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Carbonate Accumulation in the Bark of Terminalia bellirica: A New Habitat for

the Oxalate-Carbonate Pathway

Article  in  Geomicrobiology · January 2018

DOI: 10.1080/01490451.2017.1309087

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GEOMICROBIOLOGY JOURNAL
https://doi.org/10.1080/01490451.2017.1309087

Carbonate Accumulation in the Bark of Terminalia bellirica: A New Habitat for the
Oxalate-Carbonate Pathway
Vincent Herv
e a,b
, Martin Clerca,b, Guillaume Cailleaub, Matthieu Buechea, Thomas Juniera,c, Eric Verrecchiab,
and Pilar Juniera
a
Laboratory of Microbiology, Institute of Biology, University of Neuch^atel, Neuch^atel, Switzerland; bLaboratory of Biogeosciences, Institute of Earth
Surface Dynamics, University of Lausanne, Lausanne, Switzerland; cVital-IT, Swiss Institute of Bioinformatics, Lausanne, Switzerland

ABSTRACT ARTICLE HISTORY

Oxalate oxidation and carbonate precipitation associated with the oxalogenic tree Terminalia bellirica were Received 10 October 2016
investigated. Calcium oxalate crystals, oxalotrophic bacteria (dominated by genera Methylobacterium and Accepted 16 March 2017
Burkholderia), and carbonate accumulation (82% dry weight), were detected in the bark. In contrast, only a KEYWORDS
slight accumulation of carbonate was observed in soil (1.5%). The combined geochemical and Burkholderia; carbon cycle;
microbiological analyses suggest that bark of an Indian living tree represents a novel habitat for the dermosphere;
accumulation of carbonate associated with bacterial oxalate oxidation. The importance of these types of Methylobacterium; Terminalia
habitats needs to be quantified when considering the biogeochemical cycling of carbon and calcium in bellirica
tropical ecosystems.

Introduction
The oxalate–carbonate pathway (OCP) is a biogeochemical
pathway that has been hypothesized to link a flux of carbon
with pedogenic accumulations of calcium carbonate (CaCO3)
in unexpected geological settings such as acidic red tropical
soils (Braissant et al. 2004; Verrecchia et al. 2006). Indeed, the
conventional pH of such soils is around 5 (or below), preclud-
ing the abiotic precipitation of calcium carbonate. Moreover,
these red acidic soils are extremely depleted in calcium (they
are characterized by a very low cation exchange capacity), mak-
ing the precipitation of secondary calcium carbonate unlikely
(Legros 2012). In this pathway, carbon dioxide enters the sys-
tem through photosynthetic fixation by oxalogenic plants. Part
of this fixed carbon is diverted in the plant for the production
of oxalic acid, a molecule with diverse functions. One of the
most widely recognized functions of oxalic acid is the regula-
tion of intracellular levels of Ca2C, which is achieved through
the formation of calcium oxalate (Caox) salts (Franceschi and
Nakata 2005). These insoluble crystals remain trapped within
the plant tissues until they become part of the organic matter
pool by entering soils as litter (Garvie 2006). In soils, different
microbial metabolic activities determine the fate of Caox
(Martin et al. 2012). One of those is the use of Caox as carbon
and energy sources by oxalotrophic bacteria (Herv
e et al. 2016;
Sahin 2003). In addition, soil fungi have been shown to be key
in the OCP, participating in the release of Caox from plant tis-
sues by decomposition (Cailleau et al. 2011), the production of
fungal Caox (Guggiari et al. 2011), and more recently because
of their role in the transport and dispersal of oxalotrophic bac-
teria (Martin et al. 2012; Bravo et al. 2013).
The combination of a source of Caox (i.e., oxalogenic organ-
ism), the oxidation of Caox by oxalotrophic microorganisms,
and the precipitation of CaCO3 has been observed as part of an
active OCP (Cailleau et al. 2011). Although those elements
have been found in many areas around the world, the OCP has
been shown to be particularly relevant to carbon budgets in
tropical soils (Cailleau et al. 2011 2014). So far, the precipita-
tion of secondary carbonate has only been considered for the
pedogenic habitat of the ecosystem. However, numerous plants
are able to accumulate large quantities of Caox crystals in dif-
ferent tissues (Bauer et al. 2011; Franceschi and Horner 1980;
Libert and Franceschi 1987; Webb 1999). Therefore, the aim of
this study was first to describe an OCP site with carbonate
accumulation in the soil and, second, to test the existence of
other deposits of carbonate within the tree tissues. For this, the
existence of a Caox source, the presence of oxalotrophic bacte-
ria, and the precipitation of CaCO3 were investigated in the
bark of the tropical tree T. bellirica. This deciduous tree is dis-
tributed throughout tropical Asia including India (Dangi et al.
2012). Previously, calcium accumulation has been observed in
the bark (habitat also known as dermosphere; Lambais et al.
2014) of Terminalia spp. in Thailand (Stone and Boonkird
1963). Here, using a culture-independent approach, we
explored for the first time the diversity of oxalotrophic bacteria
associated with the bark surface of T. bellirica.

CONTACT Pilar Junier pilar.junier@unine.ch Laboratory of Microbiology, Institute of Biology, University of Neuch^atel, Rue Emile-Argand 11, Neuch^atel CH-2000,
Switzerland.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ugmb.
Supplemental data for this article can be accessed on the publisher’s website.
© 2017 Taylor & Francis Group, LLC
2 V. HERV

E ET AL.
Materials and methods
Field sampling
The study site was located 25 km south from the city of
Khajuraho (Madhya Pradesh State), in the North Panna Tiger
Reserve forest (24
380 1800 N/79
510 900 E), India (Figure 1). This
region belongs to the Indo-Gangetic area. The climate is defined as
a tropical monsoon with three seasons (summer, winter and mon-
soon) and with a mean annual precipitation of 1100 mm. During
the exploratory phase, 35 tree species out of the 65 species listed by
the Madhya Pradesh State were screened for the presence of car-
bonate in the surrounding soils and in the tree tissues.
These soils developed on carbonate-free parent material. Efferves-
cence using 10% HCl reaction was used as field method to evaluate
the presence of carbonate. In addition, pH of the soil under the tree
and away from the tree was also estimated with a Hellige pH indi-
cator (Eijkelkamp). Only the tree specimen presenting the highest
reaction with 10% HCl and the highest alkalinization of the soil
under the tree was selected for further analyses in this case study.
The tree selected for this study was a specimen of T. bellirica
((Gaertn.) Roxb., Combretaceae), locally known by the names
Bahera, Bastard myrobalan, or Belleric myrobalan. This specimen
was the largest in the study site with a height of 20 m and a diame-
ter at breast height (DBH) of 79 cm. The canopy was estimated at
13 m. Based on these measurements, the age of the tree was esti-
mated to be 50 years. Trunk outer bark (rhytidome) was sampled
Figure 1. Location map of the sampled site in the North Panna Tiger Reserve forest, India (24
380 1800 N/79
510 900 E). The map was generated with the QGIS software ver-
sion 2.3.
at 1.50 m height with a sterile spatula and brought back to the lab-
oratory using axenic procedures. At this height, carbonate accu-
mulations were clearly visible in the bark of the entire tree
(whitish aspect of the bark tissue). The outer bark was sampled
where carbonate deposits were the most abundant. Additionally,
branches, roots and leaves were randomly sampled and brought
back to the laboratory using axenic procedures.
Additionally, two soil profiles were dug and sampled, one at
the base of the trunk (called profile A) and another at 14 m
away from the tree (called profile C). The latter was considered
as not under the influence of other oxalogenic plants. The study
site was flat and visually homogeneous. Profile A was investi-
gated to evaluate the effect of the oxalogenic tree and its associ-
ated microbiome on the soil, and profile C was used as a
control. Each profile was 1 m deep. For microbiological analysis
the profiles were sampled every centimeter for the first 5 cm
and then every 5 cm until 1 m. Each sample consisted of
approximately 4 g of soil obtained with a sterile spatula and
immediately mixed with 5 ml of RNAlater solution (Qiagen)
and stored at 4
C until laboratory analyses. Additionally, 200 g
of soils sampled every 5 cm were used for geochemical analyses.
Tree tissue analysis
Presence of calcium carbonate (CaCO3) in the tree tissues was eval-
uated in the field directly by reaction with 10% HCl. To measure

the carbonate concentration, i.e., weight of carbonate per weight of
dried sample, 1 g of crushed bulk sample was dissolved with a mix
of 25 ml of sulfuric acid (0.5 N) and 125 ml of deionized water and
subsequently placed at 90
C in a water bath for 60 min. Then, car-
bonate concentration was measured by back titration with a
sodium hydroxide solution (0.5 N). This method was selected for
the soil and wood samples with less than 20% carbonate concentra-
tion because of the lack of accuracy and robustness of other meth-
ods at this low concentration of CaCO3 (Pansu and Gautheyrou
2007). This chemical approach ensures the total dissolution and
accurate measurement of low calcium carbonate proportions. For
the samples with concentrations higher than 20% (arbitrary limit),
carbonate concentration was measured by estimating the mass loss
of the sample after drying at 105
C and dissolution during 60 min
in 10% HCl. Oxalate concentration was measured on an Agilent
HPLC 1100 Series (Agilent Technologies, USA) instrument
equipped with a BP-100 hC column (Benson Poymeric, USA),
with 20 mM H2SO4 eluant at a flow rate of 1 ml.min¡1 and temper-
ature of 35
C. Ca-oxalate (Sigma-Aldrich, Germany) was used as
standard. Different tree tissues including bark, roots, wood and
leaves were collected for observation using scanning electron
microscopy (SEM) to verify the presence of Caox crystals. The
identification of Caox with SEM was based on the habitus of
the crystals as well as semi-quantitative information obtained with
the energy dispersive spectrometer (EDS) coupled to the SEM.
Caox are characterized by specific crystal morphologies related to
the tetragonal and monoclinic systems, which are clearly different
from the calcite trigonal-rhombohedral system. In addition, EDS
analyses provide a clear and indisputable signature of the main
chemical components of the crystals, i.e., essentially C and Ca. All
the relevant information about the identification of Caox crystals is
given by Verrecchia et al. (1993). SEM observations were per-
formed using a Tescan Mira LMU at a working distance of
10–15 mm and a 20 kV acceleration voltage. Prior to observations,
samples were coated with gold.
Soil analysis
Soil pH (pHH2O) was measured using 10 g of sieved soil mixed
with 25 ml of deionized water. Because carbonate concentra-
tion was much lower in the soil than in the tree tissues, the
back titration with sodium hydroxide (0.5 N) was used to mea-
sure carbonate content. Similarly, oxalate concentration was
measured using the method described above for the tree tissues.
DNA extraction, amplification, and cloning
Bark samples were also collected for molecular analyses. DNA
extraction was performed with a Fast DNA Spin Kit for Soil
(Bio 101 Sytems, Q-Biogene, Switzerland) following the manu-
facturer’s guidelines. Total DNA was extracted from 0.1 g of
bark previously ground in liquid nitrogen. 450 mL of DNA
lysate was purified using 450 mL of binding matrix. DNA was
then extract in a final volume of 80 mL in the DES solution.
The final DNA concentration was measured by spectropho-
tometry (Nanodrop ND-1000, Thermo Scientific, USA) to
dilute the DNA to a final concentration of 1 ng mL¡1 on MilliQ
water. Amplifications by PCR of a partial fragment of the frc
gene (473 bp) were carried out using the primers frc171-F/
GEOMICROBIOLOGY JOURNAL 3
frc627-R (Khammar et al. 2009). This gene codes for the for-
myl-coenzyme A (CoA) transferase (EC 2.8.3.16), a key enzyme
involved in bacterial oxalotrophy. The PCR reaction mix con-
tained (final concentration in 25 mL of final volume) 1X Buffer
with 2 mM MgSO4 (Standard Buffer Biolabs, New England,
Ipswich, MA, USA), 0.2 mM dNTPs mix (Promega AG,
D€ubendorf, Switzerland), 0.2 mM each primer, and 1 U of Stan-
dard DNA Taq polymerase (Biolabs, New England, Ipswich,
MA, USA). A total of 2 mL of diluted DNA was added as a tem-
plate for each reaction. The initial denaturation was carried out
at 94
C for 4 min 30 s, followed by 30 cycles consisting of dena-
turation at the same temperature for 30 s, primer annealing at
60
C for 30 s, and extension at 68
C for 1 min 30 s. A final
extension was performed at 68
C for 10 min. PCR products
were visualized by gel electrophoresis using 1% agarose gels.
PCR products from different bark samples were pooled
together and cloned with a TA PCR Cloning Kit (Invitrogen)
according to manufacturer’s guidelines. A total of 94 clones
were picked randomly and checked for inserts of the expected
size by PCR with the plasmid-specific primers M13f/M13r fol-
lowed by visualization after electrophoresis on agarose gel.
One-shot sequencing with M13f primer was performed using
the services of Microsynth AG (Switzerland).
Soil DNA was extracted similarly, using 0.8 g of soil from each
sample. Quantification of the frc and 16S rRNA genes was per-
formed using the QuantiTect SYBR Green PCR Kit (Qiagen, Ger-
many), as described by Martin et al. (2012). For the frc gene, the
frc171-F/frc306-R primers were used. To amplify the V3 region of
the 16S rRNA gene, the primer pair 338-F/520-R was used. Each
qPCR was performed in triplicate and the average value was used.
To examine relationships between gene abundances and soil char-
acteristics, Spearman’s rank correlation coefficients (rS) were com-
puted in R version 3.2.2 (R Development Core Team 2015).
Sequence analyses
In total, fifty sequences were successfully cloned and sequenced.
Among them, one was suspected to be chimeric and thus was
discarded. Sequences have been deposited in GenBank under
the accession numbers KU247480–KU247528. The 49 remain-
ing DNA sequences were translated into amino acid sequences
using the getorf function EMBOSS version 6.6.0–1 (Rice et al.
2000). Taxonomic assignment of these sequences was per-
formed using MLgsc (Junier et al. 2015) and an Frc database
composed of 661 Frc sequences extracted from UniProtKB on
July 22nd, 2015 (The UniProt Consortium 2015).
To evaluate phylogenetic relationship among the Frc pro-
teins, sequences were first aligned using the G-INS-i algorithm
of MAFFT version 7.221 (Katoh and Standley 2013). Then,
phylogenetic trees were built using maximum likelihood (ML)
estimates and Bayesian inferences (BI) with RAxML version
7.7.2 (Stamatakis 2006) and PhyloBayes version 4.1 (Lartillot
et al. 2009), respectively. The Frc sequence from the bacterial
model Lactobacillus acidophilus SC30 was used as an outgroup.
The ML tree was built using the Jones-Taylor-Thornton (JTT)
model of amino acid evolution with gamma-distributed rate
variation across sites. To obtain the statistical confidence of
internal branches, 10,000 bootstrap replicates were generated.
Regarding the BI tree, the CAT-GTR model was selected
4 V. HERV

E ET AL.

a Copy of frc gene / g of soil Copy of 16S rRNA gene / g of soil pH oxalate (mg/kg) CaCO3 (%)
0e+00 2e+10 4e+10 6e+10 8e+10 0.0e+00 1.0e+11 2.0e+11 3.0e+11 6.0 6.5 7.0 7.5 8.0 8.5 9.0 0 2 4 6 8 10 12 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0
−20

−20

−20

−20

−20
Depth (cm)

Depth (cm)

Depth (cm)

Depth (cm)

Depth (cm)
−40

−40

−40

−40

−40
−60

−60

−60

−60

−60
−80

−80

−80

−80

−80
−100

−100

−100

−100

−100
b Copy of frc gene / g of soil Copy of 16S rRNA gene / g of soil pH oxalate (mg/kg) CaCO3 (%)
0e+00 2e+10 4e+10 6e+10 8e+10 0.0e+00 1.0e+11 2.0e+11 3.0e+11 6.0 6.5 7.0 7.5 8.0 8.5 9.0 0 2 4 6 8 10 12 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0

0
−20

−20

−20

−20

−20
Depth (cm)

Depth (cm)

Depth (cm)

Depth (cm)

Depth (cm)
−40

−40

−40

−40

−40
−60

−60

−60

−60

−60
−80

−80

−80

−80

−80
−100

−100

−100

−100

−100
Figure 2. Variations of five biogeochemical parameters along the soil profiles (a, upper panel) under the influence of the oxalogenic tree Terminalia bellirica (profile A)
and (b, lower panel), 14 meters away from Terminalia bellirica (profile C).

(Lartillot and Philippe 2004) and three independent chains Indeed, HCl 10% reaction occurred with leaves found on the
starting from random trees were run using a Markov chain forest floor in a 4-m perimeter around the tree.
Monte Carlo (MCMC) sampler. Chain convergence was veri- To further explore the carbonate accumulation in the T. bellir-
fied using the bpcomp command (maxdiff<0.05) and the initial ica system, a 1-m deep profile (profile A) was dug under the tree
10,000 trees sampled in each MCMC run were discarded as the and the copy numbers of the frc (a molecular marker of oxalate-
burn-in period. Subsequently, one 50% majority-rule consensus oxidizing bacteria; Khammar et al. 2009) and 16S rRNA genes,
tree and the associated posterior probabilities were computed pH, oxalate and carbonate concentrations were measured
from 50,000 trees. Trees were visualized with TreeGraph 2 ver- (Figure 2a). As a negative control for the influence of an oxalo-
sion 2.7.1 (St€
over and M€ uller 2010). genic plant, a second profile (profile C) was dug 14 m away from
To analyze Frc diversity, amino acid sequences were clustered the tree and analyzed similarly (Figure 2b). Quantification of the
into operational taxonomic units (OTUs) at 95% sequence simi- frc gene revealed that oxalotrophic bacteria were present in both
larity (Herv
e et al. 2016) using CD-HIT version 4.6 (Fu et al. soil profiles and were particularly abundant in the upper sample
2012). Subsequently, rarefaction curve and diversity indices were corresponding to the litter. The abundance of the frc gene was
computed in R version 3.2.2 (R Development Core Team 2015) strongly correlated with the concentration of oxalate in both pro-
using the vegan package (Oksanen et al. 2012). files (rS D 0.67, p < 0.02; rS D 0.90, p < 0.005 for the profiles A
and C, respectively), indicating that oxalotrophic bacteria can be
abundant in oxalate-rich niches from forest soils. For both pro-
Results and discussion files, the highest concentration of oxalate was found in the litter
samples. This result is in accordance with a recent study showing
Identification of an active OCP in the soil
that leaf litterfall provided an important amount of Caox to forest
During the exploratory phase, 35 tree species were screened for soils (Dauer and Perakis 2014). In the litter samples, oxalate con-
the presence of carbonate in the tree tissues as well as for evi- centrations and frc copy numbers were higher in the profile C
dence of a local alkalinization in the soil below the tree. Among than in the profile A. Subsequently, we quantified more copies of
those, six species were found to be positive: Buchanania lanzan the frc gene and higher oxalate concentrations in the soil samples
(Anacardiaceae), Diospyros melanoxylon (Ebenaceae), Garde- under T. bellirica compared to profile C, especially until 60 cm
nia pinnata (Rubiaceae), Aegle marmelos (Rutaceae) and two depth. Copy number of frc gene was low and monotonic along
species of Terminalia: T. bellirica and T. tomentosa (Combreta- the profile C except for a unique sample between 65 and 70 cm
ceae). Interestingly, members of the genus Terminalia were depth (Figure 2b). On the contrary, frc abundance varied along
also found to be present in OCP ecosystem in Bolivia, with the the profile A, indicating a vertical pattern of oxalotrophic bacteria
species T. amazonia and T. oblonga (Cailleau et al. 2014). The under the influence of T. bellirica which is at least partially
species T. bellirica presented the highest reaction with HCl explained by the variations in oxalate concentration (Figure 2a).
10%, especially in the bark tissue. Thus, one specimen from Most of this oxalate is expected to originate from T. bellirica root
this species was selected for an in-depth analysis in the North exudates (Strobel 2001). The copy number of 16S rRNA genes fol-
Panna Tiger Reserve forest. During this exploratory phase, it lowed a very similar trend to the frc abundance and the quantifi-
was also observed that carbonate was not equally accumulated cation of both genes was highly correlated in both profiles
in all tree tissues. For instance, the outer bark of G. pinnata (rS D 0.94, p < 0.001; rS D 0.79, p < 0.001 for the profiles A and
showed a lower reactivity to HCl 10% compared to the leaves. C, respectively). Both pH and carbonate content showed different

trends between the two profiles. Indeed, soil pH was found to be
acidic (up to pH 6.1) in the first 50 cm of the profile C
(Figure 2b) but was alkaline (up to pH 8.4) in the first 60 cm of
the profile A. This local alkalinization in the upper soil layers
coincides with an accumulation of carbonate (3.4% of the dry
weight in the litter and 1.5% in the upper 5 cm). On the contrary,
carbonate content was lower in the profile C and showed a mono-
tonic trend.
In summary, these results revealed the presence of oxalate,
oxalotrophic bacteria and carbonate as well as a local alkaliniza-
tion in soil under T. bellirica compared to the surrounding for-
est soil. Altogether, these data indicate the existence of an
active OCP in the North Panna Tiger Reserve forest; this OCP
being, to date, the first described in India. Although carbonate
accumulation in soil was low, compared to other OCP sites
such as in Ivory Coast (Cailleau et al. 2004), and restricted to
the first 10 cm, since a strong reaction with HCl 10% was
observed on the tree tissues, the same tree specimen T. bellirica
was further investigated to address if the OCP could occur in
other habitats associated to the tree.
Biomineralization of the plant tissues
The different tissues of the T. bellirica specimen contained a vari-
able concentration of calcium carbonate. The bark displayed a
very strong reaction to HCl, and carbonate concentration corre-
sponded to 82.0% weight. In comparison, the oxalogenic tree Mil-
icia excelsa, which is the model tree in other OCP sites from
Africa concentrated calcite mainly in sap exudates (Cailleau et al.
2011). In addition, monohydrocalcite and biogenic Mg-calcite
were identified in the bark of a Pentaplaris davidsmithii in a Boli-
vian OCP site (Cailleau et al. 2014). Leaves and wooden parts
(branches) of T. bellirica contained carbonate to a lesser extent
(46.9% and 33.9%, respectively). Similarly, the presence of bio-
genic Mg-calcite was detected in leaves and wood tissues from P.
davidsmithii in Bolivia (Cailleau et al. 2014) and in the wood tis-
sues of M. excelsa in Africa (Cailleau et al. 2005, 2011). In the
present study, no Mg forms of carbonate, such as high-magnesian
calcite, were detected in T. bellirica. Regarding the roots of T. bel-
lirica, no reaction to HCl has been observed and the measured
concentration of carbonate was null. However, in Ivory Coast,
partial calcite pseudomorphosis was observed on the root and the
basal trunk (stump) of M. excelsa, and calcite blocks identified as
former root fragments were also found (Cailleau et al. 2011).
These results suggest differences in the carbonate accumulation
within oxalogenic trees of different OCP sites studied so far. Vari-
ous factors and combinations of them could explain these differ-
ences: tree taxonomy, tree age, climatic variables, edaphic
variables and/or the microbiome associated to the tree. In India,
the carbonate concentration in the upper 5 cm of soil was rela-
tively low (1.5% of the soil dry weight, Figure 2) compared to the
results of Cailleau et al. (2004) who found that carbonate concen-
tration reached 63% below the hollow trunk of a M. excelsa and
12–14% below its foliar crown in another OCP site from Ivory
Coast. Such differences might be explained by the different rain-
fall distributions between Indian and Ivory Coast sites, since the
dissolution of pedogenic carbonate is related to the intensity of
rainfall during wet periods (Violette et al. 2010). Indeed, the site
in Ivory Coast was characterized by a dry season from December
GEOMICROBIOLOGY JOURNAL 5
to January followed by important and regular precipitations dur-
ing the rest of the year (1500 mm.year¡1), whereas the Indian site
was subject to temporary and intense precipitations during the
monsoon from June to September while the rest of the year
was relatively dry (1100 mm.year¡1). Thus, monsoon likely indu-
ces the leaching of carbonate from the soil. Altogether, these
results showed that the carbonate accumulation in this Indian
OCP system does not occur mainly in the soil habitat, as previ-
ously described in African OCP systems, but in the tree habitat
especially in the bark (rhytidome).
SEM observations of bark and leaf samples revealed the
presence of numerous Caox crystals (Figure 3a–d). More
generally, Caox crystals are commonly found in tissues of
various plant species (T€ ut€
unc€
u Konyar et al. 2014; Volk
et al. 2002; Webb 1999). Caox crystals were particularly
dense in the bark tissues of T. bellirica. Specifically, calcium
oxalate druses were frequently observed in the bark
(Figure 3c). Barks of diverse tree species have been shown to
contain Caox crystals of various shapes and sizes, including
druses, e.g., in barks of Quercus robur, Populus tremula and
Betula pendula (Trockenbrodt 1995). Although a quantifica-
tion of the Caox content in the tree tissues (root, bark, leaf,
branch) was attempted, no positive signal was detected by
HPLC. Nonetheless, here we showed that the bark tissues of
T. bellirica contained both calcium oxalate and calcium car-
bonate, which are two essential compounds of the OCP. The
coexistence of Caox monohydrate (whewellite) and some
carbonate species, such as low-magnesium calcite and high-
magnesium calcite, was also observed in the bark of other
trees in Bolivian OCP sites, such as Ficus coerulescens
(Cailleau et al. 2014). In the upper 5 cm of soil, oxalate con-
centration was relatively low (1.89 mg.kg¡1 soil) compared
to the results of Cailleau et al. (2014) who found much
higher concentrations of oxalate near trunks of Ceiba spe-
ciosa (27.84 mg.kg¡1 soil) and P. davidsmithii (16.33 mg.
kg¡1 soil) in other OCP sites from Bolivia. These differences
support the assumption that in the Indian site, the OCP pri-
marily occurred in the outer bark.
According to the forest range officer of the Madhya Pradesh
State (personal communication), the bark texture of T. bellirica
gets smoother with time, which will affect the physico-chemical
properties of the bark. Indeed, bark water storage capacity is
expected to be lower for smoother bark (Levia and Herwitz
2005). Additionally, stemflow will be affected by modifications
of the bark roughness, with a stemflow being greater for
smooth barks compared to rough ones (N
avar et al. 1999).
These modifications will likely impact biogeochemical cycles of
the forest ecosystem (Levia and Frost 2003), especially regard-
ing the calcium and carbon soil inputs. Lastly, the accumulation
of calcium carbonate in the aerial parts (bark, leaves, branches)
of a living and standing tree might increase the retention time
of calcium aboveground, and thus potentially reduce the cal-
cium concentration in the soil surrounding such tree.
Diversity of oxalotrophic bacteria
The presence of both calcium carbonate and Caox crystals in
the bark of T. bellirica raised the question of the presence of
6 V. HERV

E ET AL.
Figure 3. Scanning electron microscope micrographs of (a–c) the bark tissues and of (d) a leaf of Terminalia bellirica. Druses were present in the bark (arrows).
oxalotrophic bacteria in this habitat. Their presence was con-
firmed by the amplification of the frc gene. Subsequently, using
a cloning/sequencing approach, the diversity of these oxalotro-
phic bacteria was investigated. From 49 sequences, amino-acid-
based OTU clustering at 95% sequence similarity resulted in 19
Frc OTUs. Interestingly, the rarefaction curve was unsaturated
(Figure 4), indicating that not all the bacterial oxalotrophy
diversity could be explored. The Chao1 richness estimator
(Chao 1987) indicated 31 potential Frc OTUs, revealing an
unexpected richness of oxalotrophic bacteria in the dermo-
sphere. It also suggests that the bark of oxalogenic trees is a
potential hotspot of bacterial oxalotrophic diversity. More gen-
erally, bark tissue has been shown to harbor an important bac-
terial diversity (Martins et al. 2013), however the microbial
20
15
Number of observed OTUs
10
5
0
0 10
Figure 4. Rarefaction curve of the number of observed Frc OTUs, using a 95% similarity threshold among the amino acid sequences.
diversity of this plant habitat and its ecology remains largely
unexplored (Kowallik et al. 2015).
Regarding the taxonomic diversity, 47 sequences belonged
to the Proteobacteria phylum and 2 sequences were assigned to
Actinobacteria (Table S1). These two phyla are known to con-
tain oxalotrophic bacteria (Herv
e et al. 2016). Among Proteo-
bacteria, 42 sequences belonged to the order Rhizobiales (class
Alphaproteobacteria) and 3 sequences belonged to order Bur-
kholderiales (class Betaproteobacteria). The two other Proteo-
bacteria sequences could not be assigned to a lower taxonomic
level. Regarding the three Burkholderiales sequences, they were
all assigned to Burkholderia, a genus known to harbor oxalotro-
phic bacteria associated to plants (Kost et al. 2014). Further-
more, 16S rRNA gene sequences assigned to Burkholderia were
20 30 40 50
Number of sequences

detected in the dermosphere of the tropical trees Eugenia mela-
nogyna, Ocotea dispersa and Mollinedia uleana from the pris-
tine Atlantic forest in Brazil (Lambais et al. 2014), suggesting
that this bacterial genus is frequently found in tropical dermo-
spheres. Most of Frc sequences reported here were assigned to
the Rhizobiales order. Interestingly, three unclassified members
of the Rhizobiales were found among the seventeen most abun-
dant bacterial OTUs of the trunk bark of Ginkgo biloba trees
(Leff et al. 2015). Although this study was based on 16S rRNA
gene sequences, it suggests that oxalotrophic bacteria might be
an abundant functional group on tree dermosphere. Among
our Rhizobiales sequences, three main groups can be distin-
guished: a group related to genus Methylobacterium, a group
related to family Bradyrhizobiaceae (including one sequence
assigned to genus Bradyrhizobium) and a group of unclassified
Rhizobiales sequences (Figure 5, Table S1). Remarkably, an
oxalotrophic strain belonging to genus Bradyrhizobium was
also isolated from the upper soil layer of T. bellirica, in the
same sampling site (Bravo et al. 2015). This might indicate that
the bark tissue can be a reservoir of oxalotrophic bacteria that
would later transfer to the surrounding soil during bark decay.
Sequences assigned to the Methylobacterium genus were the
most abundant (n D 20) (Table S1, Figure 5) and various Meth-
ylobacterium species are known to be oxalotrophic (Sahin et al.
2008). Additionally, Methylobacterium spp. are commonly
found in the plant phyllosphere (Knief et al. 2010), and were
isolated as plant epiphytes and endophytes (Marx et al. 2012).
Figure 5. Phylogenetic tree of the unique Frc sequences based on Bayesian inferences
(BI). Posterior probabilities of individual nodes are indicated on branches. The Frc
sequence from Lactobacillus acidophilus SC30 was used as an outgroup (LAC30SC).
GEOMICROBIOLOGY JOURNAL 7
This raises the question of the origin of the oxalotrophic
bacterial community of the T. bellirica dermosphere. On the
one hand, these bacteria could have an epiphytic/endophytic
origin, or alternatively they might have been deposited by aeo-
lian deposition (aeolian origin). On the other hand, they can
initially originate from the soil beneath the tree as suggested by
Leff et al. (2015). Recently, in a study of the dermosphere of Q.
robur in United Kingdom, four sources for the bacterial com-
munities have been suggested: the rhizosphere, between-trees
dispersal, the phyllosphere, and aeolian dispersal (Meaden et al.
2016). In this same study, no dispersal limitation for the bacte-
rial communities associated to the dermosphere could be
detected at the hectare scale. If such phenomenon also occurred
in our study site and since other trees from the same forest
showed carbonate accumulation in their tissues, one hypothesis
would be that bacterial communities (including oxalotrophic
bacteria) associated to the bark could colonize other trees via
aeolian dispersal. At the tree scale, since both Caox crystals and
carbonate were found in both the bark and the leaves of T. bel-
lirica, an aeolian dispersal of oxalotrophic bacteria could also
be hypothesized.
The phylogenetic relationship between the unique Frc
sequences is presented in Figure 5. Trees generated by ML and
BI approaches resulted in a similar topology, and thus only the
BI tree is presented here. A cluster of Methylobacterium
sequences was clearly identified (Figure 5), indicating a poten-
tially high diversity of oxalotrophic bacterial strains among
Methylobacterium. However, no cluster was observed for the
Burkholderia (Betaproteobacteria) sequences that were found
among the Alphaproteobacteria, indicating that this Frc phy-
logeny is not completely congruent with the 16S rDNA phylog-
eny. Lastly, this Frc sequence analysis provided the first insight
into the functional diversity of the dermosphere of a tropical
tree. Such molecular analyses could be integrated into larger
studies about the functional ecology of barks (Poorter et al.
2014; Rosell et al. 2014). Additionally, future studies should
integrate RNA-based community analysis to identify the active
taxa involved in oxalate oxidation.
Conclusion
For the first time, results combining geochemical and microbial
analyses revealed the existence of an active OCP in the bark of
T. bellirica, adding a habitat to the OCP primarily detected in
soils from African and Bolivian OCP sites. Additionally, bark
tissues appeared as a niche harboring an unexpected diversity
of oxalotrophic bacteria, which can contribute to carbon and
calcium cycling in tropical ecosystems. Lastly, since observa-
tions of calcium carbonate in the bark of trees are more and
more frequent, our results could also provide a mechanistic
explanation of a more general phenomenon, i.e., the presence
of carbonate in the rhytidome as the product of bacterial cal-
cium oxalate oxidation.
Acknowledgments
The authors wish to thank Audrey Taviaux for the help with the QGIS
software, as well as the anonymous reviewers for their contribution to the
improvement of the manuscript.
8 V. HERV

E ET AL.
Funding
This research was supported by the Swiss National Science Foundation
through Grants FN CR22I2-137994/1 and FN CR3212-149853/1.
ORCID
Vincent Herv
e http://orcid.org/0000-0002-3495-561X
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