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Phylogenetic Diversity of Bacteria in an Earth-Cave in Guizhou Province, Southwest of China

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The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.
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The Journal of Microbiology, April 2007, p. 105-112
Copyright 2007, The Microbiological Society of Korea
Vol. 45 , No . 2
Phylogenetic Diversity of Bacteria in an Earth-Cave in Guizhou Province,
Southwest of China
JunPei Zhou, YingQi Gu, ChangSong Zou, and MingHe Mo*
Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming 650091, P. R. China
(Received December 27, 2006 / Accepted March 20, 2007)
The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil
of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were ampli-
fied directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and
cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and repre-
sentative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190
analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high
diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%),
Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes
(2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria,
6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were
affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of
the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed
similarities in the bacterial community components, especially in the high abundance of Proteobacteria and
Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes,
Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.
Keywords:16S rRNA gene, karst, cave, bacterial community, microbial ecology
The term of “cave” is defined as any natural space below
the Earth’s surface that extends beyond the twilight zone,
and that is accessible to humans (Gillieson, 1996). Most of
common types of caves in karst regions are those formed in
limestone and other calcareous rocks, and as lava tubes in
basaltic rock. Remaining types, including those formed in
gypsum, granite, talus, quartzite, ice, and sandstone are usually
limited in extent. Caves, with relatively limited nutrient in
organic matter, stable and low temperatures, high humidity
and mineral concentrations, can be considered extreme en-
vironments for life and provide ecological niches for highly
specialized microorganisms (Schabereiter-Gurtner et al., 2003).
Dripping water, visitors and animals can provide organic
input that facilitates life of heterotrophic microorganisms in
some caves (Groth and Saiz-Jimenez, 1999; Groth et al.,
1999, 2001).
Microorganism in caves is the main biological habitant
and remarkably contributes to cave ecology (Northup and
Lavoie, 2001). Presences of microorganisms in terrestrial
and aquatic cave environments around the world have been
reported using the cultivation method (Cunningham et al.,
1995; Gonzalez et al., 1999; Laiz et al., 1999, 2000; Groth
et al., 1999, 2001; Canˇaveras et al., 2001). Many microbes
have been identified to be related to dissolution and pre-
cipitation reactions that involves carbonates, moonmilk, sili-
cates, clays, iron, manganese, sulfur, and saltpeter (Northup
and Lavoie, 2001). Enrichment-based and cultural inves-
tigations on typical heterotrophic microbes have shown that
microbes grow in proportion to less than 1% in an environ-
ment (Amann et al., 1995).
Culture-independent 16S rRNA gene sequence analysis
has been employed to study bacterial communities in envi-
ronmental samples without prior cultivation. It has signifi-
cantly revealed a broader diversity of 16S rRNA gene
sequence types than culture-based studies (Amann et al.,
1995; Head et al., 1998; Hugenholtz et al., 1998). The com-
bination of phylogenetic sequence analysis with restriction
fragment length polymorphisms (RFLPs) of PCR-amplified
bacterial 16S rRNA genes has become a powerful tool to
investigate natural bacterial communities. However, the 16S
rRNA gene-based analysis of bacterial colonization in caves
has been restricted to the samples of rocks, paintings, drip-
ping waters, springs, and underwater passages, but not on
soil (Northup et al., 2000; Vlasceanu et al., 2000; Engel et
al., 2001, 2004; Holmes et al., 2001; Schabereiter-Gurtner et
al., 2002a, 2002b, 2003).
Guizhou, a province in southwest of China, is the center
of East-Asia developing karst area. As one of the three
developing karst areas, this area has a karst area of over
5.5×105 km2 and is the largest and the most complex devel-
oping karst area in the world (Smart et al., 1986; Zhang et
al., 1992). Niu Cave, an unusual cave in this karst area, is
composed of soil other than usual limestone or alcareous
rocks. So far we have limited knowledge to understand the
microbial community in an earth-cave. The aim of the pre-
sent study was to investigate the bacterial diversity in Niu
106 Zhou et al. J. Microbiol.
Table 1. Distribution of clones and phylotypes from the bacterial 16S rRNA gene library
Putative phylogenetic affiliationbNo. of clones % of clones No. of
phylotypesa% of
phylotypes
% of sequence
similarity to its
closest relativesb
No. of phylotypes
that exhibit <90%
similarities to its
closest relativesb
1. Proteobacteria 80 42.6 43 37.4 89-100 3
1.1 Alphaproteobacteria 21 11.2 12 10.4 91-98 -
1.2 Betaproteobacteria 14 7.5 7 6.1 94-100 -
1.3 Deltaproteobacteria 20 10.6 11 9.6 89-98 3
1.4 Gammaproteobacteria 25 13.3 13 11.3 92-99 -
2. Acidobacteria 35 18.6 21 18.3 91-98 -
3. Planctomycetes 17 9.0 14 12.2 87-95 1
4. Chloroflexi 14 7.5 8 7.0 88-94 2
5. Bacteroidetes 42.1 43.592-96 -
6. Gemmatimonadetes 52.7 54.492-99 -
7. Nitrospirae 15 8.0 6 5.2 89-98 1
8. Actinobacteria 12 6.4 10 8.7 90-99 -
9. Candidate division 6 3.2 4 3.5 89-95 2
a Sequences of RFLP types differing only slightly (3%) were considered as a phylotype (Huang et al., 2004).
b Closest relatives as determined by the BLAST method (Altschul et al., 1990; Engel et al., 2004).
Cave by culture-independent method and present the first
knowledge to understand microbial composition in such
environment.
Materials and Methods
Site description and environmental sample collection
Niu Cave (25° 57 N, 107° 48 E) is located in Dushan county,
south of Guizhou province, southwest of China. As a “karst
province”, Guizhou has a karst area of 1.3×105 km2,
comprising 73.8% of its total area. Niu Cave is formed by
soil, and with 5-10 m height, 4-50 m width. After a distance
of about 2 km from the entrance, the cave is divided into
three layers. Each layer has a long distance more than 5
km. Thirty soil samples, 10 from each layer, were sampled
and mixed thoroughly for bacterial community analysis. Soil
samples were stored at -20°C and analyzed within a month.
Soil DNA extraction, PCR amplification, and cloning
Soil DNA was extracted with a soil DNA isolation kit
(Catalog #12800-50, MO BIO Laboratories, USA) following
the manufacturer’s instructions. Bacterial 16S rRNA genes
were amplified by PCR using the combination of universal
primer 1492r and bacterial primer 27f (Lane, 1991). The
PCR reaction was performed with a thermal program,
which comprised preheating at 95°C for 2 min, 25 cycles at
98°C for 1 min, 50°C for 40 s, 72°C for 2 min and a final
extension of at 72°C for 10 min. The amplified products
were purified using an agarose gel DNA purification kit
(No. DV805A, Takara, Japan). Bacterial 16S rRNA gene
amplicon (ca. 1500 bases) was then excised from a 1%
agarose gel and eluted with the same kit. Finally, the puri-
fied product was ligated into the pMD 18 T-vector (Takara)
and the ligation product was transformed into Escherichia
coli DH5α competent cells with ampicillin and blue/white
screening following manufacturer’s instructions.
Screening of rRNA gene clones
Inserts of rRNA genes from recombinant clones were
reamplified with vector primers M13-M3 and M13-RV. The
amplifications were subjected to restriction fragment length
polymorphism (RFLP) by separate enzymatic digestions
with HhaI (Takara) and MspI (Takara) endonucleases fol-
lowing the manufacturer’s instructions, and the digested
DNA fragments were electrophoresed in 3% agarose gels.
After staining with ethidium bromide, the gels were photo-
graphed using an image-capture system UVITEC DBT-08,
and scanning image analyses were performed manually.
DNA sequencing and phylogenetic analysis
One to three representative clones from each unique RFLP
type were selected for sequencing. The 16S rRNA gene
inserts were sequenced using plasmid DNA as template
and M13-20 or M13-RV-P as sequencing primer. Sequencing
was done on an automated ABI 3730 sequencer by Beijng
Genomics Institute. The resulting sequences (next to the
primer 1492r and at least 600 bp) were compared with
those available in GenBank by use of the BLAST method
to determine their approximate phylogenetic affiliation and
16S rRNA gene sequence similarities (Altschul et al., 1990;
Engel et al., 2004). Chimeric sequences were identified by
use of the CHECK-CHIMERA program of the Ribosomal
Database Project (Maidak et al., 1997), and by independently
comparing the alignments at the beginning and the end of
each sequence and the alignments of the entire sequence
with sequences from public databases. Sequences differing
only slightly (3%) were considered as a phylotype, and
each phylotype was represented by a sequence (Huang et
al., 2004). Nucleotide sequences were initially aligned using
CLUSTAL X (Thompson et al., 1997) and then manually
Vol . 4 5 , N o. 2 Bacterial diversity in Niu Cave 107
Fig. 1. 16S rRNA gene-based dendrogram showing phylogenetic
relationships of bacterial phylotypes from Niu Cave (shown in
bold) to members of the Proteobacteria from public database.
Bootstrap values (n=1000 replicates) of 50% are reported as
percentages. The scale bar represents the number of changes per
nucleotide position. Thermosipho sp. MV1063 (AJ419874) was used
as outgroup. Sequences of RFLP types differing only slightly (3%)
are shown in parentheses. Accession numbers are given at the end
of each sequence.
adjusted. Distance matrices and phylogenetic trees were
calculated according to the Kimura two-parameter model
(Kimura, 1980) and neighbor-joining (Saitou et al., 1987)
algorithms using the MEGA (version 3.1) software packages
(Kumar et al., 2004). One thousand bootstraps were per-
formed to assign confidence levels to the nodes in the trees.
The 16S rRNA gene sequences have been deposited in the
GenBank nucleotide sequence database under accession num-
bers EF141837-EF141978.
Results
A total of 190 recombinant clones were randomly selected,
and their rRNA gene inserts were subjected to restriction
endonuclease analysis (RFLP), resulting in 142 different
RFLP types. One to three representative clones of each
unique RFLP type were partially sequenced. The clones
showing sequence dissimilarity less than 3% among cloned
library were considered as a phylotype. As a result, 117
phylotypes were generated. Two chimeric sequences were
identified and excluded from subsequent analyses. Both of
them belonged to unique RFLP types represented by a single
clone. It was determined that most relatives of phylotypes
(87 sequences representing 133 clones) were related to envi-
ronmental clones and 9 phylotypes had relatively low levels
of similarity (<90%) with their closest counterparts in the
GenBank databases (Table 1). None of phylotypes were
closely related to bacterial sequences detected in other caves
or karst areas in the public databases except the clone
CV76 (DQ499315), relative of NC123. These indicated that
the bacterial community associated with Niu Cave was
novel and complex.
Phylogenetic analyses placed the 115 phylotypes in the
following 9 groups of the domain Bacteria: Proteobacteria,
Acidobacteria, Planctomycetes, Chloroflexi (Green nonsulfur
bacteria), Bacteroidetes, Gemmatimonadetes, Nitrospirae,
Actinobacteria (High G+C Gram-positive bacteria), and
candidate divisions (including the OP3, GN08, and SBR
1093). Among them, the Proteobacteria was the largest
group including 80 clones, followed by Acidobacteria (35
clones) and Planctomycetes (17 clones).
Proteobacteria
A total of 80 clones, represented by 43 phylotypes and ac-
counting for 42.6% of the clone library, were phylogenetically
associated with 4 classes of Proteobacteria with similarities
between 89%-100%: Alphaproteobacteria (number of phylotypes,
np=12, number of clones, nc=21), Betaproteobacteria (np=7,
nc=14), Deltaproteobacteria (np=11, nc=20), and Gamma-
proteobacteria (np=13, nc=25) (Table 1). Thirty-eight clones
were represented by 10 phylotypes each including at least 3
clones, while 24 clones were represented by phylotypes of
which each included a single clone.
Eight phylotypes of Deltaproteobacteria revealed less than
94% similarity. Three phylotypes (NC002, NC039, and NC
141), sharing less than 90% similarity to known sequences,
seemed to be representatives of novel taxa within Delta-
proteobacteria subdivisions, respectively (Table 1, Fig. 1).
Thirty-six clones, represented by 19 phylotypes, were
related to cultured members and belonged to putatively
108 Zhou et al. J. Microbiol.
Fig. 2. 16S rRNA gene-based dendrogram showing phylogenetic
relationships of bacterial phylotypes from Niu Cave (shown in
bold) to members of the Acidobacteria and Planctomycetes from
public database. Bootstrap values (n=1000 replicates) of 50%
are reported as percentages. The scale bar represents the number
of changes per nucleotide position. Thermosipho sp. MV1063
(AJ419874) was used as outgroup. Sequences of RFLP types
differing only slightly (3%) are shown in parentheses. Accession
numbers are given at the end of each sequence.
Sphingomonadales (NC006 and NC032), Rhizobiales (NC091
and NC076), Rhodospirillales (NC011), Nitrosomonadales
(NC017 and NC051), Burkholderiales (NC029, NC031, and
NC078), Desulfuromonales (NC002), Xanthomonadales (NC004,
NC018, NC027, NC030, NC060, NC083, and NC094) and
Oceanospirillales (NC127). The closest relatives of NC006
and NC032 were strains of Sphingomonas (Fig. 1). NC015,
NC141 affiliated to the members of Entotheonella (simi-
larity 95) which is a new genus belonging to unclassified
Deltaproteobacteria (Schirmer et al., 2005).
Acidobacteria
Thirty-five clones, represented by 21 phylotypes and account-
ing for 18.6% of the clone library, were clustered with the
uncultivated bacterial sequences of Acidobacteria with sim-
ilarities between 91%-98% (Table 1, Fig. 2). Fig. 2 showed
a phylogenetic tree of Acidobacteria, which was grouped
into at least 4 acidobacterial clusters. Acidobacteria form a
newly devised division of Bacteria, probably as diverse as
Proteobacteria or Gram-positive bacteria. The definition of
this phylum was based on the analysis of 16S rRNA gene
sequences retrieved from cloned rRNA genes and phyloge-
netically related to several cultivated species such as the
Fe(III)-reducing Geothrix fermentans (Ludwig et al., 1997;
Quaiser et al., 2003). The clones Y72 (AB116490) and Y190
(AB116442), relatives of NC065 and NC099 respectively,
were detected in coastal marine sediment beneath areas of
intensive shellfish aquaculture where sulfur cycle was accel-
erated (Asami et al., 2005).
Planctomycetes
Fourteen phylotypes, representing 17 clones and accounting
for 9.0% of the clone library, were grouped into at least 3
clusters with Planctomycetes. These clones were related with
relatively low similarities (in the range of 87%-95%) to cul-
tured and uncultured bacterial sequences listed in the
GenBank database (Table 1, Fig. 2). Molecular microbial ecol-
ogy has provided new evidence showing that Planctomycetes
bacteria are ubiquitous and constitute a representative part
of the natural bacteria population (Hugenholtz et al., 1998;
Neef et al., 1998). The clone 018 (AB252879), relative of NC057,
was a member detected in an iron-oxidation biofilm at
Shibayama lagoon. The retrieved three sequences NC010,
NC038, and NC052 were clustered with Pirellula staleyi
strain ATCC 35122 (AF399914) which can outlast periods of
nutrient depletion with the expression of genes for carbon
starvation (Glöckner et al., 2003).
Chloroflexi (Green nonsulfur bacteria)
Fourteen clones, represented by 8 phylotypes and accounting
for 7.5% of the clone library were related to the members
of the Chloroflexi phylum (88%similarity94%) (Table 1,
Fig. 3). The low similarities to closest members indicated
that the corresponding bacteria detected in Niu Cave be-
longed to putatively new taxonomic groups. The clone H5
(AF234688), close to NC130, was found in a nitrifying-
denitrifying activated sludge (Juretschko et al., 2002).
Bacteroidetes, Gemmatimonadetes, and Nitrospirae
Four phylotypes, each representing a single clone, were clus-
tered with Bacteroidetes. Five clones, each represented by a
phylotype and accounting for 2.7% of the clone library,
were grouped within Gemmatimonadetes phylum. These
clones were related to only uncultured bacterial sequences
listed in the GenBank database (Table 1, Fig. 3). Fifteen
clones, represented by 6 phylotypes and accounting for
8.0% of the clone library, were grouped with Nitrospirae.
Among them, 10 of the 15 clones were represented by 2
phylotypes (NC022 and NC132).
Vol . 4 5 , N o. 2 Bacterial diversity in Niu Cave 109
Fig. 3. 16S rRNA gene-based dendrogram showing phylogenetic
relationships of bacterial phylotypes from Niu Cave (shown in bold)
to members of the Chloroflexi, Nitrospirae, Gemmatimonadetes,
Actinobacteria, Bacteroidetes and Candidate divisions from public
database. Bootstrap values (n=1000 replicates) of 50% are reported
as percentages. The scale bar represents the number of changes
per nucleotide position. Thermosipho sp. MV1063 (AJ419874)
w
as
used as outgroup. Sequences of RFLP types differing only slightly
(3%) are shown in parentheses. Accession numbers are given at
the end of each sequence.
Actinobacteria (High G+C Gram-positive bacteria)
Ten phylotypes, representing 12 clones and accounting for
6.4% of the clone library, were associated with Actinobacteria
with 90%-99% similarities (Table 1, Fig. 3). Five phylotypes
were related to members of Actinomycetales (NC008, NC
079, and NC114) and Rubrobacterales (NC081 and NC085).
Discussion
Terrestrial subsurface environments are often inaccessible
for study, limiting our understanding of ecosystem structure
and dynamics, elemental cycling, and the impacts to earth
and atmospheric biogeochemical processes. Geomicrobiological
activities in caves are no longer underestimated, since studies
showed that bacterial metabolism remarkably contributes to
cave ecology (Northup and Lavoie, 2001). However, no study
has been initiated to investigate the phylogenetic diversity
of bacteria in caves formed in soil, such as Niu Cave. We
firstly presented the knowledge on bacterial community and
revealed a considerable number of novel and unknown bac-
terial sequences and a high diversity of putative bacterial
communities in this environment.
Using culture-independent methods, previous studies (Holmes
et al., 2001; Schabereiter-Gurtner et al., 2002a, 2002b, 2003;
Northup et al., 2003; Engel et al., 2004) have revealed that
bacteria belonging to Proteobacteria, Bacteroidetes, and Actino-
bacteria were usually found in different substances of caves
(Table 2). Among them, Proteobacteria was reported to be
the dominant bacteria and could act as a key role in the
processes of biogeochemical cycle in caves. In Tito Bustillo
Cave, Altamira Cave and La Garma Cave, Acidobacteria
was the second dominating phylogenetic group after Proteo-
bacteria. Our result supported this conclusion. Higher than
7.0% of Chloroflexi was found in NC and Tito Bustillo Cave.
And Firmicutes had been found in Llonín Cave, La Garma
Cave and Lechuguilla and Spider Caves. In our study, there
were no phylotypes belonging to Firmicutes. Orders Rhizobiales,
Sphingomonadales, Rhodospirillales, Burkholderiales, Rhodocyclales,
Desulfuromonales, Pseudomonadales and Actinobacterales were
reported to be present in caves, including Niu Cave. However,
Myxococcales was found only in Altamira Cave, and Xantho-
monadales only in Llonín Cave.
The phylum Gemmatimonadetes, and the orders Nitroso-
monadales (Betaproteobacteria), Oceanospirillales (Gamma-
proteobacteria), and Rubrobacterales (Actinobacteria) found
in Niu Cave were never reported in the previous studies.
Six clones, NC091, NC031, NC051, NC017, NC029, and
NC022, were related to Mesorhizobium, Ralstonia, Nitrosomonas,
Nitrosospira, Alcaligenes, and Nitrospira with similarities higher
than 97%, respectively (Fig. 1, 3). These genera were reported
to involve in putatively nitrogen cycle. Mesorhizobium belongs
to members of nitrogen-fixing bacteria known as rhizobia
(Bottomley, 1992). Ralstonia taiwanensis was reported being
capable of nitrogen fixation (Chen et al., 2001), and Ralstonia
eutropha containing two nitric oxide reductases (Cramm et
al., 1997). Nitrosomonas and Nitrosospira are members of the
ammonia-oxidizing proteobacteria which can convert ammonia
to nitrite (Suzuki et al., 1974; Hiorns et al., 1995). Alcaligenes
sp. STC1 (AB046605) was a C1-using aerobic denitrifier
(Ozeki et al., 2001). Nitrospira was the dominant nitrite oxi-
dizers in most environmental samples tested so far (Burrell
et al., 1998). The bacteria involved in nitrogen cycle were
reported in all studies on bacterial diversity in caves.
NC076 and NC078 were related to Ped omicrob ium and
Leptothrix respectively (Fig. 1). Ped omicr obium plays an im-
portant role in iron- and manganese-oxidization. Ghiorse
and Hirsch (1979) observed that two Pedom icr obium-like
110 Zhou et al. J. Microbiol.
Table 2. Distribution of bacteria in caves investigated by culture-independent molecular method
Putative
phylogenetic
affiliation
% of clones
Nullarbor Caves
(Holmes
et al
.,
2001)
Altamira Cave
(Schabereiter-
Gurtner
et al
.,
2002a)
Tito Bustillo Cave
(Schabereiter-
Gurtner
et al
.,
2002b)
Llonín Cave
(Schabereiter-
Gurtner
et al
.,
2003)
La Garma Cave
(Schabereiter-
Gurtner
et al
.,
2003)
Lechuguilla and
Spider Caves
(Northup
et al
.,
2003)
Lower Kane Cave
(Engel
et al
.,
2004)
Niu Cave
(this study)
Proteobacteria 40.0 52.3 48.8 59.3 32.8 35.0 92.7 42.6
Acidobacteria 23.8 29.2 24.1 5.6 18.6
Planctomycetes 5.7 4.8 2.4 9.0
Bacteroidetes 8.6 9.5 2.4 11.1 3.5 1.7 2.1
Chloroflexi 4.8 7.3 1.7 7.5
Nitrospirae 5.7 3.7 3.5 15.5 8.0
Actinobacteria 5.7 4.8 9.8 22.2 19.0 11.7 6.4
Firmicute s 3.7 13.8 37.9
Gemmatimonadetes 2.7
budding bacteria deposit Fe and Mn ions on their cell
walls. Peck (1986) reported iron-impregnated sheaths of
Leptothrix sp., which was inoculated with mud from Level
Crevice Cave. Moore (1981) found manganese-oxidizing
bacteria Leptothrix sp. in a stream in Matts Black Cave,
West Virginia, and attributed the formation of birnessite in
this cave to the precipitation of manganese around sheaths
of the bacteria. The concentrations of iron, manganese and
other elements have been found in Tito Bustillo and other
Spanish and Italian caves (Schabereiter-Gurtner et al., 2002a,
2002b, 2003).
The order Desulfuromonales included Desulfuromonas
(Liesack and Finster, 1994) related with sulfate/sulfur reduc-
tion and Geobacter with Fe(III)/Mn(IV) reduction (Nealson
and Saffarini, 1994). A pioneering study of Movile Cave,
Romania, by Sarbu et al. (1996) revealed sulfide/ sulfur oxida-
tion bacteria including species of Thiobacillus and Beggiatoa,
and sulfate-reducers including species of Desulfovibrio. Up
to now, sulfur and sulfide oxidizers, sulfate reducers, appear
abundant in caves (Schabereiter-Gurtner et al., 2003).
Acidobacteria and Planctomycetes were the second and
third dominating phylogenetic groups in Niu Cave respec-
tively. It confirmed members of the divisions are ecologi-
cally significant constituents of Niu Cave. Representatives
of the poorly studied phylogenetic divisions have been de-
tected in many clonal analyses and are thought to be of
great ecological significance to many ecosystems (Kuske et
al., 1997; Ludwig et al., 1997; Holmes et al., 2001). As limited
cultivated species, Acidobacteria and Planctomycetes’ ecologi-
cal functions, and possible impacts on caves remain unclear
at present. Two novel genera of Planctomycetes, Candidatus
Kuenenia stuttgartiensis, and Candidatus Brocadia annamoxidans
are capable of catalyzing the anaerobic oxidation of ammo-
nium (Schmid et al., 2000; Jetten et al., 2001).
All above-mentioned results in our study were ascribed
to culture-independent techniques including PCR amplifica-
tion of bacterial 16S rRNA genes and restriction fragment
length polymorphism (RFLP). The applied approach allows
the analysis of poorly studied environments where nothing
or little is known about bacterial diversity and natural
growing conditions. Our present study gave insight into the
great bacterial taxonomic diversity in Niu Cave, overcoming
some of the limiting factors of cultivation methods and allow-
ing the detection of putatively uncultivable and unexpected
bacteria.
However, studies based on culture-independent methods
make more difficult valid statements about the ecological
role that clones might play in the environment. To deter-
mine microbial contribution to cave ecology, we need to
draw on additional wealth of rigorous research done on
this system. Stable isotope techniques can provide informa-
tion on microbial contribution to mineral formation (Hose
et al., 2000) and ecosystem bioenergetics (Sarbu et al., 1996).
The PCR- and RFLP-based investigation of bacterial diversity
is not free from bias. Limitations such as inefficient cell
lysis, and the preferential and selective amplification of 16S
rRNA gene fragments may lead to an underestimation of
bacterial diversity (Liesack et al., 1997). Potentially, with
the development of molecular sequence-based techniques
and accumulation of information on the diversity and struc-
ture of bacterial communities in caves, more explanations
concerning contribution of microorganisms to cave ecosys-
tem will be presented.
Acknowledgements
This work was supported jointly by projects from NSFC
(30460078) and Department of Science and Technology of
Yunnan Province (2006XY41, 2006C0005M, 2004C0001Q,
2003RC03, and 2005NG05).
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With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.
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