Received Date: 29-Dec-2008
Accepted Date: 30-Apr-2009
Comparison of the structure and composition of bacterial communities
from temperate and tropical freshwater ecosystems
Jean-F. Humbert1,2*, Ursula Dorigo1, Philippe Cecchi3, Brigitte Le Berre1, D. Debroas4
and Marc Bouvy3
1 INRA-UMR 42, BP 511, 74203 Thonon Cedex, France, 2 Institut Pasteur-URA CNRS
2172, Unité des Cyanobactéries, 28 rue du Dr Roux, 75724 Paris Cedex 15 France, 4 IRD /
UMR 5119, Université Montpellier 2, CC093, 34095 Montpellier Cedex, France, 4.
Université Blaise Pascal, LMGE-UMR CNRS 6023, 63177 Aubière Cedex, France
*To whom correspondence should be addressed. E-mail: humbert@pasteur.fr or
humbert@thonon.inra.fr
Summary
We used a partial 16s rRNA sequencing approach to compare the structure and composition
of the bacterial communities in three large, deep sub-alpine lakes in France to those of
communities in six shallow tropical reservoirs in Burkina Faso. Despite the very different
characteristics of these ecosystems, we found that their bacterial communities share the same
composition in regard to the relative proportions of the different phyla, suggesting that
freshwater environmental conditions lead to convergence in this composition. In the same
way, we found no significant difference in the richness and diversity of the bacterial
communities in France and Burkina Faso. We defined core and satellite OTUs (sequences
sharing at least 98% identity) on the basis of their abundance and their geographical
distribution. The core OTUs were found either ubiquitously or only in temperate or tropical
and subtropical areas, and they contained more than 70% of all the sequences retrieved in this
study. In contrast, satellite OTUs were characterized by having a more restricted geographical
distribution and by lower abundance. Finally, the bacterial community composition of these
freshwater ecosystems in France and Burkina Faso were markedly different, showing that the
history of these ecosystems and regional environmental parameters have a greater impact on
the relative abundances of the different OTUs in each bacterial community than the local
environmental conditions.
Keywords: bacterial communities, 16S rRNA, freshwater ecosystems, France, Burkina Faso,
biogeography
Introduction
Since the development of molecular tools, many studies have contributed to better understand
the relative impacts of abiotic and biotic factors and processes on the species composition of
bacterial communities (see for example for marine ecosystems Morris et al. (2005) and
Fuhrman et al. (2006); and for freshwater ecosystems, Crump & Hobby 2005, Yannarell &
Triplett 2005, Schauer et al. 2005, Crump et al. 2007 and Van der Gucht et al. 2007). Several
of these studies have also highlighted the ubiquitous distribution of some taxa, and their
dominance in microbial communities. In marine ecosystems, the SAR 11 clade and the
Roseobacter-clade-affiliated cluster (Morris 2002; Selje et al. 2004) are distributed worldwide
as is the ammonia-oxidizing -Proteobacterium Nitrosococcus oceani (Ward & O’Mullan
2002). Despite the discrete distribution patterns of freshwater ecosystems, ubiquitous
actinobacterial and alphaproteobacterial clades have also been identified by Zwart et al.
(1998, 2002, 2003) in most of the 70 European lakes studied. Moreover, Van der Gucht et al.
(2007) clearly showed that the impact of spatial distance on freshwater Bacterial Community
Composition (BCC) is marginal compared to local environmental factors. These various
studies have stimulated many review papers of the presence/absence of biogeographical
patterns in bacterial communities (Finlay 2002; Horner-Devine et al. 2003; Green &
Bohanann 2006; Martiny et al. 2006).
For communities of microorganisms, Gibson et al. (1999) used the core-satellite
species hypothesis initially developed by Hanski (1982, 1991) to provide a theoretical basis to
support the classification of species as dominant, subordinate and transient. The core species
were defined as widely distributed species and were often abundant within local patches,
whereas the satellite species were mostly rare species present at only a limited number of
sites. In a recent review, Pedros-Alio (2006) suggested that microbial taxa could also be
defined as core versus occasional (= satellite) species, the core taxa being the most abundant
taxa seemingly well adapted to a given ecosystem, whereas the occasional taxa correspond to
the long tail of rare taxa in the distribution of species abundance. This concept has rarely been
exploited to date, with the exception of the work of Soininen & Heino (2005), which
demonstrated that in diatom communities in Boreal streams from Finland core species
occurring at most sites were virtually absent, whereas a large number of satellite species were
present at only a few sites. According to these authors, the very small number of core species
identified suggests that the distribution of unicellular microbial organisms may not be very
different from multicellular ones, despite marked differences in their size and the huge
abundance of the former, which facilitates their passive dispersal over large areas.
In order to find out whether this concept of core and satellite species is pertinent for
freshwater bacterial communities, we compared the structure and the composition of
freshwater bacterial communities from two very contrasted types of ecosystems: recent and
shallow, tropical reservoirs in Burkina Faso (West Africa), and ancient and deep, temperate
sub-alpine lakes in France (Europe). The composition and structure of the bacterial
communities was determined by applying a PCR-cloning-sequencing approach to a 16S
rRNA fragment in order to identify the different OTUs on the basis of a 98% sequence
identity level. In all, more than 1,100 sequences were obtained and compared both with each
other and with data available in GenBank™ database.
Results
Comparison of the global composition and structure of the French and Burkina Faso
aquatic microbial communities
In all the ecosystems, the dominant bacterial group was that of Actinobacteria, which
contained from 39 to 84% of the sequences, depending on the sampling site (Fig. 1). Except
for one sequence belonging to actinobacterial group III, all the sequences of this phylum
belonged to the actinobacterial groups I or IV defined by Warnecke et al. (2004).
Alphaproteobacteria and betaproteobacteria contain 13 and 11% respectively of all the
sequences retrieved in this study, and they were found in almost all the samples (Fig. 1). The
phylum of cyanobacteria, which contains 11% of the sequences, was found in very variable
proportions, depending on the sampling sites (Fig. 1), but most of the sequences were
retrieved from African reservoirs. Other sequences belonged to Bacteroidetes, Firmicutes,
Deltaproteobacteria,
Gammaproteobacteria,
Chloroflexi,
Green
Non-Sulfur
Bacteria,
Fibrobacteres, Nitrospirales and Plantomycetales groups, and they were always found in very
low proportions (<1%) in the various lakes.
On the basis of the proportions of each bacterial group in the different samples, no
dichotomy between the French and Burkina Faso samples was detected by hierarchical
ascendant classification based on Euclidian dissimilarities. The same kind of result was also
obtained using a k-means classification (data not shown). No significant difference was found
in the proportions of Actinobacteria, Gammaproteobacteria and Bacteroidetes when water
bodies in Burkina Faso were compared with those from the French lakes (Man-Whitney UTest). On the other hand, a significant difference (p=0.01) was found when the
Betaproteobacteria and “Others” group (p=0.03) were taken into account. For Cyanobacteria
and for Alphaproteobacteria, no significant difference was found between the two types of
ecosystems at the p level of 0.05 (the p values being equal to 0.08 and 0.06 respectively).
Comparison of the richness and diversity at the taxonomic level of OTUs in water bodies
in France and Burkina Faso
In all, 183 OTUs were identified among the 1,126 sequences retrieved in this study. The mean
estimations of the OTU richness in each sample using the ACE and the Chao 1 estimators
were equal to 48 and 52 respectively (Fig. 2A), and no significant difference (Mann-Whitney
U-Test) was detected when comparing these two estimators in the French and Burkina Faso
water bodies. The mean value of Shannon's diversity index was 2.75 (Fig. 2A). Once again,
no significant difference (Mann-Whitney U-Test) was reported when the Shannon index
values from Burkina Faso and French ecosystems were compared. In addition, there was no
significant correlation (Spearman and Kendall coefficients) between these diversity estimators
and the volumes or the maximum depths of the different lakes when data were considered
either with regard to their geographical origin or after pooling. The rarefaction curves led to
the same conclusion; showing no evident difference in the shapes of these curves when
comparing those obtained for Burkina Faso reservoirs with those for French lakes (Fig. 2B).
With regard to the OTU richness in the most abundant bacterial group (Actinobacteria,
Cyanobacteria, Proteobacteria, Bacteroidetes), it appeared that for Actinobacteria (Groups I
and IV) and for Cyanobacteria, a restricted number of OTUs were found in comparison to the
number of sequences retrieved, meaning that several OTUs contain a great number of
sequences (Fig. 3). On the other hand, for Gammaproteobacteria and Bacteroidetes, a small
number of sequences were retrieved, but most of these sequences belonged to different OTUs
(Fig. 3). The Alphaproteobacteria and the Betaproteobacteria occupied an intermediate
position between to the previous two groups (Fig. 3).
Nine of the 183 OTUs (4.9%) contain more than 50% of all the sequences identified in
this study, seven belonging to Actinobacteria, one to Cyanobacteria and one to
Alphaproteobacteria. In each sample, an average of 6 (±0.9) of these 9 OTUs were found,
representing, on average, 25% (±6%) of the total number of OTUs identified within each
sample. Similarly, 21 OTUs (12%) contained more than 70% of all the sequences obtained in
this study. On average, 10 (±1.5) of these 21 OTUs were found in each sample, representing,
on average, 43% (±8%) of the total number of OTUs identified within each sample. The
phylogenetic position of the 21 dominant OTUs in Burkina Faso reservoirs and in alpine lakes
is shown in Figure 4. From this Figure, it can be seen that most of them belonged to
Actinobacteria Groups I and IV, and were closely related to sequences from uncultured
bacteria from lakes.
Geographical distribution of the OTUs
Nine of the 21 OTUs containing >70% of all the sequences were found both in Burkina Faso
and in France, seven only in Burkina Faso, and five only in France (Fig. 5). Five of the other
162 OTUs were also found in both Burkina Faso and France. In all, the 14 OTUs that were
present both in Burkina Faso and in France, belonged to the Cyanobacteria (1 OTU), the
Actinobacteria (6 OTUs), the Alphaproteobacteria (1 OTU), the Betaproteobacteria (3 OTUs),
the Fibrobacteres (1 OTU), the Gammaproteobacteria (1 OTU) and the Bacteroidetes (1 OTU)
respectively.
As evidenced by the Blast analysis (Supplemental Tab. 1), the four most abundant
OTUs (n° 3, 4, 12 & 13 in Fig. 5) among the seven OTUs found only in Burkina Faso,
displayed an exclusively tropical and subtropical geographical distribution (Panama, Brazil
and Tanzania). Two OTUs (n°14 and n°19) were found both in tropical (Lake Gatun; Tucurui
reservoir, Lac Taihu) and temperate areas (Supplemental Tab. 1). Finally, GenBank contains
no sequence sharing ≥98% with the last OTU (OTU n° 20). Two (n° 7 and 17) of the five
OTUs present in alpine lakes but not in Burkina Faso reservoirs were found only in freshwater
ecosystems from temperate and cold areas (Supplemental Tab. 1), whereas two others (n° 10
and 16) were present in both temperate and tropical ecosystems. The remaining OTU (n°21)
was also found only in temperate and cold areas, but in very different ecosystems (freshwater,
soil, ice, sludge…).
Finally, a positive relationship (R²=0.77 using an exponential regression model) was
found between the mean local abundance of the OTUs and the number of locations where
they were found when taken in account all the OTUs (Fig. 6) or only those belonging to
Actinobacteria or Alphaproteobacteria or Betaproteobacteria or other bacteria groups (data
not shown).
Comparison of the water bodies in France and in Burkina Faso on the basis of their
composition
Correspondence analysis (Fig. 7) demonstrated a clear differentiation between the French
Alpine lakes and the reservoirs in Burkina Faso on the basis of the composition of their
bacterial communities. Similar results were obtained whether all the OTUs retrieved on this
study were taken into consideration, or only the 21 dominant OTUs. Similarly, a highly
significant (P<0.001) differentiation between the French and Burkina Faso samples was also
found by discriminant analysis when considering either all the OTUs or only the 21 dominant.
Discussion
From this study, it appears that as suggested previously (Glöckner et al. 2000; Sekar et al.
2003; Warnecke et al. 2004; Percent et al. 2008) bacterial communities of freshwater limnic
ecosystems are dominated by Actinobacteria, followed by Proteobacteria and to a lesser
extent by Bacteroidetes and Cyanobacteria. The global structure of all the bacterial
communities isolated from very contrasted aquatic systems appeared to be well conserved
(Fig. 1), in contrast with some results obtained from other natural bacterial communities (e.g.
for soils see Zhou et al. 1997; for rivers see Anderson-Glenna et al. 2008 and Beier et al.
2008). On the other hand, the structure of bacterial communities from mammal intestines
seems also to present a shared pattern characterized by the marked dominance of only two
bacterial divisions (Firmicutes and Bacteroidetes), which could be explain by the recent
colonization of the new niches offered by the recent emergence of mammals (Ley et al. 2006).
With regard to the various processes proposed by Kelt et al. (1996) to explain similarities in
mammalian community structure across widely separated geographic regions, it looks as
though the conserved structure of freshwater bacterial communities might be attributable to
the fact that aquatic environmental conditions have led to the convergence of their bacterial
community structures, regardless of the evolutionary history of lineages. Interestingly,
Pommier et al. (2007) also found a conserved structure (different from that of freshwater
ecosystems) in marine bacterioplankton communities when comparing communities sampled
in different oceans, which seems to confirm that aquatic environments may lead microbial
communities to share a common structure.
At a sequence identity level ≥98%, it appeared that in both French deep lakes and
shallow Burkina Faso reservoirs a small proportion of OTUs were abundant and widely
distributed (Fig. 5), whereas numerous OTUs were rare, and only found in a small number of
ecosystems. As positive relationship has been found between the size of the geographic range
of OTUs and their average local abundance, as had already been observed for a wide range of
taxa and habitats (see for example Gaston 1996). Different hypotheses have been proposed to
explain this relationship, such as the fact that species with extensive niches can be expected to
be more abundant and widespread than species with narrow niches (Brown 1984). Again, the
same kind of positive relationship between the mean local abundance of OTUs and their
geographical distribution was also demonstrated by Pommier et al. (2007) in marine
bacterioplankton communities.
From these observations, and by analogy with the concept of defining core (dominant)
and satellite (rare) species for eukaryote communities (Hanski, 1982, 1981), we propose that
bacterial communities from limnic freshwater ecosystems could also contain core and satellite
OTUs playing different roles in these ecosystems. We have shown that a restricted number of
OTUs widely distributed among different ecosystems, contain a large number of sequences.
These dominant OTUs can be defined as core OTUs. On the other hand, 32 OTUs only found
in one ecosystem could be considered as satellite OTUs (sensu Hanski, 1982). It is very likely
that using a larger set of sequences in each ecosystem, other OTUs should be also considered
as core OTUs, but also that new satellite OTUs would be found. Interestingly, it appeared for
example, that the 21 dominant OTUs correspond to 43% of all the OTUs recorded in each
sample. Brown (1984) has proposed that core species can be considered to be generalist
species that are able to exploit more habitats, and are thus both more widely distributed and
more locally dense. They are likely to be involved in the basic functioning of the bacterial
communities, whereas satellite species will be involved in adapting to local environmental
conditions. Due to the fact that none of these species can be cultured, the data available about
their physiological capacities and their genome are very restricted and not sufficient to make
it possible to evaluate their functional role in aquatic ecosystems.
Among the dominant OTUs, which can be considered to be core species, some were
found in both temperate and tropical areas, whereas others were found either only in the
alpine lakes or only in the Burkina Faso reservoirs. The OTUs found only in alpine lakes were
also recorded in many other temperate ponds, reservoirs and lakes from different continents,
demonstrating the existence of globally distributed bacteria in very different freshwater
ecosystems as previously described by Zwart et al. (1998). In the same way, several OTUs
found only in the Burkina Faso reservoirs appeared from Blast analyses also to occur in some
tropical and subtropical lakes in China and America, implying that they too are also widely
distributed in tropical and subtropical areas. Such tropical and sub-tropical distribution has
been also previously found in marine ecosystems for a Deltaproteobacteria bacterioplankton
clade (Brown & Donachie, 2007). All these findings suggest that many freshwater bacteria
have a cosmopolitan character, as previously found for Archaea (Massana et al. 2000), but
also that some of them have the ability to occupy preferentially temperate or tropical and sub-
tropical areas. None of the present methods used to assess microbial diversity can be used to
provide an efficient evaluation of the abundances of rare species. This means that it is not
possible to exclude the possibility that temperate OTUs are also present in tropical areas (and
vice versa) at very low abundance levels. Thus, it will be interesting in the future to develop
specific primers for these OTUs in order to be able to detect their presence even at low levels
of abundance.
There was no significant difference when richness in the alpine lakes was compared to
that in the Burkina Faso reservoirs, despite the very contrasting characteristics of these
ecosystems (in terms of volume, drainage basin, system morphology, eutrophication level..).
Two recent publications dealing with marine bacterioplankton communities (Pommier et al.
2007; Fuhrman et al. 2008) and with pathogenic bacteria and viruses (Guernier et al. 2004)
have reported latitudinal gradients of diversity in these communities like those previously
described for macroorganisms (e.g. Willig 2003), but not for soil bacteria (Fierer & Jakson,
2006). Due to the fact that we compared sharply contrasting ecosystems (large natural lakes
versus small artificial tropical reservoirs), we cannot conclude that the lack of significant
differences in the richness and diversity estimators between alpine lakes and Burkina Faso
reservoirs (Fig. 2) is indicative of a lack of latitudinal gradient in the diversity of freshwater
bacterial communities. Indeed, a taxa-area relationship, which could influence the comparison
between our ecosystems, has been reported for bacterial communities located in treeholes
(Bell et al. 2005), salt marsh sediments (Horner-Devine et al. 2004) and forest soils (Zhou et
al. 2008), but is still controversial in freshwater ecosystems (Reche et al. 2005; Lindtsröm et
al. 2007; Reche et al. 2007). In the present study, the fact that no taxa-area relationship can be
detected when taking in account only the six reservoirs from Burkina Faso or only the three
alpine lakes despite the considerable differences in their sizes, suggests that this parameter is
not determinant for the diversity of freshwater bacterial communities and so that bacterial
communities from freshwater ecosystems do not seem to display a latitudinal diversity
gradient. Fuhrman et al. (2008) has demonstrated a richness gradient in marine bacterial
communities, which is mainly explained by water temperatures and to a lesser extent by the
productivity of the ecosystems. However, no obvious difference can be detected when
comparing the diversity of bacterial communities in alpine lakes (Fig. 2) at different seasons,
despite very considerable differences in the water temperatures and in the productivity of
these ecosystems.
Our results demonstrate a clear distinction between the deep temperate lakes and
shallow tropical reservoirs on the basis of the composition of their bacterial communities (Fig.
7). These findings are interesting with regard to the question of the relative impact of the
history of the ecosystem, and of local versus continental environmental factors, on the
bacterial community composition (BCC). Van der Gucht et al. (2007) concluded that local
environmental factors and processes seem to drive the composition of bacterial communities
in lakes located along a North-South gradient in Europe, with spatial distance having only a
marginal impact. On the other hand, Yannarell and Triplett (2005) showed that differences in
the diversity of bacterioplankton communities of 30 lakes in northern and southwestern
Wisconsin were best explained by regional (northern versus southern lakes) and landscape
(seepage versus drainage lakes) factors. In the same way, Lindström and Leskinen (2002)
found area-specific taxa when comparing BCC values for several neighboring lakes located in
three different geographic regions in Scandinavia. In this study, the differentiation between
bacterial communities in France and Burkina Faso can be interpreted as the outcome of the
differing histories of these ecosystems, and also of regional or continental selection pressures.
On the other hand, local environmental parameters such as the trophic level (ranging from
oligotrophic to eutrophic in both the French and Burkina Faso ecosystems), seem to have less
influence on the BCC, because no differentiation related to this parameter was found among
the different ecosystems.
To conclude, it appears from this study that bacterial freshwater communities from
contrasted ecosystems are dominated by a restricted number of OTUs, which are distributed
over a wide spatial scale, and which can be assimilated to core species. It is now necessary to
develop metagenomic or single cell sequencing approaches for future research to make it
possible to estimate the potential physiological capacities of these dominant species in
freshwater microbial communities.
Experimental procedures
Study sites and sampling strategy. Water samples were collected in three sub-Alpine lakes,
which differed mainly by their trophic parameters, and the presence or absence of toxic
cyanobacteria proliferations. Lake Annecy is oligotrophic, whereas Lake Bourget and Lake
Geneva are both mesotrophic. Additional details about different physico-chemical and
biological parameters are given in Table 1. Sampling was carried out on three separate
occasions in 2003: during the winter stratification period (January), in spring (April-May),
when the water mass had begun to stratify, and in summer (August), when the water column
was completely stratified. Samples were collected from each lake at a specific sampling
station located above the deepest spot in the lake. In a previous study (Dorigo et al. 2006), we
demonstrated that sampling at one point provides a good evaluation of the bacterial diversity
at the scale of the whole ecosystem. At each sampling date, water samples from the surface
(~2 m) and from deeper water (~40-50 m) were collected, and put into previously autoclaved
plastic bottles that were rinsed with water from each sample. Each time, 500 mL was taken
and kept in the dark at 4°C until being processed immediately on arrival in the laboratory (3
hours later).
In Burkina Faso, the 6 reservoirs that were sampled between March 18th and April 1st
2005 lay in the Nakambé (former White Volta) basin. Two of these reservoirs (Dem and
Bamsa) are rural reservoirs, intensively used for irrigation of crops. Two other reservoirs
(Ouagadougou and Pouytenga) are located in urban areas, and are intensively used by humans
mainly for domestic purposes (watering, bathing and also drinking-water harvesting). The
Ouagadougou reservoir, located in the centre of the capital, was sampled twice (Ouaga 1 and
Ouaga 2). In contrast, Bazega is located in an isolated zone where it is probably subjected to
little anthropogenic pressure. Finally, Bagré, which is the largest reservoir studied (used for
hydropower generation and irrigation) is located far downstream, and its drainage basin
corresponds to the entire Nakambé basin. All sites except Bagré appeared to be well mixed
systems, primarily because of their shallowness. A more complete description of the sites is
proposed in Leboulanger et al. (2009).
Depth-integrated water samples were collected from the surface using a vertical 150 cm tube
to integrate the water column, except for Pouytenga (0.7 m depth), where a simple surface
sample was collected. The water samples were kept in previously autoclaved plastic bottles
that had been rinsed with water from the corresponding sample. For each reservoir, 500 mL
was taken and kept in the dark at 4°C until being processed immediately on arrival in the
laboratory (1 hour later).
Sample processing in the laboratory. For each lake-water sample, 250 ml were immediately
vacuum-filtered through a 2-µm pore-size polycarbonate membrane (Nuclepore) to eliminate
larger eukaryotes (phytoplankton and zooplankton, the chloroplastidial or mitochondrial 16S
rRNA gene of which would be amplified by the PCR primers used). This pre-filtration step
also excluded filamentous and particle-associated bacteria. Filtration through 0.2 µm poresize polycarbonate membrane filters (Nuclepore) was then used to collect and trap microbial
biomass of size <2 µm. The filters and the trapped biomass were stored at -80°C for
subsequent diversity analyses.
DNA extraction, PCR amplification, cloning and sequencing. Nucleic acid extraction was
performed on the 0.2-µm filters as described in Massana et al. (1997) with minor
modifications. Each of the 0.2-µm filters was placed in an eppendorf microtube, to which 750
µl of lysis buffer (40 mM EDTA, 50 mM Tris-HCl, 0.75 M sucrose) had been added after prewarming to 55°C. The filters were re-frozen at -80°C, and then thawed by putting the tubes
into a water bath at 55°C for 2 min, before being vortexed and placed in a sonication bath for
2 min. Lysozyme (Eurobio, 20,000 U/mg, 2.4 mg.mL-1 final concentration) was then added to
the filters, which were then incubated at 37°C for 45 min with gentle stirring. SDS (sodium
dodecyl sulfate, 1% final concentration) and proteinase K (Eurobio, 30 mU/mg, 0.2 mg.mL-1
final concentration) were then added, and the filters were incubated at 55°C for at least
90 min. The lysates were transferred to a fresh eppendorf tube, and purified twice by phenolchloroform-isoamyl alcohol. The integrity of the total DNA was checked by agarose gel
electrophoresis, and quantified from the absorbance at 260 nm. The DNA was stored at -20°C
until analyses.
PCR amplifications were performed in 50-µl volumes containing approximately 3060 ng of extracted DNA, 5 µl of 10X Taq reaction buffer (Eurobio), 1.5 mM MgCl2, 120 µM
of each deoxynucleotide, 1 µM of each primer targeting the 16S rRNA gene corresponding to
positions 358-907 of the Escherichia coli 16S rRNA, bovine serum albumin (Sigma,
0.5 mg.mL-1 final concentration), and 1.25 U Taq DNA polymerase (Eurobluetaq, Eurobio).
The primer combination of Eubacterial-specific primer 358f (Muyzer et al. 1993) and
universal primer 907rM (Schauer et al. 2003) yielded a DNA fragment of ca. 550-bp. For
each set of reactions, a negative control, in which the template was replaced by an equivalent
volume of sterile deionized water, was included. PCR reactions were carried out as described
in Schauer et al. (2000).
Amplification products were cloned into the vector pGEM T Easy (Promega)
according to the Manufacturer’s instructions. From 30 to 120 positive transformants were
randomly selected from each of the libraries, and were double strand sequenced on an Applied
Biosystems 373 automated sequencer (Perkin Elmer, Foster City, CA), according to the
Supplier's instructions.
16S rRNA gene sequences from the Alpine lakes have been added to the GeneBank™
database under accession numbers AJ965761 to AJ 966243, and those from Burkina Faso
under accession numbers (FJ185706-FJ185780; FJ207177-207344; FJ208378-FJ208589;
FJ262737-FJ262952).
Sequence analysis. The partial 16S rRNA gene sequences recovered from the clone libraries
were edited using GeneDoc (Nicholas & Nicholas 1997). Sequence alignments were
performed by ClustalW using Mega4 software (Tamura et al. 2007), and all the alignments
were corrected manually in GeneDoc. Sequences identified as chimeric sequences by
Bellerophon, freely available at http://foo.maths.uq.edu.au/~huber /bellerophon.pl (Hubber et
al. 2004), and by the Chimera Check software program of the Ribosomal Database Project II
(RDPII, freely available at http://rdp.cme.msu.edu/index.jsp (Cole et al. 2005), were
eliminated from the alignment. The percentages of identity between all sequences were
obtained using GeneDoc. Sequences sharing at least 98% identity were considered to belong
to the same OTU. An assignation was done for each OTU by searching for homologous
sequences both at the RDPII, using the Sequence Match and the Classifier tools, and at the
National
Center
for
Biotechnology
Information
(NCBI,
freely
available
at
http://www.ncbi.nlm.nih.gov/) using the BLAST (Basic Local Alignment Search Tool)
network service.
For phylogenetic analysis the new 16S rRNA gene sequences were added to a
universal tree (16S rRNA genes database provided by Greegenes) by the ARB parsimony
interactive tool (Ludwig et al., 2004). The overall tree topology was supported by maximum
parsimony and neighbour-joining analyses. The tree higlight freshwater clades defined by
Zwart et al. (2002) and Warnecke et al. (2004).
EstimateS 8.0 software (Collwell 2006) was used to estimate the richness and diversity
estimators (Chao1, ACE, Shannon index). Rarefaction curves were obtained using PAST
Software (Hammer et al. 2001). The correspondence analysis was performed on the relative
distribution of the OTUs in the different ecosystems, using ADE-4 software (Thioulouse et al.
1997).
Acknowledgements
Monika Ghosh is acknowledged for improving the English version of the manuscript. We
would like also to thank all the colleagues, which have been involved in the sampling
campaign (FACIES) in Burkina Faso in 2005 and the IRD (Research Unit 167) for funding
this campaign. The works on Alpine lakes have been funded by the program EMERGENCE
from the Région Rhône-Alpes.
References
Anderson-Glenna, M.J., Bakkestuen, V., and Clipson, N.J.W. (2008) Spatial and temporal
variability in epilithic biofilm bacterial communities along an upland river gradient. FEMS
Microbiol Ecol 64, 407-418.
Beier, S., Witzel, K.P., and Marxsen, J. (2008) Bacterial composition in central European
running waters examined by temperature gradient gel electrophoresis and sequence
analysis of 16S rRNA genes. Appl Environ Microbiol 74, 188-199.
Bell, T., Ager, D., Song, J.I., Newman, J.A., Thompson, I.P., Lilley, A.K., and van der Gast,
C.J. (2005) Larger Islands House More Bacterial Taxa. Science 308, 1884.
Brown, J.H. (1984) On the relationship between abundance and distribution of species. Am
Nat 124, 255-279.
Brown, M.V., and Donachie, S.P. (2007) Evidence for tropical endemicity in the
Deltaproteobacteria Marine Group B/SAR324 bacterioplankton clade. Aquat Microb Ecol
46, 107-115.
Cole, J.R., Chai, B., Farris, R.J., Wang, Q., Kulam, S.A., McGarrell, D.M., Garrity, G.M., and
Tiedje, J.M. (2005) The Ribosomal Database Project (RDP-II): sequences and tools for
high-throughput rRNA analysis. Nucleic Acid Res 33, D294-D296.
Colwell, R.K. (2005) EstimateS: Statistical estimation of species richness and shared species
from
samples.
Version
7.5.
User's
Guide
and
application
published
at:
http://purl.oclc.org/estimates.
Crump, B.C., and Hobbie, J.E. (2005) Synchrony and seasonality in bacterioplankton
communities of two temperate rivers. Limnol Oceanogr 50, 1718-1729.
Crump, B.C., Adams, H.E., Hobbie, J.E., and Kling, G.W. (2007) Biogeography of
bacterioplankton in lakes and streams of an arctic tundra catchment. Ecology 88, 13651378.
Dorigo, U., Fontvieille, D., and Humbert, J.F. (2006) Spatial variability in the dynamic and
the composition of the bacterioplankton community of the Lac du Bourget (France). FEMS
Microb Ecol 58, 109-119.
Finlay, B.J. (2002) Global dispersal of free-living microbial eukaryote species. Science 296,
1061-1063.
Fierer, N., and Jackson, R.B. (2006) The diversity and biogeography of soil bacterial
communities. Proc Nat Acad Sci USA 103, 626-631.
Fuhrman, J.A., Hewson, I., Schwalbach, M.S., Steele, J.A., Brown, M.V., and Naeem, S.
(2006) Annually reoccurring bacterial communities are predictable from ocean conditions.
Proc Nat Acad Sci USA 103, 13104-13109.
Fuhrman, J.A., Steele, J.A., Hewson, I., Schwalbach, M.S., Brown, M.V., Green, J.L., and
Brown, J.H. (2008) A latitudinal diversity gradient in planktonic marine bacteria. Proc Nat
Acad Sci USA 105, 7774-7778.
Gaston, K.J. (1996) The multiple forms of the interspecific abundance-distribution
relationship. Oikos 76, 211-220.
Gibson, D.J., Ely, J.S., and Collins, S.L. (1999) The core-satellite species hypothesis provides
a theoretical basis for Grime’s classification of dominant, subordinate, and transient
species. J Ecol 87, 1064-1067.
Glöckner, F.O., Zaichikov, E., Belkova, N., Denissova, L., Pernthaler, J., Pernthaler, A.,
Amann, R.I. (2000) Comparative 16S rRNA analysis of lake bacterioplankton reveals
globally distributed phylogenetic clusters including an abundant group of actinobacteria.
Appl Environ Microbiol 66, 5053-5065.
Green, J., and Bohannan, B.J.M. (2006) Spatial scaling of microbial biodiversity. Trends
Ecol Evol 21, 501-507.
Guernier, V., Hochberg, M.E., and Guégan, J.F. (2004) Ecology drives the worldwide
distribution of human diseases. PLoS Biol 2, e141.
Hammer, Ø., Harper, D.A.T., and Ryan, P.D. (2001) PAST: Paleontological Statistics
Software Package for Education and Data Analysis, p. 9, Palaeontologia Electronica, vol.
4.
Hanski, I. (1982) Dynamics of regional distribution: the core and satellite species hypothesis.
Oikos 38, 210-221
Hanski, I. (1991) Single-species metapopulation dynamics: concepts, models and
observations. Biol J Linn Soc 42, 17-38.
Horner-Devine, M.C., Leibold, M.A., Smith, V.H., and Bohannan, B.J.M. (2003). Bacterial
diversity patterns along a gradient of primary productivity. Ecol Lett 6, 613-622.
Horner-Devine, M.C., Lage, M., Hughes, J.B., and Bohannan, B.J.M. (2004) A taxa-area
relationship for bacteria. Nature 432, 750-753.
Huber, T., Faulkner, G., and Hugenholtz, P. (2004) Bellerophon: a program to detect chimeric
sequences in multiple sequence alignments. Bioinfo Appl Note 20, 2317-2319.
Kelt, D.A., Brown, J.H., Heske, E., Marquet, P.A., Morton, S.R., Reid, J.R.W., Rogovin,
K.A., and Shenbrot, G. (1996) Community structure of desert small mammals: comparison
across four continents. Ecology 77, 746-761.
Leboulanger, C., Bouvy, M., Pagano, M., Dufour, P., Got, P., and Cecchi, P. (2009)
Responses of planktonic microorganisms from tropical reservoirs to Paraquat and
Deltamethrin exposure. Arch Environ Contam Toxicol 56, 39-51.
Ley, R.E., Peterson, D.A., and Gordon, J.I. (2006) Ecological and evolutionary forces shaping
microbial diversity in the human intestine. Cell 124, 837-848.
Lindström, E.S., Eiler, A., Langenhede, S., Bertilsson, S., Drakare, S., Ragnarsson, H., and
Tranvik, L.J. (2007) Dos ecosystem size determine aquatic bacterial richness? Comment.
Ecology 88, 252-253.
Lindström, E.S., and Leskinen, E. (2002) Do neighboring lakes share common taxa of
bacterioplankton? Comparison of 16S rDNA fingerprints and sequences from three
geographic regions. Microb Ecol 44, 1-9.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar et al. (2004) ARB:
a software environment for sequence data. Nucleic Acid Res 32, 1363–1371.
Martiny, J.B.H., Bohanann, B.J.M., Brown, J.H., Colwell, R.K., Fuhrman, J.A., Green, J.L.,
Horner-Devine, C., Kane, M., Krumins, J.A., Kuske, C.R., Morin, J.P., Naeem, S., Ovreas,
L., Reysenbach, A.L., Smith, V.H., and Staley, J.T. (2006) Microbial biogeography:
putting microorganisms on the map. Nature Rev Microbiol 4, 102-112.
Massana, R., Murray, A.E., Preston, C.M., and DeLong, E.F. (1997) Vertical distribution and
phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel.
Appl Environ Microbiol 63, 50-56.
Massana, R., DeLong, E.F., and Pedros-Alio, C. (2000) A few cosmopolitan phenotypes
dominate planktonic archaeal assemblages in widely different oceanic provinces. Appl
Environ Microbiol 66, 1777-1787.
Morris, R.M., Rappé, M.S., Connon, S.A., Vergin, K.L., Siebold, W.A., Carlson, C.A., and
Giovannoni, S.J. (2002) SAR11 clade dominates ocean surface bacterioplankton
communities. Nature 420, 806-810.
Morris, R.M., Vergin, K.L., Cho, J.C., Rappé, M.S., Carlson, C.A., and Giovannoni, S.J.
(2005) Temporal and spatial response of bacterioplankton lineages to annual convective
overturn at the Bermuda Atlantic Time-series Study site. Limnol Oceanogr 50, 1687-1696.
Muyzer, G., de Waal, E.C., and Uitterlinden, A.G. (1993) Profiling of complex microbial
populations by denaturating gradient gel electrophoresis analysis of polymerase chain
reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59, 695-700.
Nicholas, K.B., and Nicholas, H.B.J. (1997) GeneDoc: a tool for editing and annoting
multiple
sequence
alignments.
Distributed
by
the
author.
(www.cris.com/~ketchup/genedoc.shtml).
Pedros-Alio, C. (2006) Marine microbial diversity: can it be determined? Trends Microbiol
14, 257-263.
Percent, S.F., Frischer, M.E., Vescio, P.A., Duffy, E.B., Milano, V., McLellan, M., Stevens,
B.M., Boylen, C.W., and Nierzwicki-Bauer, S.A. (2008) Bacterial community structure of
acid-impacted lakes: What controls diversity? Appl Environ Microbiol 74, 1856-1868.
Pommier, T., Canbäck, B., Riemann, L., Boström, K.H., Simu, K., Lundberg, P., Tunlid, A.,
and Hagström, Å. (2007) Global patterns of diversity and community structure in marine
bacterioplankton. Mol Ecol 16, 867-880.
Reche, I., Pulido-Villena, E., Morales-Baquero, R., and Casamayor, E.O. (2005) Does
ecosystem size determine aquatic bacterial richness? Ecology, 86 1715-1722.
Reche, I., Pulido-Villena, E., Morales-Baquero, R., Casamayor, E.O. (2007) Does ecosystem
size determine aquatic bacterial richness? Reply. Ecology 88: 253-255
Schauer, M., Massana, R., and Pedrós-Alió. C. (2000) Spatial differences in bacterioplankton
composition along the Catalon coast (NW Mediterranean) assessed by molecular
fingerprinting. FEMS Microb Ecol 33, 51-59.
Schauer, M., Balagué, V., Pedros-Alio, C., and Massana, R. (2003). Seasonal changes in the
taxonomic composition of bacterioplankton in a coastal oligotrophic system. Aquat Microb
Ecol 31, 163-174.
Schauer, M., Kamenik, C., and Hahn, M.W. (2005) Ecological differentiation within a
cosmopolitan group of planktonic freshwater bacteria (SOL cluster, Saprospiraceae,
Bacteroidetes). Appl Environ Microbiol 71, 2381-2390.
Sekar, R., Pernthaler, A., Pernthaler, J., Warnecke, F., Posch, T., and Amann, R.I. (2003) An
improved protocol for quantification of freshwater Actinobacteria by fluorescence in sity
hybridization. Appl Environ Microbiol 69, 2928-2935.
Selje, N., Simon, M., and Brinkhoff, T. (2004) A newly discovered Roseobacter cluster in
temperate and polar oceans. Nature 427, 445-448.
Soininen, J., and Heino, J. (2005) Relationships between local population persistence, local
abundance and regional occupancy of species: distribution patterns of diatoms in boreal
streams. J Biogeogr 32, 1971-1978.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular Evolutionary
Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.
Thioulouse, J., Chessel, D., Dolédec, D., and Olivier, J.M. (1997) Ade-4: a mulitvariate
analysis and graphical display software. Stat Comput 7, 75-83.
Van der Gucht, K., Cottenie, K., Muylaert, K., Vloemans, N., Cousin, S., Declerck, S.,
Jeppesen, E., Conde-Porcuna, J.M., Schwenk, K., Zwart, G., Degans, H., Vyverman, W.,
and De Meester, L. (2007) The power of species sorting: Local factors drive bacterial
community composition over a wide range of spatial scales. Proc Nat Acad Sci USA 104,
20404-20409.
Ward, B.B., and O’Mullan, G.D. (2002) Worldwide distribution of Nitrosococcus oceani, a
marine ammonia-oxidizing gamma-proteobacterium, detected by PCR and sequencing of
16S rRNA and amoA genes. Appl Environ Microbiol 68, 4153-4157.
Warnecke, F., Amann, R.I., and Pernthaler, J. (2004) Actinobacterial 16S rRNA genes from
freshwater habitats cluster in four distinct lineages. Environ Microbiol 6, 242-253.
Yannarell, A.C., Triplett, E.W. (2005) Geographic and environmental sources of variation in
lake bacterial community composition. Appl Environ Microbiol 71, 227-239.
Willig, M.R., Kaufman, D.M., and Stevens, R.D. (2003) The latitudinal gradient of
biodiversity: Pattern, process, scale and synthesis. Ann Rev Ecol Evol Syst 34, 273-309.
Zhou, J., Davey, M.E., Figueras, J.B., Rivkina, E., Gilichinsky, D., and Tiedje, J. (1997)
Phylogenetic diversity of a bacterial community determined from Siberian tundra soil
DNA. Microbiol UK, 143, 3913-3919.
Zwart, G., Hiorns, W.D., Methé, B.A., van Agterveld, M.P., Huismans, R., Nold, S.C., Zehr,
J.P., and Laanbroek, H.J. (1998) Nearly identical 16S rRNA sequences recovered from
lakes in North America and Europe indicate the existence of clades of globally distributed
freshwater bacteria. Syst Appl Microbiol 21, 546-556.
Zwart, G., Crump, B.C., Kamst-van Agterveld, M.P., Ferry, H., and Suk-Kyun, H. (2002)
Typical freshwater bacteria: an analysis of available 16S rRNA sequences from plankton
of lakes and rivers. Aquat Microb Ecol 28, 141-155.
Zwart, G., van Hannen, E.J., Kamst-van Agterveld, M.P., Van der Gucht, K., Lindström, E.S.,
Van Wichelen, J., Lauridsen, T., Crump, B.C., Han, S.-K., and Declerck, S. (2003) Rapid
screening for freshwater bacterial groups by using reverse line blot hybridization. Appl
Environ Microbiol 69, 5875-5883.
Tab 1. Main characteristics of the Burkina Faso reservoirs (Ouagadougou, Pouytenga, Dem,
Bamsa, Bagré and Bazega) and of the French lakes (Annecy, Bourget and Geneva) studied in
this work
Location
Max volume
Max depth
(Mm3)
(m)
pH
Temperature
(°C)
Bagré
11°29’N ; 0°32’W
1,700
9.7
7.0
27.0
Bamsa
12°15’N ; 1°06’W
2.0
3.1
6.9
26.3
Bazega
11°44’N ; 1°21’W
11.2
3.2
6.7
28.8
Dem
13°11’N ; 1°09’W
4.0
1.5
7.0
27.0
Ouagadougou
12°23’N ; 1°30’W
1.5
1.6
7.6
30.4
Pouytenga
12°12’N ; 0°25’W
0.6
0.7
7.4
29.4
Annecy
45°52’N ; 6°09’E
1,124
65
7.7-8.4*
5.7-25*
Bourget
45°42’N ; 5°52’E
3,600
145
7.7-8.4
6.2-25.7
Geneva
46°26’N ; 6°33’E
8,900
310
7.7-8.6
6.4-23.7
* Minimum and maximum values
Supplemental Tab 1. Results of the blast analysis (sequence identity ≥98%) performed on
the 12 first OTUs from the figure 4 only distributed in France or in Burkina Faso
Ø: No sequence sharing ≥98% sequence identity
Tropical and
Temperate and cold
subtropical areas
areas
OTUs only found in BF
OTU n°03 (Actinobacteria)
OTU n°04 (Actinobacteria)
OTU n°12 (Actinobacteria)
OTU n°13 (Actinobacteria)
OTU n°14 (Actinobacteria)
OTU n°19 (Actinobacteria)
OTU n°20 (Actinobacteria)
Lake Gatun (Panama)
Tucurui reservoir (Brazil)
Lake Tanganyika
(Tanzania)
Lake Gatun (Panama)
Tucurui reservoir (Brazil)
Lake Gatun (Panama)
Tucurui reservoir (Brazil)
Lake Gatun (Panama)
Tucurui reservoir (Brazil)
Lake Gatun (Panama)
Tucurui reservoir (Brazil)
Lake Taihu (China)
Lake Gatun (Panama)
Lake Taihu (China)
Ø
Ø
Ø
Ø
Ø
Lake Fuchskuhle (Germany)
Lakes Michigan and Adirondack (USA)
Chesapeake Bay (USA)
Bohai Bay (China)
Ø
OTUs only found in France
OTU n°07 (Actinobacteria)
OTU n°10 (Betaproteobacteria)
OTU n°16 (Betaproteobacteria)
OTU n°17 (Chloroflexi)
OTU n°21 (Betaproteobacteria)
Ø
Lake Taihu (China)
Lake Gatun (Panama)
Tucurui reservoir (Brazil)
Ø
Ø
Several lakes from Wisconsin, lakes Adirondack
and Michigan (USA)
Chesapeake and Delaware bays (USA)
Several lakes in Europe (Germany,
Denmark, Sweden)
Nam Co Lake (Tibet)
Circumpolar river (Russia)
Lake Michigan (USA)
Chesapeake and Delaware bays (USA)
Several lakes in Europe (Germany, Austria,
Switzerland, France)
Several lakes in Japan and South Corea
Circumpolar river (Russia)
…
Lake Michigan (USA)
Chesapeake and Delaware bays (USA)
Several lakes in Europe (Germany, Austria,
Switzerland)
Several rivers in China, USA and Alaska
Other lakes (Hawaï, South Corea)
Crater lake (USA, only one sequence)
Lakes Adirondack and Michigan (USA)
Chesapeake and Delaware bays (USA)
Several lakes and rivers in Europe (Germany,
Switzerland, Denmark)
Polluted soils (France, Japan)
Glacier (Antartica, Switzerland), Snow (Tibet)
Soil glacier (Antartica, Canada, India)
Altitude soil (Andes, France, Switzerland)
Sludge (Japan, New Zealand)
…
Fig. 1 Comparative structure of the bacterial communities in six reservoirs located in Burkina
Faso, and in three sub-alpine lakes located in France
Ouaga = Ouagadougou; Ann = Annecy; Bou = Bourget; Gen = Geneva
Sum = Summer; Spr = Spring; Win = Winter
100%
80%
Others
70%
Bacteroidetes
60%
Cyanobacteria
50%
Gammaproteobacteria
40%
Betaproteobacteria
30%
Alphaproteobacteria
20%
Actinobacteria
10%
ua
O
ga
ua 1
Po ga
uy 2
te
ng
a
D
em
Ba
m
s
Ba a
g
Ba ré
ze
An ga
n
An Sp
n r
S
An um
n
W
Bo in
u
Bo Sp
u r
S
Bo um
u
W
G in
en
G Sp
en r
S
G um
en
W
in
0%
O
Proportions
90%
Fig. 2 Comparative analysis of the bacterial diversity in six reservoirs located in Burkina Faso
and in three French sub-alpine lakes.
A: Comparison of the OTU (sequence identity ≥ 98%) richness (Chao 1) and diversity
(Shannon index) in all bacterial communities.
White histogram: Chao 1; Black triangle: Shannon index
Ouaga = Ouagadougou; Ann = Annecy; Bou = Bourget; Gen = Geneva; Sum = Summer; Spr
= Spring; Win = Winter
B: Comparison of the rarefaction curves obtained from bacterial communities of sub-alpine
350
3,5
300
3
250
2,5
200
2
150
1,5
100
1
50
0,5
0
O
ua
ga
1
O
ua
g
Po a 2
uy
te
ng
a
D
em
Ba
m
sa
Ba
gr
é
Ba
z
An ega
ne
c
An y S
ne p
cy
S
An
ne u
c
Bo y W
ur
ge
Bo t S
p
ur
ge
Bo t S
u
ur
ge
t
G
en W
ev
a
G
en Sp
ev
a
G
Su
en
ev
a
W
0
B
45
40
30
25
20
15
10
5
Number of sequences
95
10
0
10
5
11
0
11
5
12
0
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
5
10
0
0
Number of OTUs
35
Shannon values
Chao 1 values
lakes (gray curves) and of Burkina Faso reservoirs (dark curves).
Fig. 3 Distribution of the proportions of OTUs, of sequences and of the ratio OTU/sequence
in the different bacterial phyla retrieved in this study
80
70
Proportions (%)
60
50
% OTUs (±95% confidence limit)
40
Nb OTUs*100/ Nb sequences
30
20
10
G
Be
t
ph
ap
r
ot
eo
ba
ap
ct
r
o
er
am
te
ia
o
m
ba
ap
ct
ro
er
te
ia
ob
ac
Ba
te
ria
ct
er
oi
Ac
de
tin
te
ob
s
Ac
ac
tin
te
ria
ob
ac
I
te
C
ria
ya
IV
no
ba
ct
er
ia
0
Al
% sequences (±95% confidence limit)
Fig. 4 Phylogenetic relationships between the 21 dominant OTUs in bacterial communities
from three alpine lakes and six reservoirs located in Burkina Faso
SEE PDF FILE
Fig. 5 Frequency distribution (±95% confidence limits) and geographical origin (Black:
France; White: Burkina Faso; Striped: France + Burkina Faso) of the 9 OTUs containing >50
% of all the sequences obtained in this study and of 21 OTUs containing >70% of all the
sequences obtained in this study
12
Frequency (%)
10
8
6
4
2
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
Fig. 6 Mean local abundance of each OTU as a function of the range of occupation
Mean local abundance
100
10
1
0,1
0
1
2
3
4
5
6
Number of locations
7
8
9
10
Fig. 7 Correspondence analysis performed on the distribution of abundances of all OTUs in
the different sampling sites
(Spr=Spring; Sum=Summer; Win=Winter)
Bazega
Pouytenga
Bourget Win
Annecy Spr
Annecy Sum
Bourget
Annecy Win
Spr
Geneva Sum
Geneva Spr
Bourget Sum
Geneva Win
Bagré
Ouaga 2
Axis 1 (19%)
Ouaga 1
Axis 2 (11%)
Bamsa
Dem