and a minimum alignment of 15
amino acids (for a summary, see Table S1). Both annota-
tion tables were then exported to Statistical Analysis of
Metagenomic Profiles (STAMP) (Parks and Beiko, 2010)
to identify the differentially abundant features using Fish-
er’s exact test; confidence intervals were calculated by
Newcombe–Wilson’s method.
The phylogenetic and functional annotation tables were
used in canonical correspondence analysis (CCA) fol-

lowed by forward selection (FS) within the CANOCO v.4.5
software package. Also, analysis of similarity (ANOSIM)
and Mantel tests were performed within Past software
(Hammer et al., 2001); for this tests, all annotation tables
were converted into relative abundance tables and envi-
ronmental tables which were normalized by the Box–Cox
method. ANOSIM was performed using a Bray–Curtis
similarity matrix. For the Mantel test, Bray–Curtis similarity
matrices were constructed from annotation tables and
Euclidian similarity matrixes were constructed from the
environmental data.
Results and discussion
Rhizosphere soils collected from the three different sites
during the dry and rainy seasons along the Brazilian semi-
arid environment were sequenced by Ion Torrent (PGM)
using a shotgun metagenomic approach. Sequences
classified as bacterial represented about 83–98% of the
annotated sequences. This ratio was similar to those pre-
viously observed for other soil metagenomic studies
(Delmont et al., 2012; Fierer et al., 2012). The most abun-
dant phyla in both small subunit (SSU) rRNA libraries and
gene annotation were Actinobacteria and Proteobacteria;
however, there were some discrepancies between the
abundance observed for the gene and SSU classifi-
cation. The 16S rRNA-based classification revealed
Actinobacteria as the dominant phylum in all libraries
(data not shown), whereas the gene classification placed
the Proteobacteria as dominant (Supporting Information
Fig. S1A). These differences must be due to the more
comprehensive nature of Silva SSU database and/or dif-
ferent number of rRNA copies per functional gene ratio
between these phyla. These groups have been previously
identified as major components of the microbial commu-
nity in arid and semiarid environments (Barnard et al.,
2013; Kavamura et al., 2013b) which are abundant in the
majority of the soils studied up to now (Janssen, 2006;
Fierer and Ladau, 2012; Fierer et al., 2012). Sequences
from archaeal origin were rare in all samples from 0.05%
to 1.75% according to functional classification and from
undetected to 1.89% on SSU libraries, which is most likely
due to the lower coverage of the 16S rRNA of sequences.
The high variability of the communities observed in differ-
ent sites of semiarid soils have been observed in previous
studies (Fierer et al., 2012; Barnard et al., 2013;
Kavamura et al., 2013b) and was highlighted by Barnard
and colleagues (2013).
In terms of functional categories, all libraries have
shown similar patterns among them (Supporting Informa-
tion Fig. S1B). The most abundant category observed
was carbohydrate metabolism followed by clustering
based subsystems and protein metabolism. All of the 11
most abundant categories were related to basic cellular
© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Functional congruence in semiarid 3
functions. Only the minor groups represented ecological
functions such as nutrient cycles, including those related
to the nitrogen cycle that we were expecting to find in
higher abundance due to the ecological role of these
plants. Such distribution was expected and has been
observed in several other metagenomes from natural
environments (Delmont et al., 2012; Fierer et al., 2012). In
dry areas such as deserts, function related to nutrient
cycling metabolism (nitrogen, potassium and sulphur)
tend to be lower than in non-desert environments and the
opposite is observed for genes related to the metabolism
of carbohydrates, amino acids, proteins and dormancy/
sporulation genes (Fierer et al., 2012). The presence of
higher numbers of carbon and protein metabolism genes
might be linked to the ability of the soil microbiota of the
semiarid biome to rapidly respond to the rewetting
(Borken and Matzner, 2009; Inglima et al., 2009). These
organisms increase their biological activity in a matter of
hours and increase in numbers by several fold during the
rainy period (Kavamura et al., 2013a). Also, since this
environment is subjected to high heat, osmotic and oxi-
dative stresses, the large abundance of stress response
genes is not surprising and this has also been observed
by Fierer and colleagues (2012) when comparing desert
soils to non-desert soil samples.
The alpha diversity indexes varied greatly across
samples. In terms of diversity, dry samples presented a
higher Shannon diversity index (H′) (based on class com-
position) and evenness [Buza and Gibson’s evenness
(Buzas and Gibson, 1969)] (Fig. 1A). However, although
the average of these indexes were different, this was not
significant due to the large variation observed. The rank
abundance curve (RAC) (Fig. 1B) of the taxonomic ranks
shows that there is no particular pattern of dominance or
evenness when comparing dry versus rainy samples.
Nevertheless, these results indicate that the diversity is
not specially affected by lack of rainfall. Although previous
studies have shown that the dry season leads to a
decrease in microbial counts, diversity and richness
(Kavamura et al., 2013b), in our study this pattern was not
observed; however, it is important to note that the three
selected sites are not homogeneous in terms of soil nor
vegetation. The presence of a rich and diverse plant com-
munity is considered an important factor in the resilience
to desiccation (Bloor and Bardgett, 2012; Vogel et al.,
2012); this might also be true to the rhizosphere commu-
nity. The presence of a large number of species might
benefit the functional redundancy, which would aid the
activity resumption upon the return of rain.
This functional redundancy can be accessed by the
means of the functional diversity. The observed H′, based
on the functional diversity, was higher during rain as
opposed to what was observed for the class composition.
However, the evenness was not different between the
4 R. G. Taketani, V. N. Kavamura, R. Mendes and I. S. Melo

Fig. 1. Alpha diversity analysis of the Caatinga metagenomes.

A. Diversity indexes, Buza and Gibson’s Evenness and Shannon’s H′ based on the annotation by MG-RAST, i.e. function annotation by
hierarchical classification and taxonomic assignment by best hit classification.
B. Rank abundance curve of the taxonomic classification of the Caatinga metagenomes.
C. Rank abundance curve of the functional classification of the Caatinga metagenomes.

seasons, indicating that rain might have a positive effect
over the functional diversity. The RAC (Fig. 1C) also
shows that there is a very different profile between
seasons. Also, the present result indicates that there is a
contradiction between the effect of rain/dry over commu-
nity structure and function; these community characteris-
tics may respond differently when challenged by the
changes in the environment. This also led us to believe
that there might be an important role of functional re-
dundancy among different taxa (even different phyla)
that allows the discrepancy between the functional/
phylogenetic results.
Due to the large variation observed between samples
from the same season and low correlation between them,
the comparisons based on the phylogenetic assignment
of the sequences did not produce any taxon that could be
marked as significantly different. However, it does not
mean that there is no taxon that is selected by either
season; it just means that the variation was large enough
to prevent the identification of such taxa. It is also impor-
tant to note that the samples were collected with a long
distance between them and this site variation in samples
collected across large distances and that present physical
and chemical differences are expected and has been
seen previously in studies that used 16S gene libraries
(Barnard et al., 2013; Kavamura et al., 2013b). Therefore,
this observation suggests that if we had a larger number
of libraries (like in the mentioned studies), we could be
able to detect such features as seen previously (Barnard
et al., 2013; Kavamura et al., 2013b).
On the other hand, since the variation between samples
in the functional assignment of the reads was less pro-
nounced, the Welch’s t-test (implemented in STAMP)
could identify different functional groups that were
selected by each season (Fig. 2). The central carbohy-
drate metabolism cluster was the most significantly abun-
dant function to be negatively affected by desiccation.
Since during this period, the organic matter and the micro-
bial activity are decreased (Inglima et al., 2009) and there
is a paucity of plant biomass that reduces the inputs of
organic carbon (Fierer et al., 2012) due to the loss of
leaves by the majority of plants; the low abundance

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

Fig. 2. Differentially abundant taxa as identified by STAMP of the comparison of dry (black bars) and rainy season (white bars) metagenomes
error bars indicate the 95% confidence intervals. ammon, ammonia assimilation.
of microorganisms carrying a high genomic content
related to the carbohydrate metabolism is expected. Also,
three other groups differentially abundant between
seasons were associated with amino acids and deriva-
tives, i.e. arginine; urea and polyamines; glutamine,
glutamate, aspartate, asparagine and ammonia assimila-
tion; and proline and 4-hydroxyproline. The first cluster is
related to the production of urea that would lead to the
loss of nitrogen, and the remaining two clusters are within
the central amino acid metabolism and the assimilation of
ammonia. Interestingly, the first cluster is increased in the
dry season while the other two are increased during rain,
which is concordant to the pattern of soil’s nitrogen
content in this forest (Trovão et al., 2007), i.e. nitrogen is
lost during the dry season and accumulated in rain.
Furthermore, the higher abundance of cell division,
polyhydroxybutyrate metabolism, purines and folate and
pterines reads during rain also indicates that population
with higher metabolic activity are selected during this
season.
The application of CCA (a direct gradient analysis,
i.e. uses only operational taxonomic unit (OTU) data that
correlates with environmental data) also showed that dry
samples had lower similarity among them compared with
samples obtained from the rainy season (Supporting
Information Fig. S2A and B). Although CCA allows the
correlation between the OTU tables and environmental
variables, automatic FS with Monte Carlo permutation
(999 permutations) indicates that none of them had a
significant effect over the community structure. Despite
the season, phylogenetic differences had been observed
among samples from the three sites. Although there has
not been a significant effect of environmental variables
over the community structure through CCA, soil variabil-
ity might have influenced this variation, since some of
these characteristics (i.e. temperature, moisture content,
nutrient availability) have been pointed out as the main
drivers of soil bacterial communities variability (Fierer
et al., 2012; Pasternak et al., 2013) as observed for the
© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Functional congruence in semiarid 5
diversity in the Caatinga based on gene markers (SSU
and amoA gene) (Kavamura et al., 2013b; Lançoni et al.,
2013).
Functional redundancy has been often seen as a
crucial factor on the resilience of an ecosystem to any
disturbance. In the case of microbial communities, due to
its overwhelming magnitude and richness, taxon diversity
has always been used as a proxy to functional diversity.
This study represents one of the first analyses of Brazilian
semiarid metagenomes conducted to date with over
250 Mb of metagenomic data obtained from six different
libraries. Nevertheless, we could detect some patterns in
the datasets that highlight the same trend in the shifts of
communities when compared with studies of the same
biome (Kavamura et al., 2013b; Lançoni et al., 2013).
Furthermore, microbial communities found in semiarid
soils are known to display two different life moments, one
of high activity (Barnard et al., 2013) and increased diver-
sity characterized by the presence of Proteobacteria due
to the presence of rain, and another one of lower activity
and abundance of Actinobacteria, in a strategy of prepar-
edness. However, despite the differences observed on
the studied taxa, due to the variability of the sites, in
general, the functional traits detected were generally
stable between sites and seasons suggesting that the
native microbial communities of semiarid rhizosphere
soils of the Caatinga might have selected soil organisms
to display the same functional roles to cope with stress
despite their phylogenetic assignment. Therefore, we
believe that this might be responsible for the resilience
expected from these communities.
Acknowledgements
The authors acknowledge the support of Cosme Corrêa dos
Santos (UEFS) on identifying the plant species Mimosa
tenuiflora. RGT was a recipient of a postdoctoral grant from
FAPESP (2010/50799-7). This study was supported by
Embrapa. The authors also thank João Luiz da Silva and
Suikinai Nobre Santos for the support during sampling.
6 R. G. Taketani, V. N. Kavamura, R. Mendes and I. S. Melo
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Fig. S1. Metagenomic community profiles obtained by the
phylogenetic and functional classification of the caatinga
metagenomes.
A. Phylogenetic classification represents the affiliation up to
the level of phylum by the best hit classification tool.
B. Functional classification represents the MG-RAST classi-
fication as assigned by the hierarchical classification tool.
© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Functional congruence in semiarid 7
Fig. S2. Multivariate statistics analysis of the classification
of metagenomic reads from the rhizosphere of Mimosa
tenuiflora.
A. Canonical correspondence analysis of the class-level
phylogenetic classification of metagenomic reads.
B. Canonical correspondence analysis of the functional
classification.
Table S1. Overview of the Mimosa tenuiflora rhizosphere
metagenomes.