Methods
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.
