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Bacterial diversity in the waterholes of the Kruger National Park: an eDNA metabarcoding approach
Journal: Genome
Manuscript ID gen-2018-0064.R1
Manuscript Type: Article
Date Submitted by the Author: 08-Jul-2018
Complete List of Authors: Farrell, Maxwell; McGill University, Department of BiologyGovender, Danny; Scientific Services, Kruger National Park, SANParks; University of Pretoria, Department of Paraclinical Sciences, Faculty of Veterinary ScienceHajibabaei, Mehrdad; University of GuelphVAN DER BANK, Michelle; University of Johannesburg, African Centre for DNA BarcodingDavies, T; University of British Columbia, Botany, Forestry & Conservation Sciences; University of Johannesburg, African Centre for DNA Barcoding
Keyword: microbiome, conservation, biomonitoring, 16S, watering holes
Is the invited manuscript for consideration in a Special
Issue? :7th International Barcode of Life
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Bacterial diversity in the waterholes1
of the Kruger National Park:2
an eDNA metabarcoding approach3
Maxwell J. Farrell1∗, D. Govender2,3,M. Hajibabaei4, M. van der Bank5, T. J. Davies5,6
1Department of Biology, McGill University2Scientific Services, Kruger National Park, SANParks
3Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria4Integrative Biology & Biodiversity Institute of Ontario, University of Guelph
5African Centre for DNA Barcoding, University of Johannesburg6Botany, Forest & Conservation Sciences, University of British Columbia
∗To whom correspondence should be addressed; e-mail: [email protected]
4
September 21, 20185
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Abstract6
Bacteria are essential components of natural environments. They contribute to ecosystem functioning7
through roles as mutualists and pathogens for larger species, and as key components of food webs8
and nutrient cycles. Bacterial communities respond to environmental disturbances, and the tracking9
of these communities across space and time may serve as indicators of ecosystem health in areas10
of conservation concern. Recent advances in DNA sequencing of environmental samples allow for11
rapid and culture-free characterization of bacterial communities. Here we conduct the first metabar-12
coding survey of bacterial diversity in the waterholes of the Kruger National Park, South Africa.13
We show that eDNA can be amplified from waterholes and find strongly structured microbial com-14
munities, likely reflecting local abiotic conditions, animal ecology, and anthropogenic disturbance.15
Over timescales from days to weeks we find increased turnover in community composition, indicating16
bacteria may represent host-associated taxa of large vertebrates visiting the waterholes. Through taxo-17
nomic annotation we also identify pathogenic taxa, demonstrating the utility of eDNA metabarcoding18
for surveillance of infectious diseases. These samples serve as a baseline survey of bacterial diversity19
in the Kruger, and in the future, spatially distinct microbial communities may be used as markers of20
ecosystem disturbance, or biotic homogenization across the park.21
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Introduction22
Traditional programs that monitor for early signs of ecosystem degradation require baseline data on the23
distributions and ecology of species in an ecosystem. DNA barcoding uses differences in conserved24
regions of genomes to classify sequences as belonging to particular taxonomic units, regardless of25
whether or not they have been described formally by taxonomists (Hebert et al. 2003; Blaxter et al.26
2005; Ratnasingham and Hebert 2013). DNA barcoding is thus a particularly powerful tool for explor-27
ing microbial diversity, where there are many undescribed taxa that cannot be cultured using traditional28
methods (Rappe and Giovannoni 2003). Molecular barcoding coupled with recent advances in genetic29
sequencing have allowed for unprecedented exploration of microbial communities and the ability to30
characterize organisms of interest from environmental samples with great sensitivity (Shokralla et al.31
2012). In particular, sequencing of cellular and extracellular DNA that can be extracted from envi-32
ronmental samples, collectively known as environmental DNA (eDNA) (Taberlet et al. 2012), is an33
emerging approach for exploring diversity in aquatic ecosystems (Rees et al. 2014; Lodge et al. 2012).34
Microbial diversity in freshwater systems responds to environmental conditions (Lozupone and35
Knight 2007), and perturbations (Zeglin 2015) including multiple anthropogenic impacts such as ur-36
banization (Fisher et al. 2015) and pollution (Bouskill et al. 2010). In addition to acting as indicators of37
ecosystem health, changes in microbial diversity may be important in themselves. Bacteria are essen-38
tial components of ecosystems and play important roles in food webs, nutrient recycling, disease, and39
as important mutualists for larger multicellular species. Bacteria are thus integral to maintaining the40
natural balance of ecosystems and shifts in taxonomic composition due to environmental change may41
severely impact connectivity, functioning (Laforest-Lapointe et al. 2017; Delgado-Baquerizo et al.42
2016) and increase exposure to pathogens (Cabral 2010). However, in most ecosystems of conserva-43
tion priority, microbial diversity is poorly described.44
Surface waters are a vital resource for savannah ecosystems (Redfern et al. 2005; Owen-Smith45
1996), but frequent use by a large variety of species means they can also be a source of cross-species46
infection and spread of harmful pathogens (Bengis and Erasmus 1988). These ecosystems provide47
an ideal context for refining eDNA metabarcoding approaches as they act as sources and sinks of48
microbial species for larger animals, however baseline information about microbial diversity in these49
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systems is lacking. Here we conduct a survey of bacterial diversity among watering holes of Kruger50
National Park, South Africa (KNP) through spatio-temporal sampling and sequencing of the V3-V451
region of 16S genes present in water. Water can be scarce in the KNP throughout the dry season and52
periods of drought (Redfern et al. 2005), and the park has a long history of water provisioning that53
included the construction of a series of more than 300 artificial waterholes beginning in the 1930s54
(Smit et al. 2007). These waterholes were intended to increase game numbers by stabilizing water55
availability year-round and are frequently visited by a diversity of birds and mammals (Smit and56
Grant 2009). However, they have proven to alter the distributions of wildlife, which in turn have57
negative impacts on vegetation dynamics and the park-wide ecosystem (Smit et al. 2007; Smit and58
Grant 2009). As a result, a number of artificial waterpoints have been closed since 1994 as the park59
began reverting to a more natural cycle of water availability (Smit et al. 2007; Van Wyk 2011). A60
subset of the waterholes still open are small concrete troughs which are well mixed, largely mud and61
silt-free, and experience limited inflow from nearby surface waters. This means that eDNA samples62
will largely represent microbes in sourcewater and those dispersed by air and animals, allowing us to63
capture snapshots of local bacterial diversity across the park.64
This study provides the first survey of bacterial diversity in the waterholes of the KNP, and is65
among the first studies using next-generation sequencing to describe aquatic microbial diversity in66
Africa (see Jordaan and Bezuidenhout (2016; 2013); Tekere, M., Prinsloo, A., Olivier, J., Jonker, N.,67
Venter, S. (2012); Mwirichia, R., Cousin, S., Muigai, A. W., Boga, H. I., Stackebrandt, E. (2011);68
Tekere, M., Lotter, A., Olivier, J., Jonker, N., Venter, S. (2011)). Here we explore bacterial diversity69
across the southern half of the park and describe variation across, space, time, sample volume, and70
abiotic influences.71
Methods72
Study Site73
Waterholes were sampled in June and July of 2015 in the Kruger National Park, South Africa (KNP), a74
large protected savannah ecosystem and a global diversity hotspot (Lahaye et al. 2008). Sampling was75
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conducted during the dry season when natural sources of surface water are largely dry and watering76
hole visitation rates by medium and large vertebrates are highest. The park is divided into twenty-two77
ranger sections, which range in size from roughly 520 to 1,170 square kilometers. Across the southern78
half of the reserve below the Olifants river, ten concrete bottom artificial waterholes were selected79
from five of these sections (Table 1, Fig. 1). The waterholes varied in shape with some mimicking the80
contours of natural pans, making volume estimations difficult. However, the generic design included81
longer and shorter axes, with comparable dimensions across waterholes. Each waterhole is equipped82
with a ball-valve, which regulates water levels and re-fills the trough from nearby reservoirs when83
water levels drop. Water is sourced predominantly from groundwater via boreholes, but three sites use84
pipeline troughs filled with diverted river water.85
Water Sampling and Processing86
At each site, samples were taken once per week for three weeks. For one site (NWA), samples were87
also taken every day for five consecutive days. Sampling consisted of two 1L water samples collected88
in autoclaved, UV sterilized glass jars from opposite ends of the waterhole, approximately one foot89
from the nearest edge. These two within-waterhole samples (A/B) were taken along the waterhole’s90
longest axis that maximized the distance and upwind-downwind gradient between them, if a strong91
wind was present. Water samples were placed on icepacks in a cooler and kept between 4-8◦C until92
returning to the laboratory, where they were placed in the fridge.93
Water quality parameters were taken during each sampling period using a YSI 650QS multi-94
parameter sonde. Temperature (◦C), conductivity (mS/cm), dissolved oxygen (in mg/L and % sat-95
uration), and pH were recorded. Three measurements were taken along the same axis that the A/B96
water samples were drawn, and then averaged to measure quality per sample-time.97
In the lab, the outside of water sample collection bottles were washed with ELIMINase (Decon98
Labs) and rinsed with deionized (DI) water to limit contamination. For each A/B sample, 150 mL of99
water was sub-sampled and filtered through gamma-irradiated 0.2 µm Supor hydrophilic polyether-100
sulfone membranes (Pall no. 66234). The filtration apparatus consisted of three 300 mL Advantec101
polysulfone 47mm filter funnels fitted to a Pall vacuum manifold with vacuum pressure maintained by102
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a Pall filtration vacuum/pressure pump (model no. 13158). After filtration, filters were stored in sterile103
15 mL Falcon tubes and placed in a freezer at -60◦C. On one sampling date for six sites, additional104
volumes of 50 ml and 15 ml were filtered from each 1L sample to asses the impact of sample volume105
filtered. Twice throughout sampling, BLANK samples were generated by filtering 1L of deionized106
water used in the laboratory.107
Prior to and between filtrations, all funnel components and tweezers used to manipulate the filters108
were sterilized by soaking with 10% bleach for 10 minutes, rinsing with DI water, washing with109
ELIMINase, rinsing with DI water, and subsequent exposure to UV radiation for a minimum of 30110
minutes. Gloves were worn at all times and changed between samples to minimize cross-sample111
contamination. To avoid sample freezing and bacterial growth in collection jars, all samples were112
processed within 12 hours of collection. Frozen 50 mL unfiltered voucher samples were kept and113
placed at -80◦C at the University of Johannesburg’s African Centre for DNA Barcoding for long term114
storage.115
DNA Extraction, Amplification, and Sequencing116
DNA was isolated from filter papers using MO BIO PowerWater DNA Isolation Kits. Universal bac-117
terial primer sets designed by Sundquist et al. (2007) (V3-F: 5’ACTCCTACGGGAGGCAGCAG 3’;118
V4-R: 5’GGACTACARGGTATCTAAT 3’) tagged with an Illumina adapter sequence were used to119
amplify the V3-V4 hypervariable region of the 16S ribosomal RNA gene through polymerase chain120
reaction (PCR). The PCR used a standard mix of 17.8µL molecular grade water, 2.5µL 10 reaction121
buffer (200mM Tris HCl, 500mM KCl, pH 8.4), 1µL MgCl2 (50mM), 0.5µL dNTP (10mM), 0.5µL122
forward primer (10mM), 0.5µL reverse primer (10mM), 0.2µL Platinum Taq DNA polymerase (Invit-123
rogen), and 2µL DNA as template for a total volume of 25µL. PCRs underwent the following cycler124
conditions: initial 94◦C for 5 minutes, then 30 cycles of 94◦C for 40 seconds, 46◦C for 1 minute, 72◦C125
for 30 seconds, and a final temperature of 72◦C for 2 minutes. Amplification success was confirmed126
through gel electrophoresis, using a 1.5% agarose gel. PCR products were purified using MinElute127
PCR purification kit (Qiagen), and quantified through flurometry using a Quant-iT PicoGreen dsDNA128
assay kit (Invitrogen). Samples were normalized, then multiplexed with the Nextera XT Index kit (96129
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indexes) (Illumina) and sequenced on an Illumina MiSeq flowcell using a V2 sequencing chemistry130
kit (2 x 250) making up approximately 1/8th of the run.131
Sequence Processing, Taxonomy Assignment, and Phylogeny Construction132
Across all samples, we generated a total of 2,164,262 Illumina reads. Primer sequences were removed133
using the trim.seqs function in mothur (Schloss et al. 2009). Reads were then processed in R (version134
3.4.3) (R Development Core Team 2008) using the package dada2 version 1.6.0 (Callahan et al. 2016)135
following a modified version of the DADA2 Bioconductor workflow (Callahan et al. 2017) and online136
tutorials v1.6 and workflow for big data v1.4 (benjjneb.github.io/dada2/tutorial.html). Reads were137
filtered by quality, removing sequences with maximum expected error (maxEE) greater than 6 for both138
forward and reverse reads, and reads with any base pair having Q of 6 or lower. Reads were truncated139
to a length of 230bp and 220bp for forward and reverse reads respectively, consistent with dropoffs in140
quality profiles, and reads shorter than this were removed. Since the samples were sequenced across141
four different runs, subsequent steps of learning error rates, dereplication, denoising and Amplicon142
Sequence Variant (ASV) calling (Callahan et al. 2017) using pooled samples, and merging of paired143
reads were performed separately for each run. Tables of ASV sequences per sample within each run144
were then combined and chimera detection using all pooled samples was performed (see SM Table S2145
for the number of reads retained across each step). In total 1,184,831 reads were retained, representing146
3533 ASVs.147
Taxonomy assignment from Kingdom to Genus was performed using the RDP classifier and148
SILVA nr v128 reference database (Quast et al. 2013) formatted for DADA2 (benjjneb.github.io/dada2/training.html),149
using the assignTaxonomy function (Fig. 5). ASVs assigned as Archaea, Eukarya, Chloroplast, or Mi-150
tochondria were removed. Species level assignments were added by exact sequence matching using151
the addSpecies function. ASV sequences were aligned with the pynast algorithm via align seqs.py in152
QIIME (Caporaso et al. 2010) and sequences with poor alignment automatically removed. A phylo-153
genetic tree was constructed using the GTRCAT model in FastTree version 2.1.3 (Price et al. 2010)154
after filtering nucleotides with greater than 90% gap fraction and removing the 5% highest entropy155
positions with filter alignment.py in QIIME (Caporaso et al. 2010). This resulted in a phylogenetic156
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tree of 3393 ASVs which were used in subsequent community analyses.157
Community Analyses158
The ASV sequence table was merged with the phylogeny and sample metadata using the R pacakge159
phyloseq version 1.22.3 (McMurdie and Holmes 2013). Negative controls (BLANK samples) used to160
investigate contamination during sample filtration contained 43 ASVs collectively (with 9 ASVs found161
in both samples). The sequence reads in each filtration blank were both dominated by the same ASV162
(53% and 86% respectively), however none of the 43 ASVs identified in the blanks were identified in163
any of the other samples. These control samples were removed prior to community analyses.164
A subset of core samples was created by removing the first four daily NWA samples and samples165
of differential volume (S & XS samples), resulting in 54 samples of 150 mL each (Table 2). An ASV166
accumulation curve for core samples was generated using the specaccum function in the R package167
vegan version 2.4.6 (Oksanen et al. 2018) using the “exact” method, and extrapolated to total ASV168
richness using the Chao and Bootstrap methods in vegan’s specpool function. Alpha diversity was169
calculated for the core samples as observed ASV richness and Shannon diversity using the phyloseq170
package. Additive partitioning of Shannon diversity across core samples was investigated using the171
adipart function in vegan (Table 3).172
Taxonomic composition was assessed by merging core samples at each site, and plotting rela-173
tive abundances of reads for the most common taxa at levels of phylum, class, and order (Fig. 6).174
Temporal variation in taxonomic composition across core samples was assessed by merging A and B175
samples and plotting relative abundances of reads for sites with two or more weekly samples, for the176
levels of phylum (Fig. 7), and class (Fig. S6). To further investigate fine-scale temporal variation in177
taxonomic composition (phylum, class, and order), relative abundance of reads were plotted across178
the daily samples at site NWA (Fig. 8). We also explored temporal turnover among samples with179
Sorensen’s dissimilarity calculated using the beta.pair function from the betapart package (Baselga180
et al. 2018) (Fig. S9) and significant differences among daily and weekly samples was assessed using181
permutationsl multivariate analysis of variance (adonis in vegan) with 999 permutations each.182
Community composition across sites in the core samples was described with nonmetric multidi-183
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mentional scaling (NMDS) ordinations on relative ASV abundances per sample using the Bray-Curtis184
dissimilarity, and the abundance weighted Unifrac dissimilarity (Figs. 9, S10 & S11). Statistically,185
associations between dissimilarities and both water quality properties and common taxonomic groups186
were assessed using the envfit function in vegan for bacterial classes (Fig. 10) and orders (Fig. S12).187
Phylogenetic community structure across core samples was calculated using standardized effect188
sizes of mean pairwise phylogenetic distances (MPD) and mean nearest taxon distances (MNTD) in189
the R package picante version 1.6.2 (Kembel et al. 2010) using the abundance weighted “richness”190
null model and 999 randomizations in the ses.mpd and ses.mntd functions (Fig. S13). For a given sam-191
ple, MPD calculates the mean phylogenetic distance among each pair of taxa present, while MNTD192
calculates the mean phylogenetic distance from each taxa to its closest relative. These raw metrics193
give an estimate of how closely related community members are to each other, and are then compared194
to randomized communities to determine whether the observed metrics are different than what would195
expected if communities were assembled at random from taxa pooled across all samples.196
To assess the effect of differential sample volumes, S (50 mL) and XS (15 mL) samples were subset197
along with their corresponding full volume samples (150 mL). Alpha diversity, calculated as observed198
ASV richness and Shannon diversity were calculated as described above (Fig. S14). Variation in199
taxonomic composition was investigated by comparing relative read abundances of bacterial phyla in200
A/B samples across sites and differential volumes (Fig. S15).201
Raw reads with primers removed are available via the NCBI Sequence Read Archive BioProject202
PRJNA490450 (accession numbers SRR7822814 to SRR7822901). The ASV table, taxonomic as-203
signments, phylogenetic tree, sample metadata, and scripts necessary to reproduce the results can be204
found at 10.6084/m9.figshare.7119668.205
Results206
Our sampling design aimed to sequence a core set of samples with all ten sites being sampled once207
per week for three weeks. Due to logistic constraints of sample storage and extremely low water208
levels from drawdown by animals, we were only able to process 27 of the planned 30 weekly samples209
(Table 2). In addition to this core sampling, we sequenced differential volumes for six samples, and210
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an additional four daily samples from Nwaswitshaka (site NWA). A/B samples were taken at each211
site-time, resulting in a total of 88 sequenced samples, including the two filtration blanks. Across all212
88 samples, we identified a total of 3393 ASVs. Roughly 15% of ASVs (n=524) were represented213
by a single read, together comprising fewer than 0.05% of all reads. The DADA2 approach infers the214
biological sequences in the sample prior to the introduction of amplification and sequencing errors,215
and can distinguish sequence variants differing by as little as one nucleotide. As such, we included all216
ASVs, including those represented by single reads, in subsequent analyses of biodiversity.217
The ASV accumulation curve generated for the core sample set (2603 ASVs) does not appear218
to saturate (Fig. 2). Estimates of total richness using the Chao estimator predicts 6164 ASVs (+/-219
262 SE) among the core samples, indicating we may be capturing less than half of the total bacterial220
diversity present among our sites. However, estimates of total diversity using the Bootstrap method221
were more conservative, with 3260 (+/- 146 SE) estimated ASVs. ASV diversity varied across sites222
(Figs. 3, Fig. 4), but the largest turnover (β diversity) was observed among park sections (Table223
3). Variation among A/B samples contributed very small amounts to β diversity, indicating that at a224
particular time, microbial diversity within each waterhole was fairly well mixed.225
In terms of taxonomic composition, 99.2% of ASVs were assigned to a known phylum, with the226
proportion of assignments decreasing at lower taxonomic levels (Fig. 5). The majority of bacterial227
ASVs were classified as Proteobacteria (∼ 59%), followed by Bacteroidetes (∼ 14%), Firmicutes (∼228
9%), Actinobacteria (∼ 6%), and Verrumicrobia (∼ 2%). For bacterial classes, ASVs were largely229
classified as Betaproteobacteria (∼ 34%), Alphaproteobacteria (∼ 11%), Gammaproteobacteria (∼230
10%), and Sphingobacteriia (∼ 6%). Among core samples, relative abundances of phyla, classes,231
and orders varied across sites (Figs. 6, S4, S5). Across weeks, relative abundances of phyla varied232
within each site (Fig. 7), with some sites displaying more stability (IMB & HOY) compared to others233
(NYA & NGO). Patterns among bacterial classes (Fig. S6) largely reflected variation among phyla,234
though one site (HOY) displayed much more variation in relative abundances among classes, reflecting235
substantial turnover within Proteobacteria. Comparing weekly turnover with the five daily samples236
taken at Nwaswitshaka (NWA) (Fig. 8), taxonomic composition appeared more stable across days than237
weeks. Using hierarchical clustering of Sorensen’s dissimilarity, we find that samples taken within238
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a single week cluster together (Fig. S9). Permutational multivariate ANOVAs on these distances239
revealed a significant difference in beta diversity among weekly samples (NWA 2,7,8; p = 0.02), with240
49% of the variance explained by sample date, but no significant difference among the additional daily241
samples (NWA 3,4,5,6; p = 0.54), with 14% of the variance explained by sample date.242
Community composition visualized through NMDS ordinations reflected results from the additive243
partitioning of diversity, with core samples clustering by site (Fig. 9) and section (Fig. S10) for both244
Bray-Curtis and abundance weighted UniFrac dissimilarities. Interestingly, waterholes filled by water245
from pipeline troughs (NGO, NYA, WIT) grouped together (Fig. S10), although these three sites are246
situated on a different geological type than sites fed by boreholes, making us unable to differentiate247
the effects of each factor (Fig. S11). Bacterial community composition was significantly structured248
by conductivity and pH for both Bray-Curtis and UniFrac dissimilarities, and dissolved oxygen also249
had an influence on UniFrac dissimilarity (Figs. 10 & S12).The dissimilarity of high conductivity250
sites (particularly HOY & IMB in the Kingfisherspruit section) was associated with high abundances251
of Clostridia, Gammaproteobacteria, and Bacteroidia, while sites with high pH and dissolved oxygen252
were positively related to the abundances of Actinobacteria and Alphaproteobacteria (Fig. 10).253
Reflecting the NMDS structure of the abundance weighted UniFrac dissimilarities, MNTD, which254
is most sensitive to phylogenetic structure towards the tips of the tree (Mazel et al. 2015), indicated255
strong phylogenetic clustering within the majority of samples (Fig. S13). The strength of clustering256
was weaker for MPD, which is more sensitive to phylogenetic structure deeper in the tree (Mazel et al.257
2015).258
We did not find any clear decrease in alpha diversity with smaller sample volumes (Fig. S14),259
and one of the 15 mL samples returned the largest richness of ASVs, though the median value for260
15 mL samples was lower and had a larger interquartile range than the 50 mL and 150 mL samples.261
The major phyla detected within samples was also relatively consistent, with most groups represented262
across different sample volumes, though not always in the same proportions (Fig. S15).263
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Discussion264
Biological monitoring is an essential aspect of conservation for tracking contemporary changes in265
ecosystems as well as providing a historical baseline for making management decisions. The Kruger266
National Park, established in 1898, has a long history of management practices revolving around267
maintenance of large mammals (Venter et al. 2008). While bacterial diversity has been explored for268
important infectious agents in the system (Michel et al. 2007; Bengis and Erasmus 1988; Smith et al.269
2000), recent advances in next generation sequencing methods now allow for the rapid and culture-free270
description of bacterial diversity throughout the park.271
Here we present the first description of bacterial diversity in the waterholes of the Kruger Na-272
tional Park. In total we identified over 3000 unique taxa (referred to as amplicon sequence variants, or273
ASVs), only about half of which could be assigned to a previously described genus. The relative domi-274
nance of bacterial phyla was consistent with bacterial surveys of the Vaal River in central South Africa275
(Jordaan and Bezuidenhout 2016). However, bacterial diversity was strongly structured across space,276
with the largest turnover in diversity occurring among park sections. This is not surprising consider-277
ing the distances from site to site range from 3km to 115km and represent a gradient in large animal278
density, rainfall, vegetation, and major subsurface geology (Chirima et al. 2012; Van Wilgen et al.279
2000; Smit and Grant 2009; Smit et al. 2013). Samples also clustered by site, displaying substantial280
variation in taxonomic composition across sites. This variation was associated with physico-chemical281
properties of the water, with conductivity and pH being important explanatory variables. In addition282
to water quality, variability in taxonomic composition is likely influenced by the origin of the water283
used to fill each waterhole, differences in the surrounding soil and vegetation types, and the particular284
species and populations of animals using the waterholes.285
We assessed daily turnover in composition at Nwaswitshaka, which appeared to be more stable286
over this shorter timescale when compared to turnover across weeks. However, Nwaswitshaka was287
less variable across weeks than other sites, indicating that daily variation in bacterial communities288
could be greater in other locations. Important water quality variables (conductivity and pH) were289
largely consistent across weeks (Table S1), suggesting that the observed temporal heterogeneity may290
be driven by differences in external factors influencing bacterial input and removal from the system,291
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such as variation in animal visitation throughout the sampling period. Between sampling events,292
water levels would sometimes drop substantially, indicating major drawdown by animals and likely293
removing bacteria deposited by animals visiting earlier in the week. Large mammal communities294
vary across the sampled regions of the park (Chirima et al. 2012), which may contribute to observed295
spatial variation in bacterial communities. However, different species also differ in their dependence296
on water, which is reflected in their rates of visitation to water points (Redfern et al. 2005). Variation297
in samples taken across subsequent weeks may therefore reflect different components of local animal298
communities, each with their unique host-associated bacterial taxa (Ley et al. 2008). By pairing299
bacterial composition with animal visitation prior to sampling (either through direct observation, or300
presence of genetic material), it may be feasible to build statistical probabilities of associations using301
co-occurrences of microbial and animal signatures.302
Across samples, patterns of phylogenetic clustering were consistent with observed taxonomic vari-303
ation. Multiple phyla were present in all samples, consistent with an even representation of deep304
bacterial lineages. However, turnover at lower taxonomic levels shown by significantly low mean305
nearest taxon distances indicate that there are distinct subsets of closely related taxa present at each306
site. This structuring may reflect filtering of bacterial communities by local environmental conditions,307
or the deposition of microbes by particular animal populations or individuals. Many vertebrate species308
have expansive home ranges, but during the dry season drought-intolerant animals will restrict their309
movement so as to stay close to permanent water bodies (Redfern et al. 2005). Thus the maintenance310
of major bacterial taxa may reflect both free-living environmental bacteria, and the core microbiome311
of water-dependent species. By taking repeat temporal samples, it may be possible to build associ-312
ation networks between bacterial taxa and host species, or even their local populations, solely from313
environmental DNA.314
Through examination of taxonomic assignments we identified taxa belonging to genera that in-315
clude important pathogens (Arcobacter, Bacillus, Burkholderia, Coxiella, Legionella, Neisseria, Pas-316
teurella, Rickettsia, and Yersinia). While many of these genera include both pathogenic as well as be-317
nign species found in environmental samples, some of these genera are comprised solely of pathogenic318
species. For example, the genus Coxiella is represented by one species, Coxiella burnetii, the causative319
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agent of Q fever, which has previously been documented as causing disease in the park (Van Heerden320
et al. 1995). Additionally, taxa in the order Chlamydiales are all obligate intracellular pathogens of321
eukaryotes (Ball et al. 2015), and taxa in the genus Neisseria colonize mucosal surfaces of animals,322
some of which are pathogenic in humans (Liu et al. 2015). Interestingly, we also identified sequences323
classified as Streptococcus urinalis, a recently described species linked to urinary tract infections in324
humans (Peltroche-Llacsahuanga et al. 2012). While hypervariable regions of the 16S gene may not325
be the optimal genomic regions for detecting the presence of particular pathogenic species or strains,326
our findings indicate that broad scale surveys of microbial diversity may be useful in determining the327
presence of potential pathogens across vastly divergent groups of bacteria. This can in turn guide more328
targeted sampling of both pathogenic and commensal bacteria across the park.329
In addition to the presence of pathogens, surveys of bacterial diversity may be used to detect330
anthropogenic influences in the park. For example, we found that bacterial diversity was quite different331
for the two sites sampled in the Kingfisherspruit section (Hoyo Hoyo and Imbali). These sites were332
dominated by an ASV assigned to the genus Arcobacter, but which did not exactly match any sequence333
in the SILVA reference database. Three of the five described members of this genus are known to334
be pathogenic (Fera et al. 2004) and include A. butzleri, which can cause severe diarrhea (Lerner335
et al. 1994) and was detected in two of the weekly samples at Hoyo Hoyo. The two waterholes in336
Kingfisherspruit are fed in part with greywater that is passed through reed beds. Greywater is untreated337
household wastewater that typically has not been contaminated by toilet waste and is often used as338
year-round sources of water, especially in water scarce areas (Nganga et al. 2012). Compared to source339
water, kitchen and laundry greywater can have elevated conductivity (Nganga et al. 2012), which may340
explain the high conductivity of water at these sites, and present a strong selective environment driving341
their unique bacterial communities.342
We did not have sufficient sampling of sites to explore all possible drivers of differences in bacte-343
rial communities. Nonetheless, some features differed obviously among sites. For example, Witpens344
is a heavily vulture-dominated site. Bathing by vultures likely results in large influxes of nutrients345
such as blood, and vulture feces has been found to alter soil bacterial communities through elevated346
nitrogen and decreased pH (Ganz et al. 2012). Visually, water from Witpens was bright green, indi-347
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cating high abundance of photosynthetic species and consistent with the large variations observed in348
dissolved oxygen (Table S1). However, we found no evidence of elevated abundances of Microcystis349
or other Cyanobacteria, though samples from Witpens strongly clustered together and had relatively350
high abundances of Rhizobiales and Rhodocylales, both of which include species known to fix nitro-351
gen (Carvalho et al. 2010; Loy et al. 2005).352
Witpens, along with Nyamarhi and Ngotso North are filled by pipeline troughs that divert river353
water to waterholes many kilometers away. While pipeline troughs are likely to reflect a subsample of354
the diversity found in river water, the acts of pipeline transport and storage themselves may have strong355
filtering effects on bacterial communities. Our results indicate that waterholes represent locally unique356
bacterial communities, thus the practice of diverting river water to waterholes kilometers away may357
homogenize microbial diversity across the landscape, ultimately disrupting local communities. The358
consequences of such shifts in community structure are difficult to assess without comparing pipeline359
troughs with their source waters, but the diversion of river water may have unintended impacts on360
microbial diversity. For example, genes conferring antimicrobial resistance have been shown to spread361
from river water to impala in the Kruger National Park (Mariano et al. 2009).362
Here we show that eDNA can be amplified from waterholes in the Kruger National Park, and find363
strongly structured microbial communities, likely reflecting local abiotic conditions, animal ecology,364
and anthropogenic disturbance. We suggest that disruption of spatially distinct microbial communi-365
ties may be used as a marker of ecosystem disturbance, or biotic homogenization across the park.366
We find that for artificial waterholes, bacterial diversity is surprisingly insensitive to sample volume,367
with even small volumes useful for capturing major components of bacterial communities, though368
larger volumes are necessary to detect rare taxa. Replicating this study across different seasons, and369
expanding sampling to include natural waterpoints will provide improved understanding of the roles370
micro-organisms play in ecosystem stability and resilience, and offer an effective method for mon-371
itoring of shifting species interactions in the face of environmental change. Just as studies of the372
microbiome have revolutionized our understanding of human health, metagenomic analysis of envi-373
ronmental DNA have the potential to revolutionize our understanding of ecosystem health. Tracking374
of bacterial communities can provide a template for monitoring ecosystem disturbance through their375
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response to biological contaminants, documenting the spread of invasive species or infectious organ-376
isms, and better understanding the impacts ecological disturbances have on the composition of native377
communities.378
Acknowledgements379
This work would not have been possible without the logistical support of the African Centre for DNA380
Barcoding and Purvance Shikwambana, molecular work by Shannon Eagle, encouragement and sup-381
port from Lea Berrang-Ford, and the incredible knowledge and protection provided by Kruger game382
guards Thomas, Velly, and Martin. We also thank Steven Kembel, his lab, Alexis Carteron, and383
Benjamin Callahan for constructive feedback on bioinformatic analyses. Special thanks are due to384
B.S. Bezeng for volunteering his friendship, dedication, and long days in the field. This work was385
performed with the support and permission of SANParks (project DAVIJ1256). MJF was supported386
by a Vanier NSERC CGS and the CIHR Systems Biology Training Program, with project funding387
supported by the Quebec Centre for Biodiversity Science, the McGill Biology Department, and an388
NSERC Discovery Grant awarded to TJD.389
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Figures & Tables536
Section Site Type GeologyTshokwane (TSH) Nhlanguleni (NHL) Borehole GraniteSkukuza (SKZ) Nwaswitshaka (NWA) Borehole GraniteSkukuza (SKZ) De LaPorte (DLP) Borehole GraniteSkukuza (SKZ) Kwaggas Pan (KWA) Borehole GraniteSatara (SAT) Girivana (GIR) Borehole GraniteSaTara (SAT) Witpens (WIT) Pipeline trough BasaltKingfisherpruit (KFI) Imbali (IMB) Borehole GraniteKingfisherpruit (KFI) Hoyo Hoyo (HOY) Borehole GraniteHoutboschrand (HOU) Nyamarhi (NYA) Pipeline trough BasaltHoutboschrand (HOU) Ngosto North (NGO) Pipeline trough Basalt
Table 1: Sample locations
●●●●
-24.5
-25.0
31.25 31.5 31.75 32.0Longitude
Latit
ude
KWA
NWA DLP
NHL
HOY IMB
GIRWIT
NYA
NGO
Figure 1: Map of site locations with park boundary indicated by dashed line. Circles represent sitesfilled by boreholes while triangles represent sites filled by river water via pipeline troughs.
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Genome
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Draft
Site Weeks S XS Daily A/B TotalNhlanguleni (NHL) 3 0 0 0 Yes 6Nwaswitshaka (NWA) 3 1 1 4 Yes 18De LaPorte (DLP) 1 1 1 0 Yes 6Kwaggas Pan (KWA) 2 1 1 0 Yes 8Girivana (GIR) 3 0 0 0 Yes 6Witpens (WIT) 3 0 0 0 Yes 6Imbali (IMB) 3 0 0 0 Yes 6Hoyo Hoyo (HOY) 3 1 1 0 Yes 10Nyamarhi (NYA) 3 1 1 0 Yes 10Ngosto North (NGO) 3 1 1 0 Yes 10BLANK 2 0 0 0 No 2
29 6 6 4 88
Table 2: Samples sequences, broken down by number of weekly samples, number of site-times forwhich S (50 mL) and XS (15 mL) samples were filtered, additional daily samples taken, whether A/Bsamples were taken, and the resulting total number of samples sequenced per site.
0 10 20 30 40 50
050
015
0025
00
Number of 150mL samples
Num
ber
of B
acte
rial A
SV
s
Figure 2: ASV accumulation curve of bacterial ASV richness using the “exact” method. Bars representtwo standard deviations around mean estimates.
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0.955 0.9700.6200.000 0.9850.7800.8200.000 1.0000.9160.865 0.9370.8630.9330.0000.8060.9450.7360.9280.7710.9770.9120.0000.3000.9740.000
0.906
0.9850.9010.2510.9640.9700.955 0.172 0.9250.8840.8060.461 1.0000.0000.534 0.958 0.9750.0000.8560.9350.6290.9480.9450.9440.796 0.9560.7670.9390.6840.9410.8770.8150.9380.8670.511
0.8520.7830.8560.8510.7810.7660.8260.0000.9120.912
0.923 0.958 0.988 1.0000.8170.9630.4460.7400.789
0.741 0.914 1.000 0.9550.8790.8410.7250.6900.5421.0000.9370.7780.9050.9780.0000.9310.8710.0000.8950.0000.3940.594 0.998 1.0001.000
0.8810.8320.827 0.9760.7750.967 0.949 0.9970.7780.7740.0000.8490.8010.000
0.947 0.640
0.987 0.878 0.992
0.846 0.9280.7500.368 0.991 0.9050.9750.5430.8990.8170.8010.7670.737
0.7950.9270.956 0.0970.985 0.9860.788 0.994 0.9720.9080.8460.9570.860 1.0000.792 0.9850.940 0.9270.969 1.0000.281
0.691
1.000
0.0000.947 0.7660.8950.8610.797 0.981 0.907 1.000 0.9980.2950.8290.9670.9080.6450.9140.8330.8650.9330.7650.9310.758
0.7450.7810.7530.8690.8620.9650.8730.0000.9410.8500.9760.6960.6840.8360.876 0.9150.912
0.9740.8700.922
0.8610.8680.657 0.861 0.9970.8880.9050.916 0.992 0.9930.4550.065 0.9980.7410.9820.0000.925
0.735 0.9960.9260.8570.775
0.9040.000
0.9500.9450.6860.624 0.9840.4730.9280.650 0.9300.8460.9150.9560.990 0.942 1.000
0.9540.901 0.941
0.1910.832 0.987 0.9460.5550.311 0.9840.9440.9540.7980.9830.9470.1530.874
0.8930.7640.9600.9090.856
0.7470.9480.9250.9530.8110.552 0.9730.9460.8440.0000.464
0.7610.814 0.9690.9120.874 0.9730.6280.8890.8540.873
0.2740.436
0.916 0.9980.302
0.996 0.996
0.737 0.994 0.9770.8580.0000.9360.8510.880 0.918 0.9400.4940.7970.7970.3650.9940.0450.8130.6230.9990.8510.8460.916 0.971
0.465
0.8200.900
0.753
0.944
0.8570.9700.000 0.9910.9790.5500.8870.783
0.9820.734
0.9960.853
1.000
0.9630.450
0.860
0.859
0.8450.0000.8230.0000.8690.7750.0000.8030.0000.7960.9740.7140.8980.8580.930
0.7980.9010.0000.8430.0001.0000.0000.9310.0000.8950.9520.6770.6930.715
0.7970.9190.8390.0000.7670.8700.909 0.9910.8700.8740.0000.7500.0000.0000.8870.6490.8800.9560.0000.8540.0000.858
0.0000.8710.9560.0000.0000.5150.000
0.8840.0000.9310.9430.0000.6680.8500.0000.000
0.9130.0000.866
0.854 0.9820.0000.9100.5330.4560.7930.9370.939
0.0000.8460.8820.8580.0000.8500.935
0.0000.000
0.000
0.9520.0000.8390.877
0.959
0.775
0.0000.729 0.9170.7700.343 0.909 0.9660.885 0.9880.8020.5970.894 0.9950.7640.977 0.9830.4220.9000.0000.531 0.939 0.937 0.9930.721
0.7700.432 0.9920.921 0.552 0.9530.7450.903 0.873 1.0000.9400.9970.655 0.9620.862 0.987 0.992
0.997 0.9790.909 0.988
0.7600.538
0.978 0.983
0.2660.883
0.879 1.0000.326
0.8610.8980.9210.918 0.9830.9110.792
0.7780.7810.9600.952
0.744
0.983
0.741
0.9010.8200.9330.9100.9770.9130.0000.0000.7360.897
0.886
0.8790.860 0.911 0.999
0.9580.235 0.9940.5090.8370.8900.000 0.9940.740 0.9570.9310.7640.9180.5180.8560.9790.915
0.8940.725 0.9990.0000.7990.883
0.803 0.9540.8760.832 0.975
0.7540.8060.0000.7260.7930.9420.8290.0000.9360.9970.7750.9510.0000.5110.8700.6530.9030.7960.9580.9240.0000.9280.0000.9670.8460.000
0.778
0.750
0.9100.3710.880
0.9730.769
0.8240.8560.9240.0000.8430.1590.0000.0000.8730.9420.0000.7570.7680.7970.8240.0000.7550.9221.0000.8300.0000.9060.924
0.7820.729 0.9430.7110.0000.0000.9770.9640.9130.3180.9540.0000.0000.903
0.7740.9740.8470.8210.0200.9140.7830.9130.934
0.8900.944 0.997
0.8080.8750.6430.733 0.9350.983 0.985
0.7520.8860.848 0.9490.925 0.925 0.9110.840
0.9730.871
0.9380.8500.8980.7620.9620.9140.914 0.9830.7430.0000.9750.9670.9870.000
0.3150.575 0.9640.7610.8600.7730.9320.9410.9800.9360.946 0.9930.1860.8470.0000.8640.7430.893
0.8990.7210.8730.9640.826 0.9880.8960.7460.000 0.9810.9970.9040.851
0.9840.7430.9410.8530.6870.8840.478 0.943 0.931 0.9990.7410.9800.7680.2680.9580.8900.9280.9340.9180.8700.7740.7480.988
0.980 0.973
0.9490.8790.9370.8280.9060.8370.7620.7360.763
0.7390.904 0.9840.4520.9320.7150.9880.8020.8750.0000.4020.8310.214
0.9440.8390.6720.9920.9070.000
0.8900.9500.3540.8670.8370.0000.7830.8920.8480.8390.8630.9140.943
0.9630.0000.8480.0000.9220.8650.0000.8580.9570.0000.8990.7570.9740.3940.9010.9770.7890.789
0.9980.0000.769
0.787
0.0000.8840.7830.8870.9100.0000.7480.723
0.9920.7950.3000.0000.8800.509 0.9940.7450.7280.000
0.9350.9220.7550.873 0.9680.740 0.958 0.9470.8610.8700.8380.9610.7990.0000.0000.9650.8850.0000.7510.9600.7410.8790.3060.7580.5890.995
0.9980.743
0.8930.8800.809 0.9650.755 0.9940.765 0.989 0.8860.267 0.930 0.7640.396 0.999 0.9840.8270.3950.521 0.9590.8730.9670.864 0.928 0.9170.8680.9140.000
0.7610.5110.9780.7490.8840.8870.7600.0000.810
0.9700.7750.8210.2040.9340.000 1.0000.7880.7950.7450.9350.000
0.8250.916
0.976 0.9970.729 0.359 0.9390.9760.9420.7510.8050.8800.7660.7920.7790.339
0.901 0.998
0.9440.855
0.5040.602
0.9660.750 0.965 0.928 0.9440.9410.803 0.960 0.995 0.219 0.9870.924
0.1080.7230.8850.774
0.917 0.9670.0800.7880.9450.7850.9820.9650.8750.8940.7560.7140.857 0.956 0.944 1.0000.8120.871 0.9600.8150.1140.772 0.9870.874 0.991
0.8140.0000.789
0.999
0.9720.1300.9710.788 1.0000.9800.8310.8850.8670.7930.929
0.9790.0000.7560.880 0.998 0.984 0.8750.000 0.9790.8680.7780.765 0.9770.9140.3770.980 0.8550.622 0.9450.8360.7500.9550.0000.7770.7510.9010.738 0.824 0.832
0.9680.9060.9040.7700.9720.9840.8150.000
0.8670.836 0.952 0.9360.7270.9420.2770.5180.8670.747 1.0000.000
0.9420.7500.9580.0000.910
0.0000.8560.9270.7840.8000.8680.802 0.9990.8540.7820.0000.8580.0000.9250.926
0.9830.0000.7160.9120.8320.8540.7620.8460.8860.0000.000
0.8540.0000.8570.0000.000
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NHL
NWA
DLP
KWA
GIR
WIT
IMB
HOY
NYA
NGO
SectionTSH SKZ SAT KFI HOU
Figure 3: Phylogenetic tree of 16S ASV sequences in the core samples, paired with their relativeabundances at each site. Sites are ordered by section.
Diversity Level Shannon SES 2.5% 97.5% Pr(sim.)α A/B samples 2.93 -9335.9 5.10 5.10 0.01α Temporal samples 2.99 -12535.9 5.13 5.13 0.01α Sites 3.39 -16873.6 5.15 5.15 0.01α Sections 3.94 -14919.9 5.16 5.16 0.01γ (Total) 5.17 0.0 5.17 5.17 1.00β A/B samples 0.05 153.3 0.027 0.027 0.01β Temporal samples 0.40 2688.2 0.022 0.022 0.01β Sites 0.55 6528.7 0.008 0.008 0.01β Sections 1.23 14919.9 0.008 0.008 0.01
Table 3: Additive partitioning of Shannon diversity into α, β, and γ diversities across sections, sites,temporal samples, and within site-time samples (A/B) as components of the total diversity observedacross all core samples. Observed diversity is compared to 99 simulations and the standardized effectsize (SES) using “r2dtable” null model with the adipart function in the R package vegan.
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Draft
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type●● Borehole
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section●●
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HOU
KFI
SAT
SKZ
TSH
Figure 4: Plots of observed ASV richness and Shannon diversity across samples. Samples are groupedand coloured by park section and with shape indicating waterhole type.
Phylum Class Order Family Genus
Cla
ssifi
ed s
eque
nces
up
to ta
xono
mic
leve
l (%
)
020
4060
8010
0
Figure 5: The proportion of ASVs assigned a given taxonomic level using the SILVA database v128and a pre-trained RDP classifier with minimum 50% bootstrap support.
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ativ
e ab
unda
nce
(%)
Phylum
Actinobacteria
Bacteroidetes
Chlamydiae
Cyanobacteria
Firmicutes
Other
Proteobacteria
Verrucomicrobia
Figure 6: Relative abundances of bacterial phyla across sites. Sites are ordered by relative abundanceof phylum Proteobacteria.
NHL NWA KWA GIR WIT IMB HOY NYA NGO
0%
25%
50%
75%
100%
Rel
ative
abu
ndan
ce (%
) PhylumActinobacteria
Bacteroidetes
Chlamydiae
Firmicutes
Other
Proteobacteria
Verrucomicrobia
Week1 2 3 1 2 3 1 2 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Figure 7: Relative abundances of bacterial phyla across weekly samples.
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Draft0%
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3A −
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e 29
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e 30
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e 30
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Rel
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e ab
unda
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(%) Phylum
Actinobacteria
Bacteroidetes
Chlamydiae
Firmicutes
Other
Proteobacteria
Verrucomicrobia
Figure 8: Relative abundances of bacterial phyla across five days at a single site (NWA).
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NM
DS2
site●●●●●●●●●●●●●●●●●●●●
DLP
GIR
HOY
IMB
KWA
NGO
NHL
NWA
NYA
WIT
a) b)
Figure 9: NMDS plots of a) Bray-Curtis and b) abundance-weighted UniFrac distances. Coloursrepresent site.
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Draft−3 −2 −1 0 1 2
−2−1
01
2
NMDS1
NM
DS2
Actinobacteria
AlphaproteobacteriaBacteroidia
Betaproteobacteria
Clostridia
Flavobacteriia
Gammaproteobacteria
Sphingobacteriia
Conductivity
pH
(a) Bray-Curtis NMDS
−0.03 −0.01 0.01 0.02 0.03
−0.0
20.
000.
010.
020.
03
NMDS1
NM
DS2
ActinobacteriaAlphaproteobacteria
Bacteroidia
Clostridia
Gammaproteobacteria
Sphingobacteriia
Conductivity
Dissolved Oxygen
pH
(b) Weighted UniFrac NMDS
Figure 10: Nonmetric multidimensional scaling (NMDS) ordination of variation in bacterial com-munity structure across 54 samples based on a) Bray-Curtis and b) abundance-weighted UniFracdistances. Arrows indicate the direction of significant (p < 0.05) correlations among variables andthe NMDS axes, with arrow length indicating the strength of the correlation. Blue arrows indicateenvironmental variables, while black arrows indicate relative abundances of sequences from differ-ent microbial classes. The ordination axes explain 96.8% (a) and 98.1% (b) of the variance in thedissimilarities (Fig. S17).
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