US Long-Term Ecological Research Network

Spatial heterogeneity of within-stream methane concentrations North Temperate Lakes LTER, 2014

Streams, rivers, and other freshwater features may be significant
sources of CH4 to the atmosphere. However, high spatial and temporal
variabilities hinder our ability to understand the underlying
processes of CH4 production and delivery to streams and also challenge
the use of scaling approaches across large areas. We studied a stream
having high geomorphic variability to assess the underlying scale of
CH4 spatial variability and to examine whether the physical structure
of a stream can explain the variation in surface CH4. A combination of
high-resolution CH4 mapping, a survey of groundwater CH4
concentrations, quantitative analysis of methanogen DNA, and sediment
CH4 production potentials illustrates the spatial and geomorphic
controls on CH4 emissions to the atmosphere.
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LTER Keywords
We determined stream CH4 concentrations at a large spatial extent (10
km) and fine grain (35,000 total measurements) by using a
biogeochemical mapping platform on a small boat [Crawford et al.,
2015]. Water was pumped on board where gases were stripped from the
water by using a sprayer-type equilibrator and analyzed with a Los
Gatos Research ultraportable greenhouse gas analyzer (using a
cavity-enhanced absorption technique). Survey speeds were very slow
(less than 3 kph) to enable the detection of small-scale changes in
CH4 concentrations over short distances. CH4 concentrations were
corrected for hydraulic and equilibrator lags by using first-order
step-change experiments detailed in Crawford et al. [2015] following
the outline provided in Fozdar et al. [1985]. Lag-corrected CH4 values
were georeferenced by using concurrent Global Positioning System
readings with the Wide Area Augmentation System capability enabled.
The highresolution transect was sampled on 24 and 25 July 2014 (two
morning to afternoon segments were combined into one data set). Both
days were similar in terms of weather and in-stream conditions.
Maximum daily air temperatures were 22.4degC and 20.9degC. Mean daily
air temperatures were 18.5deg and 19.1degC. At the middle site, daily
mean discharges were 3.3 and 3.7 × 10 2 m3 (Julys mean Q is 3.7 × 10 2
m3). Mean water temperatures were 18.1degC and 17.8degC (Julys mean
temperature is 19.6).
Using the high-resolution spatial CH4 data sets, we assessed the
degree of spatial autocorrelation by using semivariograms (spherical
model, using the function autofitVariogram in the R package automap).
We focused on the semivariogram range parameter which describes the
average scale of autocorrelation (i.e., the average patch size). We
also assessed the structure of spatial autocorrelation by using the
global Morans I statistic. The Morans I statistic evaluates whether a
series of geospatial observations are randomly distributed in space
(the null model), clustered, or dispersed. Statistically significant
positive values indicate spatial clustering, whereas negative values
indicate dispersed patterns. We used the Anselin Local Morans I
statistic for spatial cluster analysis of high-resolution CH4 data
[Anselin, 1995]. Statistically significant values of Local Morans I
identify regions (clusters) of high or low values relative to the
global data set, in addition to outliers (e.g., low outliers
surrounded by high values). The analysis was executed by using the
Spatial Statistics toolbox in ArcMap 10.2.
Groundwater Methane Sources
We analyzed groundwater CH4 from a series of wells near the middle and
lower sites and from wells at the head of the drainage near the spring
ponds (upper site) during the time frame of this study by using a
headspace equilibration method [Striegl et al., 2001]. Wells were
developed by using a peristaltic pump, and a minimum of two well
volumes were purged before sample collection. The goal was to evaluate
additional (external) lateral and vertical sources of CH4 beyond the
hyporheic zone. Despite a relatively homogenous sand aquifer,
groundwater flow paths and residence times are complex in this
catchment [Pint et al., 2003; Walker et al., 2003]. A combination of
flow paths including deep groundwater derived from meteoric recharge,
deep groundwater derived from lakes, and meteoric riparian water all
contribute to surface flow in the catchment. These water sources and
flow paths have been studied for over a decade as part of the USGS
WEBB program [Pint et al., 2003; Walker et al., 2003]. Differences in
substrate, organic matter availability, oxygen conditions, and other
metrics of redox state were previously shown to relate to the
concentrations of dissolved gases in wells at the middle site based on
historical data [Crawford et al., 2014b]. Here we expand the survey to
correspond to the timing of surface water mapping and to determine
whether patterns previously observed at the middle site held for the
catchment in general.
Sediment Methanogen Distribution and Abundance
CH4 production potential within stream sediments was first determined
by extracting DNA from stream sediment cores and quantifying the
abundance of methanogenic Archaea. We collected 14 cores approximately
22 cm long in sand and organic-rich wetland locations near the middle
site (locations correspond to odd numbered transect locations in
Figure 1; also corresponding to CH4 bubble trap locations described in
Crawford et al. [2014b]). Sediment cores were collected by using a 2.5
cm diameter, 30 cm length, stainless steel corer with an internal
polycarbonate tube attached to a one-way flow valve and a PVC
extension. Intact cores were transported to the laboratory within 2 h
and immediately frozen. Sediment cores were split into 2 cm segments
followed by DNA extraction by using a PowerSoil DNA isolation kit
(MoBio Laboratories Inc., Carlsbad, CA). We used quantitative
polymerase chain reaction (qPCR) targeting the gene encoding the alpha
subunit of methyl coenzyme-M reductase (mcrA) to quantify both
longitudinal and vertical distributions of methanogens. The mcrA gene
encodes a component of the terminal enzyme complex in the methane
generation pathway and is thought to be unique to methanogens and well
conserved [Thauer, 1998]. Many previous studies have used mcrA as a
genetic marker to determine methanogen abundance and community
composition [Luton et al., 2002; Earl et al., 2003; Freitag et al.,
2010; West et al., 2012]. Each extracted sample containing mcrA was
amplified in a 20 uL qPCR reaction in an ep gradient s realplex2
master cycler (Eppendorf), using SYBR Green as the reporter dye. Each
reaction contained 1 uL of 1/10 diluted sample DNA template, 1× iQ
SYBR Green Supermix (Biorad), and 0.25 uM of each primer targeting
mcrA: mcrAqF (50-AYGGTATGGARCAGTACGA-30) and mcrAqR
(50-TGVAGRTCGTABCCGWAGAA-30) [West et al., 2012]. Thermocycling
conditions for the mcrA qPCR were as follows: an initial denaturation
at 94degC for 1 min, followed by 40 cycles of 94degC denaturation for
40 s, 54degC annealing for 30 s, 72degC elongation for 30 s, and a
fluorescent detection at 85degC for 20 s. Melting curves were run to
ensure absence of nonspecific amplification. Amplification,
fluorescence data collection, and initial data analysis were all
performed by using the Eppendorf realplex2 software (Eppendorf,
Hauppauge, NY, USA).
Despite collecting greater than 20 cores, we were not able to perform
cluster analysis similar to that for CH4 concentrations on the genetic
data because the sample size was too low. Instead, we elected to
compare organicrich versus sand sediment mcrA gene abundance by using
a t test. To determine if methanogen abundance was correlated with CH4
production, we fit a linear model (log transformed) of mcrA abundance
versus average CH4 ebullition documented in the same year [see
Crawford et al., 2014b] with the R statistical programming language [R
Core Team, 2014]. We contend that the comparison between microbial
communities and integrated CH4 bubble flux over time is a stronger
comparison than that of point measurements of CH4 concentration.
Sediment Methane Production Potential
We collected surface sediments from Allequash Creek and placed them in
sealed jars the morning of the start of laboratory experiments. These
sediments were presumed to be mostly anoxic per the oxygen profile
study by Crawford et al. [2014b]. In the lab, about 75 mL of water
saturated surface sediments was transferred into a 150 mL glass
container, flushed with N2, sealed with a gas-tight lid equipped with
a butyl rubber septum for headspace gas sampling, and placed on a
shaker table in the dark at room temperature (about 22degC). Gas
samples were collected after 24 h for CH4 determination by using a
Shimadzu GC-2014 gas chromatograph. Headspace volume of each sample
was determined after gas sampling, then sediments were transferred to
a preweighted aluminum pan for drying (72 h at 50degC) and ashing (4 h
at 500degC). CH4 production potential was determined as headspace CH4
accumulation per gram of dry sediment and per gram of ash-free dry
mass (AFDM) per hour. Production rates were based on two-point
measurements of CH4 concentration and are thus presumed to be linear
over time. Because gas production rates could not be transformed to
meet assumptions of normality, significant differences among
treatments were assessed by using a Kruskal-Wallis test followed by
Wilcoxon rank tests for pairwise comparisons with Bonferroni-adjusted
P value using R.
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North Temperate Lakes LTER Regional Survey Water Chemistry 2015 - current

The Northern Highlands Lake District (NHLD) is one of the few regions in the world with periodic comprehensive water chemistry data from hundreds of lakes spanning almost a century. Birge and Juday directed the first comprehensive assessment of water chemistry in the NHLD, sampling more than 600 lakes in the 1920s and 30s. These surveys have been repeated by various agencies and we now have data from the 1920s (UW), 1960s (WDNR), 1970s (EPA), 1980s (EPA), 1990s (EPA), and 2000s (NTL). The 28 lakes sampled as part of the Regional Lake Survey have been sampled by at least four of these regional surveys including the 1920s Birge and Juday sampling efforts. These 28 lakes were selected to represent a gradient of landscape position and shoreline development, both of which are important factors influencing social and ecological dynamics of lakes in the NHLD. This long-term regional dataset will lead to a greater understanding of whether and how large-scale drivers such as climate change and variability, lakeshore residential development, introductions of invasive species, or forest management have altered regional water chemistry. The regional lakes survey in 2015 followed the standard LTER protocol for standard water chemistry and biology. Samples were taken as close to solar noon as possible. Seven lakes had replicates performed, which were chosen at random.
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Inorganic and organic carbon
Inorganic carbon is analyzed by phosphoric acid addition on a Shimadzu TOC-V-csh Total Organic Carbon Analyzer.
Organic carbon is analyzed by combustion, on a Shimadzu TOC-V-csh Total Organic Carbon Analyzer.
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Molecular composition of dissolved organic matter in NTL-LTER lakes detected by Fourier-transform ion cyclotron resonance mass spectrometry

The composition of dissolved organic matter (DOM) varies widely in the environment due to distinct sources of the material and subsequent processing. DOM composition drives its reactivity in terms of many processes including photochemical reactions, microbial metabolism, and carbon cycling within water bodies. This study uses ultra-high resolution mass spectrometry via a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to evaluate DOM composition at the molecular level to determine differences in DOM composition among the NTL-LTER lakes. Whole water samples were collected from the surface of each lake near the shore on August 18th and 19th in 2016 in. Ultraviolet-visible spectra were recorded as light absorbance can also give information about DOM composition. Additionally, concentrations of anions, cations, and pH were measured waters because these can all alter DOM reactivity in the environment. Both water chemistry and DOM composition vary widely among the lakes with the bogs displaying the most terrestrial-like signature in DOM and the oligotrophic lakes show more microbial-like or environmentally processed DOM.
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Molecular Composition

Water was acidified to pH = 2 with concentrated hydrochloric acid and organic matter was extracted from the water using Agilent PPL cartridges. Extracts were diluted 100x in 50:50 acetonitrile to ultra-pure water and directly injected into a Bruker SolarX 12T Fourier-transform ion cyclotron resonance mass spectrometer. Ionization was achieved with electrospray ionization by an Advian NanoMate delivery system in both positive and negative mode.

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Cascade project at North Temperate Lakes LTER - High-resolution spatial analysis of CASCADE lakes during experimental nutrient enrichment 2015 - 2016

This dataset contains high-resolution spatio-temporal water quality data from two experimental lakes during a whole-ecosystem experiment. Through gradual nutrient addition, we induced a cyanobacteria bloom in an experimental lake (Peter Lake) while leaving a nearby reference lake (Paul Lake) as a control. Peter and Paul Lakes (Gogebic county, MI USA), were sampled using the FLAMe platform (Crawford et al. 2015) multiple times during the summers of 2015 and 2016. In 2015 nutrient additions to Peter Lake began on 1 June, and ceased on 29 June, Paul Lake was left unmanipulated. In 2016 no nutrients were added to either lake. Measurements were taken using a YSI EXO2 probe and a Garmin echoMap 50s. Sensor- data were collected continuously at 1 Hz and linked via timestamp to create spatially explicit data for each lake.

Crawford, J. T., L. C. Loken, N. J. Casson, C. Smith, A. G. Stone, and L. A. Winslow. 2015. High-speed limnology: Using advanced sensors to investigate spatial variability in biogeochemistry and hydrology. Environmental Science & Technology 49:442–450.
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In two consecutive years, we measured lake-wide spatial patterning of cyanobacteria using the FLAMe platform (Crawford et al. 2015). To evaluate early warning indicators of a critical transition, in the first year we induced a cyanobacteria bloom through nutrient addition in an experimental lake while using a nearby unmanipulated lake as a reference ecosystem (Pace et al. 2017). During the second year, both lakes were left unmanipulated. Proposed detection methods for early warning indicators were compared between the manipulated and reference lakes to test for their ability to accurately detect statistical signals before the cyanobacteria bloom developed.
Peter and Paul Lakes are small, oligotrophic lakes (Peter: 2.5 ha, 6 m, 19.6 m and Paul: 1.7 ha, 3.9 m, 15 m, for surface area, mean, and max depth respectively) located in the Northern Highlands Lake District in the Upper Peninsula of Michigan, USA (89°32’ W, 46°13’ N). These lakes have similar physical and chemical properties and are connected via a culvert with Paul Lake being upstream. Both lakes stratify soon after ice-off and remain stratified usually into November (for extensive lake descriptions, see Carpenter and Kitchell, 1993).
In the first year, Peter Lake was fertilized daily starting on 1 June 2015 (DOY 152) with a nutrient addition of 20 mg N m-2 d-1 and 3 mg P m-2 d-1 (molar N:P of 15:1) through the addition of H3PO4 and NH4NO3 until 29 June (day of year, DOY 180). The decision to stop nutrient additions required meeting four predefined criteria based on temporal changes in phycocyanin and chlorophyll concentrations indicative of early warning behavior of a critical transition to a persistent cyanobacteria bloom state. (Pace et al. 2017). Nutrients uniformly mix within 1-2 days after fertilization based on prior studies (Cole and Pace 1998). No nutrient additions were made to Paul Lake. In the second year (2016), neither lake received nutrient additions.
We mapped the surface water characteristics of both experimental lakes to identify changes in the spatial dynamics of cyanobacteria. In 2015, mapping occurred weekly from 4 June to 15 August (11 sample weeks). In 2016, when neither lake was fertilized, the lakes were mapped three times in early to mid-summer. In both years, mapping occurred between the hours of 07:00 to 12:00 (before the daily nutrient addition). We rotated the order that we sampled the lakes to avoid potential biases due to differences in time of day. Each individual lake sampling event was completed in approximately one hour.
The FLAMe platform maps the spatial pattern of water characteristics. A boat-mounted sampling system continuously pumps surface water from the lake to a series of sensors while geo-referencing each measurement (complete description of the FLAMe platform in Crawford et al. 2015). For this study, the FLAMe was mounted on a small flat-bottomed boat propelled by an electric motor and was outfitted with a YSI EXO2™ multi-parameter sonde (YSI, Yellow Springs, OH, USA). We focused for this study on measures of phycocyanin (a pigment unique to cyanobacteria) and temperature. Phycocyanin florescence was measured using the optical EXO™ Total Algae PC Smart Sensor. The Total Algae PC Smart Sensor was calibrated with a rhodamine solution based on the manufacturer’s recommendations. Phycocyanin concentrations are reported as ug/L; however, these concentrations should be considered as relative because we did not calibrate the sensor to actual phycocyanin nor blue-green algae concentrations. Geographic positions were measured using a Garmin echoMAP™ 50s. Sensor- data were collected continuously at 1 Hz and linked via timestamp to create spatially explicit data for each lake. Each sampling produced approximately 3500 measurements in the manipulated lake and 2000 in the reference lake. The measurements were distributed by following a gridded pattern across the entire lake surface to characterize spatial patterns over the extent of the lake.
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LTREB experimental chironomid mesocosms at Myvatn, Iceland

During the summer of 2014, we conducted experiments testing whether increasing numbers of chironomid larvae would increase primary production and standing chlorophyll a concentrations. We incubated experimental mesocosms with varying numbers of chironomid larvae for 12 days in July. We tested sediments for chlorophyll a concentrations, as sediments are primarily composed of benthic diatoms. We tested the oxygen production in these mesocosms. We did this by sealing the mesocosms and incubating them in Lake Myvatn for 3 hours, and taking measurements of dissolved oxygen before and after the incubations.
We were also interested in whether this increase in food resources might translate to increased growth rates of chironomid larvae at high larval densities. After stocking experimental mesocosms with varying numbers of chironomid larvae, we set these mesocosms in Lake Myvatn for 12 days. We collected the larvae at the end of the 12 day experiment and obtained the average dry weights of the Chironomus islandicus larvae in each mesocosm.
We hypothesized that the tubes that chironomid larvae build would be a superior substrate for algal growth, as compared to loose sediments. Because there are two taxa (Chironomus islandicus and Tanytarsus gracilentus) that are overwhelmingly dominant at our study site, we wondered whether there would be differences in this effect between the two species. We stocked mesocosms with larvae from one of the two species, and mesocosms were then incubated in Lake Myvatn. We collected sediments and larval tubes from each mesocosm and tested their chlorophyll a concentrations.
We hypothesized that one mechanism that chironomid larvae might alleviate algal nutrient limitation by depositing concentrated nutrients near algae in the form of larval excretions. We collected chironomid larvae from Lake Myvatn and placed them in distilled water. We then sieved out the larvae and their fecal passings, and transported the water samples to Madison, WI, USA, where nutrient concentrations were analyzed
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Please refer to the following manuscript for a description of methods:
Herren, Cristina M., Webert, Kyle C., Drake, Michael D., Vander Zanden, M. Jake, Einarsson, Árni, Ives, Anthony R., Gratton, Claudio. 2016. Positive feedback between chironomids and algae creates net mutualism between benthic primary consumers and producers, Ecology, DOI: 10.1002/ecy.1654
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Myvatn midge experiment chlorophyll
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Long-term trends and synchrony in dissolved organic matter characteristics in Wisconsin, USA lakes: quality, not quantity, is highly sensitive to climate

Dissolved organic matter (DOM) is a fundamental driver of many lake processes. In the past several decades, many lakes have exhibited a substantial increase in DOM quantity, measured as dissolved organic carbon (DOC) concentration. While increasing DOC is now widely recognized, fewer studies have sought to understand how characteristics of DOM (DOM quality) change over time. Quality can be measured in several ways, including the optical characteristics spectral slope (S275-295), spectral ratio (SR), absorbance at 254 nm (a254), and DOC-specific absorbance (SUVA; a254:DOC). However, long-term measurements of quality are not nearly as common as long-term measurements of DOC concentration. We used 24 years of DOC and absorbance data for seven lakes in the North Temperate Lakes Long Term Ecological Research site in northern Wisconsin, USA to examine temporal trends and synchrony in both DOC concentration and quality. We predicted lower SR and S275-295 and higher a254 and SUVA trends, consistent with increasing DOC and greater allochthony. DOC concentration exhibited both significant positive and negative trends among lakes. In contrast, DOC quality exhibited trends suggesting reduced allochthony or increased degradation, with significant long-term increases in SR in three lakes. Patterns and synchrony of DOM quality parameters suggest they are more responsive to climatic variations than DOC concentration. SUVA in particular tended to increase with greater moisture and decrease with drier conditions. These results demonstrate that DOC quantity and quality can exhibit different complex long-term trends and responses to climate components, with important implications for aquatic ecosystems.
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Data contained within is derived from data publicly available through the North Temperature Lakes Long Term Ecological Research (NTL-LTER) website at the following url: All data is for the upper meter of the water column, collected in the deepest point of the lake. For DOC concentration data and ancillary data such as pH, iron, and total N, please see the NTL-LTER website.
The file dat.w.blank.fin 7_29_16.csv contains the absorbance scans that were used to calculate the spectral metrics (NTL LTER 2012). Samples for absorbance scans were collected approximately quarterly. Absorbance scans were run on a spectrophotometer over the wavelengths from 200-800 nm. In this file, the column value is the raw value for absorbance read directly off the instrument for the corresponding wavelength. The column blank.value is the value of the DI blank taken nearest in time for the corresponding wavelength and cuvette width. The column cor.value is the blank.value column subtracted from the value column. The cor.value column was used in all subsequent analyses and calculations.
The file metrics for_analysis.csv contains the spectral metrics calculated from the absorbance scans as well as select meteorological metrics. ab.254 is raw absorbance measured by the instrument (after correcting for DI blanks), corrected for a 1 m pathlength. lin.slope.275.295 is the slope for the log transformed absorbance scan over the wavelengths 275-295 nm, determined by linear regression. nlin.slope.275.295 is the spectral slope (S275-295) over the wavelengths 275-295 nm, calculated using non-linear regression in R version 3.2.3 (R Core Team 2015), fitting the following equation:
alambda = alambdarefe-S(lambda-lambdaref)
In this equation a is the Naperian absorption coefficient (see below), lambda is the wavelength, lambdaref is the reference wavelength, and S is the spectral slope (nm-1) (Twardowski et al. 2004; Helms et al. 2008). The initial estimate supplied to the non-linear regression procedure was supplied by the value for lin.slope.275.295. lin.slope.350.400 and nlin.slope.350.400 were calculated in a similar fashion, but over the wavelengths 350-400 nm. This calculation yields the spectral slope over the wavelengths 350-400 nm (S350-400). slope.ratio is the slope ratio (SR), the ratio of S275-295 to S350-400 (Helms et al. 2008). ab.254.1cm is similar to ab.254, but is corrected for a 1 cm cuvette width. a254.nap is the Naperian absorption coefficient and is calculated from the equation:
a = 2.303A/l
In this equation, a is the Naperian absorption coefficient, A is raw absorbance measured by the spectrophotomer, and l is the path length (Green and Blough 1994). SUVA was calculated by dividing raw absorbance at 254 nm by the DOC concentration of the DOC sample collected nearest in time to the absorbance sample. SUVA is reported as Ltimesmg C-1timesm-1 (Weishaar et al. 2003).
wk.prcp, mth.prcp, and ninety prcp are precipitation totals for the 7, 30, and 90 days up to and including the sampling date (mm). These data come from the Minocqua, WI station in the Global Historical Climatology Network (GHCN) dataset (available at (Menne et al. 2010).
wk.tmp, mth.tmp, and ninety.tmp are mean values for the mid-daily temperature in the 7, 30, and 90 days up to and including the sampling date (°C). These are from the same data source as the precipitation data.
The l.lev column contains lake level from the NTL LTER website in meters above sea level. wk.insol, mth.insol, and ninety.insol are mean solar insolation incident on a horizontal surface in the 7, 30, and 90 days up to and including the sampling date (kWh/m2/day) (these data were obtained from the NASA Langley Research Center Atmospheric Sciences Data Center NASA/GEWEX SRB Project).
Monthly Palmer Drought Severity Index (PDSI) data corresponding to the study period can be accessed at: using StateCode=47 and Division=2. These data were retrieved from the US National Oceanic and Atmospheric Administration (NOAA) National Center for Environmental Information (NCEI). These data were used to relate trends and synchrony in spectral metrics to moisture conditions.
The DOC column contains DOC concentration (mg L-1) for the corresponding sample date, obtained from the NTL LTER website.

Short Name
DOM trends data
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Saint Louis River Estuary Water Chemistry, Wisconsin, Minnesota, USA 2012 - 2013

These data pertain to water and sediments collected from the Saint Louis River Estuary (SLRE) and its nearby water sources by Luke Loken and collaborators for his Masters thesis and additional publications. In brief, we sampled SLRE surface waters and sediments for a variety of physical, chemical, and biological attributes. Ten estuary stations were sampled approximately monthly from April 2012 through September 2013. On four of the sampling campaigns, water was collected from an additional 20 sites. Sites were selected to represent a gradient from the Saint Louis River to Lake Superior and included several tributaries that drain directly into the estuary. This design aimed to understand the spatial and temporal mixing pattern of the estuary as it receives water from several rivers, 2 waste water treatment plant, and Lake Superior. We sampled the estuary to assess the magnitude and timing of source water contributions to the estuary and establish a baseline of chemical and physical measurements to aid in future limnological research. Additionally, we performed nitrogen and carbon cycling rate experiments to determine the estuary-wide influence on nitrate, ammonium, and dissolved organic carbon. This included 8 sediment denitrification, 1 nitrification, and 2 breakdown dissolved organic carbon (BDOD) surveys. This work was funded by the Minnesota and Wisconsin Sea Grant and in coordination with the establishment of the Lake Superior National Estuary Research Reserve (LSNERR).
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We collected water samples from 10 estuary stations to represent a gradient from river to lake on 13 dates between April 2012 and September 2013. Stations 1-5 represented upper estuary sites, while stations 6-10 were lower. Stations were situated near the thalweg, but were shifted laterally to avoid traffic within the shipping channel. Sampling occurred approximately monthly during the open water season when sites were accessed by boat, and once during winter ice cover when a subset of sites were visited on foot. In addition to the core 10 stations, we sampled an additional 20 sites, four times over the two-year study during a high flow and baseflow period. These sites include 7 end members (Saint Louis River, Nemadji River, Bluff Creek, Kinsbury Creek, Pokegama River, and Lake Superior) and an additional 15 in-estuary sites (i.e., stations 16-30). Additional sites were occasionally visited and geographic locations to all stations are provided in SLRESitesTable.Physical LimnologyWe used a YSI EXO2 or 6-Series sonde (Yellow Springs, OH) to measure temperature, specific conductivity, dissolved oxygen, pH, turbidity, and algae fluorescence. Briefly, the sonde was lowered to appr. 0.5 m depth and allowed to stabilize. The sonde was calibrated in the lab that morning according to Lake Superior National Estuary Research Reserve (LSNERR) protocols.Light extinction was determined by lowering a photosynthetically active radiation (PAR) sensor (Licor model 192 or 193) attached to a light meter (Licor model 250A) through the water column. The sensor was allowed to stabilize at 0.25 m depth intervals. We linearly regressed the natural log of the measured light intensity against depth. The slope of this regression is the negative light extinction coefficient (k). Briefly k values closer to zero indicate clearer waters that transit more light.Water ChemistrySurface water from each station was collected into an HDPE carboy and processed in the lab within 10 h of collection. We processed samples in the lab (instead of on the boat) to expedite sample collection so that all stations could be visited within a single day (or within 2 days for spatial intensive surveys). Integrated water samples were taken from 0-2 m using a peristaltic pump or an integrated water sampler and stored in a cooler to maintain ambient temperature. Samples for dissolved solute analysis were filtered through a 0.45 microm Geotech capsule filter. All samples were refrigerated, frozen, or acidified (dependent on the analysis in question) within 12 h of collection. See meta data for SLREWaterChemTable for specifics regarding lab responsible for analyses.Samples for major cations (Calcium (Ca), Iron (Fe), Potassium (K), Sodium (Na), Magnesium (Mg), and Manganese (Mn)) were filtered upon collection into 60 mL acid-washed HDPE bottles, acidified to 1 percent ultrapure hydrochloric acid (HCl) and stored at room temperature until analysis (within 6 months). Cations were analyzed simultaneously on an optical inductively-coupled plasma emission on a Perkin-Elmer model 4300 DV ICP spectrophotometer according to methods outlined at the North Temperate Lakes- Long Term Ecological Research site.Samples for major anions (Chloride (Cl) and sulfate (SO4)) were filtered into a new 20 mL HDPE scintillation vials and stored at 4degree C until analysis (within 3 months). Anion samples were analyzed simultaneously by Ion Chromatography, using a hydroxide eluent determined by a Dionex model ICS 2100 using an electro-chemical suppressor.Samples for dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC) were analyzed on a Shimadzu TOC analyzer. DOC and DIC samples were filtered into acid-washed 24 mL glass vials and capped with septa, leaving no headspace. DOC samples were acidified with 100 microL of 2 M HCL upon collection. Both DOC and DIC were stored at 4 degreeC, and then analyzed within three weeks at the University of Minnesota-Twin Cities. Both DOC and DIC were collected in duplicate and reported as means.Samples for UV absorbance were filtered into ashed 40 mL glass amber vials and stored at 4degree C until analysis (within 2 months). We measured UV absorbance at 254 nm (Abs254) using a spectrophotometer (Cary 50 UV-Vis Spectrophotometer, Varian, Palo Alto, CA). Specific UV absorbance at 254 nm (SUVA254) was then calculated by dividing Abs254 by the DOC concentration of the water sample.Nitrate plus nitrite nitrogen (referred to as NO3-N), ammonium plus ammonia nitrogen (referred to as NH4-N), and soluble reactive phosphorus (SRP) were analyzed colormetrically. Samples were filtered into new 20 mL plastic scintillation vials and frozen within 8 h of collection. Samples were thawed within 4 months and were analyzed in parallel by automated colorimetric spectrophotometry, using an Astoria-Pacific Astoria II segmented flow autoanalyzer. NO3-N was determined using the automated cadmium reduction method with absorption monitored at lambda=520 nm. NH4-N was determined using the Berthelot Reaction, producing a blue colored indophenol compound, where the absorption was monitored at lambda=660 nm. SRP was determined by forming a phosphoantimonymoledbeun complex and was measured as lambda=880nm.Samples for total and dissolved nitrogen and phosphorus analysis were collected together and in-line filtered (dissolved nitrogen and phosphorus only) into 60 ml LDPE bottles and acidified to a 1 percent HCl. Once acidified, the samples were stored at room temperature until analysis, which occurred within one year. The samples were first prepared for analysis by adding a NaOH&ndash;Persulfate digestion reagent and heated for 1 h at 120 degreeC and 18-20 pounds per square inch (psi) in an autoclave. The samples were analyzed for total nitrogen and total phosphorus simultaneously by automated colorimetric spectrophotometry, using a segmented flow autoanalyzer. Total nitrogen is determined by utilizing the automated cadmium reduction method where the absorption is monitored at 520 nm; total phosphorus is determined using ascorbic acid-molybdate method where the absorption is monitored at 880 nm. Both are described in LTER standard methods.We determined dual isotopic natural abundance of nitrate (NO3) and water (H2O) from a subset of collected water samples. Samples for delta18O-NO3 and delta15N-NO3 were filtered into acid-washed 60 mL HDPE bottles and frozen within 8 h of collection. Nitrate isotope samples were analyzed using the denitrifier method at the Colorado Plateau Stable Isotope Laboratory. delta18O-NO3 and delta15N-NO3 isotopes were reported as the per mil (per-mille) deviation from Vienna Standard Mean Ocean Water (VSMOW) and air standards, respectively. Samples for isotopes of water (delta18O-H2O and delta2H-H2O) were collected without headspace in glass vials and measured using isotope ratio infrared spectroscopy at the University of Minnesota &ndash; Biometeorology lab. Six replicates were run per sample, and delta18O-H2O and delta2H-H2O were determined relative to VSMOW.Chlorophyll ALaboratory analysis of chlorophyll A (ChlA) uses the Turner Designs model 10-AU fluorometer, following improvements described in Welschmeyer (1994). In this method, ChlA in 90percent acetone is separated from other pigments by the use of specialized optical filters. ChlA samples were preserved within 24 h of water sampling, by collecting filtrand on a 0.2 microm cellulose nitrate filter, placing the filter in a 15 mL falcon tube, and freezing it. Between 200 and 1000 mL of sample was based through the filter until the filter was moderately stained and filtering speed slowed. Within three weeks of collection, filters were thawed, and 12.0 mL of acetone was added to tube, which was allowed to steep for 18-24 h in the dark at 4 degreeC. After steeping, samples were centrifuged at high speed in Sorvall GLC-2B centrifuge for 20 min and warmed to room temperature. Sample fluorescence was then measured on a calibrated Turner Designs model 10-AU fluorometer (excitation 436 nm, emission 680 nm). Sample fluorescence was then converted to a water column concentration by multiplying by the extract volume (i.e., 12 mL) and divided by the volume of water that passed through the filter (i.e., 200-1000 mL).ParticulatesSimilar to ChlA, particulate carbon, nitrogen, and phosphorus samples were collected by passing 200-1000 mL of water through a pre-combusted 0.7 glass fiber filter (GFF) and analyzing the filtrand. Filters were frozen immediately after filtration, and then dried at 60 degreeC for at least 48 hours. Particulate carbon and nitrogen was measured using a Thermo Fisher Flash 2000 elemental analyzer. Particulate phosphorus was determined from a separate filter. Filters were digested in 5 mL potassium persulfate and phosphorus was analyzed spectrophotometrically using the ascorbic acid-molybdate method (Menzel and Corwin 1965).NitrificationWater column nitrification rates were determined on 30 July 2013 for a subset of the water chemistry sampling stations (n = 15) that represented the full spatial extent and previously observed NH4-N range of the estuary. Water from each station was transferred to 333 mL polycarbonate bottles within 10 h of collection and spiked with 15NH4Cl to achieve a concentration of 0.03 micromol 15NH4 L-1. Samples were incubated at ambient temperature (20 degreeC ) in a dark cooler for 20 h. Pre- and post-incubation samples were filtered through 0.45 microm filters and analyzed for NO3-N, NH4-N and delta15N-NO3. Nitrification rates were determined based on changes in NO3-N, NH4-N, and delta15N-NO3 according to methods outlined in Small et al. (2013). Analysis for each station was performed in duplicate and reported as the mean.SedimentsSediments were collected on 8 of the water chemistry survey dates from stations 2-9 to determine spatial and temporal patterns of denitrification and sediment organic content. We also collected a single sediment sample from additional lower (n = 17) and upper (n = 6) stations on 19 June 2012 and 24 June 2013, respectively, to increase the spatial extent of our survey. In total, 56 and 42 individual sediment collections were made in 2012 and 2013, respectively. Sediments were collected from the upper 5-20 cm of the benthic zone using an Ekman dredge. At least 500 mL of benthic material was transferred to 1-L widemouth Nalgene containers and used in denitrification rate experiments. Fifteen mL of the uppermost sediment layer was transferred into sterile 100 mL disposable plastic screw-top containers to be analyzed for sediment composition content. Sediments were stored in a cooler while on the boat and transferred to 4 degreeC within 6 h.To assess the effects of sediment composition on denitrification, dry:wet ratios, bulk density, particle size distributions, loss-on-ignition (LOI), percent carbon, and percent nitrogen were determined from the 15 mL sediment subsamples. Sediments were weighed before and after drying at 60 degreeC for at least 48 h to determine dry:wet ratios and bulk density. Sediment particle size composition was determined optically using a Coulter LS-10 particle size analyzer and sizes were binned into percent clay (0-2.0 microm), silt (2.0-63.0 microm) and sand (63-2000 microm) (Scheldrick and Wang 1993). LOI was determined by the loss in mass of 2.0plus/-0.2 g dried homogenized sediment combusted at 550 degree Celsius for 4 h. Sediments were ground and analyzed for percent carbon and nitrogen using a Thermo Fisher Flash 2000 elemental analyzer.Sediment denitrificationWe determined actual (DeN) and potential (DEA) sediment denitrification rates in the laboratory using the acetylene block technique modified from Groffman et al. (1999) within 48 h of collection. We incubated 40&plusmn;2 g of wet sediment saturated with 40&plusmn;0.1 mL of estuary water in 125 mL glass Wheaton bottles at 20 degreeC. DEA incubations were spiked with glucose and NO3-N to a final concentration of 40 mg C L-1 and 100 mg N L-1, respectively; DeN incubations were given no amendments. All incubations were augmented with 10 mg L-1 chloramphenicol to inhibit microbial proliferation (Smith and Tiedje 1979). Samples were capped with rubber septa, flushed with helium (He) for 5 min to remove oxygen (O2), and injected with 10 mL acetylene. We allowed the acetylene 30 min to fully diffuse into the sediment slurry before taking the initial headspace sample (T0). Samples were placed on a shaker table in the dark for 2.6 h then sampled the final headspace (T1). The change in headspace N2O partial pressures (pN2Ofinal - pN2Oinitial) was used to determine the denitrification rate using the Bunsen correction and the ideal gas law. For both T0 and T1 samples, 10 mL of headspace was withdrawn from incubation bottles and injected into a He-flushed 12 mL gas-tight glass vials (Exetainers) sealed with rubber septa. We determined pN2O and pO2 in parallel on a gas chromatograph equipped with an electron capture detector (ECD) and thermal conductivity detector (TCD) using methods outlined in Spokas et al. (2005). Gas samples with O2 concentrations greater than 5percent were removed from analysis due to potential gas leakage. Denitrification rates were standardized to sediment dry mass. Samples collected on or before 6 June 2013 were incubated in triplicate; samples collected after were incubated in duplicate.Denitrification controls were further investigated by amending sediments with combinations of NO3-N and two types of organic carbon: glucose and natural organic matter (NOM; supplied by the International Humic Substance Society). On two dates in 2013, we incubated sediments from five of our core stations that spanned a gradient of sediment organic content with the following amendments: NO3-N only, NO3-N and glucose (DEA), NO3-N and NOM, glucose only, NOM only, and no amendments (DeN). The two carbon treatments were intended to test for possible effects of carbon quality, with NOM representing a recalcitrant, humic-rich carbon source similar to allochthonous materials in the SLRE to contrast the labile glucose treatment. Both carbon sources were amended to 40 mg C L-1, and NO3-N was amended to 100 mg N L-1. Sediments were incubated in parallel (see above).Breakdown Dissolved Organic Carbon (bDOC)Breakdown of DOC (bDOC) was determined from core stations (1-10) from water collected on 23 April and 30 July 2012. Briefly, 250 mL of estuary water was filtered through a 0.45 microM Geotech flow-through filter using a peristaltic pump into sealable glass jars. 25 mL of 2.0 microm filtered water from a common estuary source was added to the filtered jars. DOC samples were collected after 0, 1, 2, 4 ,8, 16, and 32 d and analyzed for DOC (see above). A linear model was fit between time since inoculation and DOC concentration to determine the breakdown of DOC from water column microbes.ReferencesMeyers PA, Teranes JL. 2001. Sediment organic matter. Pages 239-269, In: Track Enviornmental Change Using Lake Sediments Vol 2 Phys Geochemical Methods. Dordrecht: Kluwer Academic Publishers.Groffman, Peter M, Holland EA, Myrold DD, Robertson GP, Zou X. 1999. Denitirification. Pages 272-288 in Standand Soil Methods Long-Term Ecological Research, Oxford University, New York.Menzel DW, Corwin N. 1965. The measurement of total phosphorus in seawater based on the liberation of organically bound fractions by persulfate oxidation. Limnol and Oceanogr 10: 280&ndash;282.Scheldrick HB, Wang C. 1993. Particle size distribution. Pages 499-512 In: Soil Sampling and Methods of Analysis. Boca Raton: CRC Press LLC.Small GE, Bullerjahn GS, Sterner RW, Beall BFN, Brovold S, Finlay JC, McKay RML, Mukherjee M. 2013. Rates and controls of nitrification in a large oligotrophic lake. Limnol Oceanogr. 58:276&ndash;86.Smith MS, Tiedje JM (1979) Phases of denitrification following oxygen depletion in soil. Soil Biol Biochem 11:261-267Spokas K, Wang D, Venterea R. 2005. Greenhouse gas production and emission from a forest nursery soil following fumigation with chloropicrin and methyl isothiocyanate. Soil Biol Biochem. 37:475&ndash;85.Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnol Oceanogr 39:1985-1992.&nbsp;
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A Global database of methane concentrations and atmospheric fluxes for streams and rivers

This dataset, referred to as MethDB, is a collation of publicly available values of methane (CH4) concentrations and atmospheric fluxes for world streams and rivers, along with supporting information on location, geographic, physical, and chemical conditions of the study sites. The data set is composed of four linked tables, corresponding to the data sources (Papers_MethDB), the study sites (Sites_MethDB), concentrations (Concentrations_MethDB), and influx/efflux rates (Fluxes_MethDB). Information was extracted from journal articles, government reports, book chapters, and similar sources that were acquired before 15 September 2015. Concentrations and fluxes were converted to a standard unit (micromoles per liter for concentration and millimoles per square meter per day for flux) and both the author-reported and converted data are included in the database. MethDB was assembled as part of a larger synthesis effort on stream and river CH4 dynamics, and assembled data were used to identify large-scale patterns and potential drivers of fluvial CH4 and to generate an updated global-scale estimate of CH4 emissions from world rivers.
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CH4 data from streams and rivers are widely scattered, as values are often included as end-member in studies focused on other processes or types of ecosystems. Thus, while we sought to be as complete as possible in compiling existing data, some sources have undoubtedly been overlooked. Sources included journal articles, book chapters, dissertations, USGS open file reports, meeting proceedings, and unpublished results provided by individual investigators. Data incorporated into MethDB were strictly limited to surface waters of rivers and streams; values for groundwater, porewater, saturated soils, lakes, reservoirs, wetlands, estuaries, and floodplains were not included. Some papers were excluded because essential supporting information was missing (e.g., units), or extracting data from complex graphs was considered to be unwise. Data sources are listed in the Notes and Comments section below.
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WSC - Leaf area index (LAI) at various points within Wibu field site, 2012-2014

Leaf area index (LAI) measurements collected at various points within the Wibu field site between 2012-2014. Measurements were collected approximately weekly from plant emergence until appr. 1 month past the onset of senescence. The Wibu field site is a commercial agricultural field, which grew corn in the 2012, 2013, and 2014 growing seasons; therefore, these are all LAI values for corn. See Zipper and Loheide (2014) Ag. For. Met. for more information about the field site and use of the LAI data.
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Measurements were made using a Licor-brand LAI-2200 device at approximately weekly intervals during the 2012, 2013, and 2014 growing seasons. At each point, the measurements are the average LAI value calculated from 20 subcanopy measurements ranging from in-line with planted rows to the area between rows. In 2012 and 2013, measurements were taken exclusively during diffuse light conditions (sunrise, sunset, or full cloud cover). In 2014, measurements were taken during daylight hours and corrected for light scattering using the LAI-2200C correction described on their website. This means that 2014 data is less accurate than 2012-2013 data. Note that, for each growing season, a date is listed with 0 LAI &ndash; this corresponds to the date prior to emergence at each site.After installation of wells and soil monitoring gear in each of the 2012, 2013, and 2014 growing seasons, we used a Topcon GR5 RTK-GPS to collect the location of each relevant point within the field. In 2014, a gridded set of soil sampling points was also measured.
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WSC - Water surface elevation (WSE) and water table depth (WTD) from 14 points at the Wibu field site, 2012-2013 growing seasons

Observation wells were installed for the purpose of continuously monitoring the water table level during the 2012 and 2013 growing seasons at the Wibu field site. These data were then used to study the yield response of corn to water table depth, soil texture, and growing season weather conditions (Zipper et al., in prep). The Wibu field site is a commercial agricultural field, which grew corn in the 2012, 2013, and 2014 growing seasons. See Zipper and Loheide (2014) Ag. For. Met. for more information about the field site. The 2012 growing season was characterized by severe drought, and the water table fell below the bottom of most wells in late June/early July.
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Each well consisted of a casing and a pressure transducer. Casings were made of 1.5 diameter PVC pipe. Each casing consisted of a screened interval, approximately 0.61 m in length, and continuous casing from the top of the screened interval to the land surface. Wells were installed via hand augering to greater than 1.5 meters below the water table at the time of installation. Where augering was impeded by subsurface rocks and gravel, steel drive point wells were used. Pea gravel was used to backfill the hole to the top of the screened interval, after which soil removed during the well installation process was re-packed to approximately the same density as prior to installation. A 5 cm layer of bentonite was installed at the land surface to prevent preferential flow down the borehole. After installation, wells were pumped to reduce the risk of fine sediment clogging the well screen, and then Onset HOBO U20 water level loggers were installed in each well. Of the 14 total wells, 9 wells were installed during the 2012 growing season (WIBU-1,2,3,5,7,8,9,10,11) and an additional 5 wells installed during the 2013 growing season (WIBU-4,12,13,14,15). Wells within the field were installed following planting and uninstalled prior to harvest for each growing season; the individual installation and start/end dates are evident in the data. Locations of individual wells are available in the Point Locations dataset. Note that some wells move between years due to uninstallation prior to harvest.
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