Supplementary Materials
Bacterioplankton community sampling and fingerprinting
121 bacterioplankton community (BC) samples were collected from the integrated
upper water column (0-12m) on monthly- to twice-monthly basis from 2000-2009 under
ice-free conditions on Lake Mendota, Wisconsin, USA. Sample dates are listed in
Supplementary Table 1 (S1). Samples were collected from the location of maximum depth
in Lake Mendota at 43° 06´ N, 89° 24´W, 327 m.s.l. Seasons were defined by the
astronomical March and September equinox and the June solstice.
Samples were filtered (0.2 m Pall Supor filters) and stored at -80 C until
subsequent processing. Deoxyribonucleic acid (DNA) was extracted with QBiogene Bio101
FastDNA extraction kit and modifications to manufacturer’s protocol described previously
(Yannarell et al., 2003; Yannarell & Triplett, 2005), and followed by amplification by
polymerase chain reaction and automated ribosomal intergenic spacer analysis (ARISA) as
described by Shade and colleagues (Shade et al., 2007). ARISA profiles were analyzed using
Genemarker v 1.5 software (SoftGenetics, LLC) and a custom R script developed in the R
statistics package (R Development Core Team, 2011) to aid in high-throughput ‘binning’ of
peaks, as previously described (Jones & McMahon, 2009).
Environmental variables
Thirty-three physical, chemical and biological water quality parameters were
collected at biweekly frequency during ice-free seasons by the North Temperate Lakes
Long Term Ecological Research (NTL LTER) program at the same location as microbial
sample collection; a complete listing of variables, protocols, and observational data is
available at www.lter.limnology.wisc.edu and variables and abbreviations are listed in
Supplementary Tables 1 and 2 (NTL-LTER, 2011a, 2011b, 2011c). Data underwent quality
control as described in NTL LTER protocols, all flagged data were removed, and replicates
averaged. Phytoplankton samples were collected across a vertically averaged integrated
depth 0-8m and biomass was summed at the division level for functional groups
Cyanophyta, Bacillariophyta, and Chlorophyta. For zooplankton, organism density was used.
For all other variables, discrete depth measurements were typically made every 1-4 m from
0-20 m depth; all variables measured between 0-12m depths were vertically-averaged.
When NTL LTER observations did not co-occur with microbial sampling, timeweighted averages of observations within +/- 17 days were assigned to microbial
observations. For >90% of samples, LTER environmental observations occurred within 7
days of bacterioplankton sampling. For time-weighted interpolation, nearby observations
were averaged after being weighted by a value inversely proportional the absolute value of
the number of days between water quality observations and microbial observations:
Yint = [∑ni=1 wi *yi] / ∑ni=1 wi
wi= 17-|xi-x0|
Where Yint is the interpolated value used in subsequent analyses for microbial samples
corresponding to day of year x0, n is the number of observations made within +/- 17 days of
x0 for any given year, xi is day of year that variable yi was collected on. wi is the ‘weighted’
factor which is proportional to the temporal proximity of the water quality observation to
the microbial observation. For example: a microbial sample was collected on day 50 (x0 =
50), and NTL LTER water quality samples were collected on day 40 (x1) and 54 (x2). If
temperature is unknown on day 50, but had values of 15 (y1) and 18 (y2),
Yint = [((17-|40-50|)*15)+ ((17-|54-50|)*18)] / ((17-|40-50|) +(17-|54-50|)) = 17.0
Table S1. Bacterioplankton community sampling dates
Microbial sampling dates (mm/dd/yy)
3/15/00
8/29/02
8/2/05
7/22/08
3/30/00
9/12/02
8/19/05
8/7/08
4/14/00
9/24/02
8/31/05
8/22/08
4/27/00
10/8/02
9/18/05
9/5/08
5/11/00
10/22/02
10/10/05
9/15/08
6/6/00
11/7/02
11/8/05
10/2/08
6/20/00
11/20/02
4/6/06
10/17/08
7/17/00
5/13/03
4/21/06
2/24/09
8/1/00
5/28/03
5/4/06
4/29/09
8/17/00
6/9/03
5/15/06
6/9/09
9/12/00
6/27/03
6/9/06
6/18/09
9/26/00
7/23/03
7/13/06
6/26/09
10/10/00
8/7/03
7/17/06
7/7/09
10/24/00
9/8/03
8/3/06
7/30/09
11/28/00
9/22/03
8/22/06
8/10/09
3/13/01
10/15/03
9/8/06
8/26/09
4/23/01
11/11/03
10/6/06
9/13/09
5/21/01
5/19/04
10/20/06
9/27/09
6/4/01
5/26/04
11/3/06
10/9/09
7/2/01
6/11/04
11/16/06
10/26/09
7/5/01
6/25/04
5/11/07
11/14/09
7/16/01
7/8/04
5/29/07
12/4/09
7/30/01
7/22/04
6/11/07
8/13/01
9/2/04
7/23/07
8/28/01
9/16/04
8/6/07
9/10/01
9/30/04
8/20/07
9/26/01
10/12/04
9/27/07
10/20/01
9/16/04
10/12/07
11/6/01
9/30/04
11/1/07
11/26/01
10/12/04
5/10/08
5/24/02
4/11/05
6/5/08
7/1/02
5/25/05
6/25/08
8/1/02
6/22/05
7/11/08
Table S2. Environmental variables and abbreviations
Environmental variables
Abbreviation
Month
Month
Year
Year
Day of year
Day of year
Day length
Day Length
Alkalinity
Alk
Calcium
Ca
Chlorophyll-a
Chl
Chloride
Cl
Dissolved inorganic carbon
DIC
Dissolved oxygen
DO
Dissolved organic carbon
DOC
Dissolved oxygen percent saturation
DOsat
Dissolved reactive silica
DRSilica
Iron
Fe
Potassium
K
Magnesium
Mg
Manganese
Mn
Ammonium
NH4
Nitrate+ nitrite
NO3NO2
Sodium
Na
Sulfate
So4
Soluble reactive phosphorus
SRP
Total inorganic carbon
TIC
Total kjeldhal nitrogen
TKN
Total organic carbon
TOC
Total phosphorus
TP
Temperature
Temp
Zooplankton density
Zoop
pH
pH
Secchi depth
Secchi
Bacillariophyta biomass
Bacillarophyta
Chlorophyta biomass
Chlorophyta
Cyanophyta biomass
Cyanophyta
Figure S1 Co-occurrence network properties for a ten-year time series of environmental variables in Lake
Mendota, WI, USA (number of nodes (S), edges (L), clustering coefficient (Cl), and characteristic path length
(D)). Open circles indicate properties calculated for networks that include all observations for a given season.
Boxplots represent observation-normalized results: we performed LSA (LSA R > 0.3, p < 0.001, no time lag)
and network analysis on networks generated from Monte Carlo simulations of 30 randomly drawn
observations from each season. Results of 1000 simulations for each season were aggregated and compared
to test the effect on network properties of the number of observations included in the analysis. Boxes
represent inter-quartile range and whiskers indicate 10th and 90th percentiles, red pluses indicate outliers.
Significant differences exist between observation-normalized distributions of network properties from each
season, for all properties (Wilcoxon rank sum test: α=0.01, p<0.0001).
Figure S2 Box and whisker plots of co-occurrence network properties using same bootstrapping technique as described in main text, but with a range
the correlative local similarity score (R values): 0.1 (upper plots) and 0.5 (lower plots). Plus signs indicate outliers, while boxes represent 10th and 90th
percentiles, and median values. Network trends in clustering coefficient and average path length reported in text (decreasing cluster coefficient and
increasing path length) were generally found in this sensitivity analysis. One exception to these trends is the average path length for seasonal
distributions with higher (0.5) R-value (L-score).
Citations
Jones, S. E., & McMahon, K. D. (2009). Species-sorting may explain an apparent minimal
effect of immigration on freshwater bacterial community dynamics. Environmental
microbiology, 11(4), 905-13. doi:10.1111/j.1462-2920.2008.01814.x
NTL-LTER. (2011a). Physical Limnology Dataset. North Temperate Lakes Long Term
Ecological Research Program. Madison, WI.
NTL-LTER. (2011b). Chemical Limnology Dataset. North Temperate Lakes Long Term
Ecological Research Program. Madison, WI. Retrieved from
http://www.lternet.edu/sites/ntl/
NTL-LTER. (2011c). Biological Limnology Dataset. North Temperate Lakes Long Term
Ecological Research Program. Madison, WI.
Shade, A., Kent, A. D., Jones, S. E., Newton, R. J., Triplett, E. W., & McMahon, K. D. (2007).
Interannual dynamics and phenology of bacterial communities in a eutrophic lake.
Limnology and Oceanography, 52(2), 487-494. doi:10.4319/lo.2007.52.2.0487
R Core Development Team. (2011). R: A Language and Environment for Statistical
Computing. Retrieved from http://www.r-project.org
Yannarell, a C., Kent, a D., Lauster, G. H., Kratz, T. K., & Triplett, E. W. (2003). Temporal
patterns in bacterial communities in three temperate lakes of different trophic status.
Microbial ecology, 46(4), 391-405. doi:10.1007/s00248-003-1008-9
Yannarell, A. C., & Triplett, E. W. (2005). Geographic and Environmental Sources of
Variation in Lake Bacterial Community Composition. Applied and environmental
microbiology, 71(1), 227-239. doi:10.1128/AEM.71.1.227