DOI: 10.5061/dryad.s4mw6m99r
title: Data from: A test of the seasonal availability of water hypothesis in a C3/C4 mixed grassland

description: Understanding how cool-season C3 and warm-season C4 grasses will respond
 to climate change is critical for predicting future grassland functioning.
 With warming, C4 grasses are expected to increase relative to C3 grasses.
 But, alterations in the seasonal availability of water may also influence
 C3/C4 dynamics because of their distinct seasons of growth. To better
 understand how shifts in the seasonal availability of water can affect
 ecosystem function in a northern mixed grass prairie in southeastern
 Wyoming, we reduced early season rainfall (April – June 2021) using
 rainout shelters and added the amount of excluded precipitation during the
 latter half of the growing season (July-September), effectively shifting
 spring rainfall to summer rainfall.  As expected, this shift in
 precipitation seasonality influenced patterns of soil water availability,
 leading to increased soil respiration in the summer months and sustained
 canopy greenness throughout the growing season. Despite these responses,
 there were no significant differences in C3 aboveground net primary
 production (ANPP) between the seasonally shifted treatment (SEAS) and the
 plots that received ambient (AMB) precipitation. This was likely due to
 the high levels of spring soil moisture present before rainout shelters
 were deployed that sustained C3 grass growth.  However, in plots
 with high C4 grass cover, C4 ANPP increased significantly in response to
 increased summer rainfall. Overall, we provide the first experimental
 evidence that shifts in the seasonality of precipitation, with no change
 in temperature, will differentially impact C3 vs. C4 species, altering the
 dynamics of carbon cycling and canopy albedo in this extensive semi-arid
 grassland.
description: <em>Experimental Design</em>
 Before the 2021 growing season, we established twenty 1
 m<sup>2</sup> plots (n=10 per treatment). Plots were separated
 by at least 3 m, and aluminum flashing was installed (10 cm belowground
 and 5 cm aboveground) 20 cm outside of the plot perimeter to reduce
 surface and shallow soil water movement into and out of each plot. 
 Rainout shelter roofs (2.44 m × 3.05 m made of clear corrugated
 polycarbonate, Suntuf, Palram Americas) that were larger than the 1
 m<sup>2</sup> plots were then placed over ten of the plots.
 Roofs were initially installed 80 cm above the ground at a slight angle to
 allow water to drain away from the plot; later in the season, the shelters
 were raised to 100 cm. Although previous work has demonstrated that these
 shelters have minimal influence on the microclimate (Loik et al. 2019;
 Post and Knapp 2020; Hoover et al. 2022), we monitored soil temperatures
 at 10 cm weekly and evaluated light transmission under the roofs using a
 1-m linear quantum light sensor (Decagon AccuPAR, model LP-80).
 We altered the seasonal dynamics of soil water availability
 (seasonally shifted treatment, SEAS) by using the clear roofs to exclude
 all precipitation from April 10 – June 30 (some minor blow-in of
 precipitation during storms was inevitable). We removed the shelters July
 1 and added the equivalent amount of water excluded in the spring to these
 plots in addition to the ambient precipitation received.  The additional
 precipitation was applied manually throughout July – September replicating
 the distribution of rainfall events from the spring
 (<em>i.e.</em>, additions were similar in event frequency and
 magnitude, Table S1). This ensured that total growing season precipitation
 was similar for the SEAS and the ambient (AMB) treatment, which received
 natural precipitation for the entire growing season. Precipitation was
 recorded at a nearby NOAA weather station (Cheyenne Weather Forecast
 Office, (41.1516, -104.80622)). Treatments (n=10) were randomly assigned,
 and because there was some minor topographical variation in the landscape
 (10 plots were slightly uphill from the others), we assigned treatments
 within two blocks to control for any effects topography. Block effects
 were non-significant in our analyses, but after plants became active, we
 noted that the cover of C<sub>3</sub> and
 C<sub>4</sub> functional groups varied widely among plots.
 Thus, we estimated total plant cover by species in each plot to account
 for this variation in our analyses. <em>Measured
 Responses</em> We measured soil moisture
 (volumetric water content, VWC) weekly in all plots throughout the
 experiment (April 10 – September 23) with a 20 cm handheld soil moisture
 time-domain reflectometry probe (Campbell-Scientific Hydrosense II). This
 instrument integrates soil moisture in the top 20 cm of soil where most of
 the root biomass in this grassland is located (Sun et al. 1997; Carillo et
 al. 2014). To assess treatment effects on plant water status, we estimated
 mid-day (12:00 - 14:00hr MST) leaf water potential with a Scholander
 pressure chamber (PMS instruments) for a dominant
 C<sub>3</sub> grasses, <em>Pascopyrum
 smithii</em>, and the dominant C<sub>4</sub> grass,
 <em>Bouteloua gracilis</em>. Fully expanded, mature canopy
 leaves (1-2 leaves per plot, n=6 per treatment) were collected each week.
 Because the C<sub>3</sub> and C<sub>4</sub>
 grasses become active at different times of the growing season, we
 measured <em>P. smithii </em>water status from May 27 – Sept.
 16 and <em>B. gracilis</em> from June 15 - Sept. 16.
 <em>            </em>To evaluate how differences in
 the seasonal availability of water influenced C<sub>3</sub>
 and C<sub>4</sub> dynamics and ecosystem function, we measured
 canopy greenness and soil CO<sub>2 </sub>efflux weekly,
 photosynthetic rates for the primary C<sub>3</sub> and
 C<sub>4</sub> species in June and July, and ANPP at the end of
 the growing season (September).             Canopy
 greenness, measured to assess canopy-scale phenological responses and to
 serve as a proxy for potential ecosystem carbon uptake, was estimated with
 repeat digital photography (following the methods of Post and Knapp 2020,
 Hoover et al. 2022). Briefly, an iPhone camera was positioned directly
 above a marked 50 cm x 50 cm frame in a corner of each plot, and each
 photograph was then cropped to contain only the interior area of the
 frame. These cropped photos were processed using the R package EBImage
 (Pau et al. 2010) to calculate the average green chromatic coordinate
 (GCC) index (Filippa et al. 2016). The GCC index accounts for variation in
 pixel brightness (Filippa et al. 2016), thus avoiding background light
 levels and potential infrastructure impacts. Soil
 respiration was measured weekly (May 5 – Sept. 23) to quantify how the
 treatments influenced this important carbon flux (Hashimoto et al. 2015).
 Permanent PVC collars (10 cm in diameter, n=6 per treatment) were
 installed in locations between grasses at the end of April (2.4 cm
 belowground and 2 cm aboveground), and all vegetation within the collars
 was removed (clipped at the base). Before each measurement, any new
 vegetation growth was also gently removed.  Soil respiration was measured
 using a 6400-09 soil flux chamber attached to an LI-6400XT (LiCor., Inc,
 Lincoln NE, USA). Measurements were taken mid-day (between 8:30hr –
 12:30hr MST) at ambient CO<sub>2</sub> concentration,
 humidity, and temperature.             Leaf
 gas-exchange was measured (June 23-24) prior to roofs coming off and after
 the roofs were removed (July 24-25). On each date, a portable
 photosynthesis system (LI-6400, LiCor., Inc, Lincoln NE, USA) was used to
 measure the CO<sub>2</sub> uptake (net photosynthesis, or A)
 on 12 fully expanded mature upper canopy leaves for both
 C<sub>3</sub> (<em>P. smithii</em>) and
 C<sub>4</sub> (<em>B. gracilis</em>) individuals
 in each treatment. The LI-6400 was fitted with a 3×2 cm
 cuvette head and a red-blue LED light source. For all measurements, flow
 rate was held constant at 600 μmol s<sup>-1</sup>. The
 temperature exchanger was set to an average midday summer temperature of
 30 °C. Leaf temperature (T<sub>leaf</sub>) was measured with a
 thermocouple and averaged 31 ± 1.7 °C (standard deviation) across all
 measurement dates. Relative humidity conditions in the chamber were
 controlled near ambient levels but did vary slightly depending upon water
 vapor fluxes from the leaf. Photosynthetic photon flux density in the
 chamber was set at 1800 μmol m<sup>-2</sup>
 s<sup>-1</sup>, approximating full-sun conditions to measure
 light-saturated net photosynthesis (A<sub>sat</sub>) and
 stomatal conductance to water vapor (g<sub>s</sub>, Fig. S1)
 at a chamber reference [CO<sub>2</sub>] of 420 μmol
 mol<sup>-1</sup>. All measurements occurred between 10:00hr
 and 15:00hr MST Finally, ANPP was estimated near the
 end of the growing season (Sept. 1-2) in all plots as plants began to
 senesce.  For each plot, all aboveground vegetation within two 0.1
 m<sup>2</sup> subplots was harvested to ground height, sorted
 by functional group (C<sub>3</sub> grass,
 C<sub>4</sub> grass, forb, woody, or annual grass), and then
 dried at 60 °C for 48 hours before being weighed to the nearest 0.01g.
 Previous year’s growth was easily distinguished from current year growth
 and was not included.
description: # Data from: A test of the seasonal availability of water hypothesis in a
 C3/C4 mixed grassland Abstract: To better understand how shifts in the
 seasonal availability of water can affect ecosystem function in a northern
 mixed grass prairie in southeastern Wyoming, we reduced early season
 rainfall (April June 2021) using rainout shelters and added the amount of
 excluded precipitation during the latter half of the growing season
 (July-September), effectively shifting spring rainfall to summer rainfall.
 As expected, this shift in precipitation seasonality influenced patterns
 of soil water availability, leading to increased soil respiration in the
 summer months and sustained canopy greenness throughout the growing
 season. Despite these responses, there were no significant differences in
 C3 aboveground net primary production (ANPP) between the seasonally
 shifted treatment (SEAS) and the plots that received ambient (AMB)
 precipitation. This was likely due to the high levels of spring soil
 moisture present before rainout shelters were deployed that sustained C3
 grass growth. However, in plots with high C4 grass cover, C4 ANPP
 increased significantly in response to increased summer rainfall. Overall,
 we provide the first experimental evidence that shifts in the seasonality
 of precipitation, with no change in temperature, will differentially
 impact C3 vs. C4 species, altering the dynamics of carbon cycling and
 canopy albedo in this extensive semi-arid grassland. Contact: Olivia Hajek
 () Data collection location: USDA-ARS
 High Plains Research Station (41.20, -104.88) Data collection time period:
 2021-04-10 2021-09-23 File information: 10 files are included in this
 folder. 1. README. Contains detailed information regarding files 2-9. File
 format: .pdf 2. ANPP. Aboveground net primary production of the
 experimental plots collected at the beginning of September. File format:
 .csv Variables: Plot: Plot number of sample (Plots 1-20) Trt: Treatment
 (AMB = Ambient, SEAS = Seasonally shifted treatment) Cover_C4:
 Categorization of percent C4 grass cover for the plot (high corresponds to
 &gt; 25%; low corresponds to &lt; 5%) Rep: Experimental replication of
 sample (0.1 m2, 2 reps/plot) C3: Mass of C3 perennial grasses in sample,
 in grams Understanding how cool-season C3 and warm-season C4 grasses will
 respond to climate change is critical for predicting future grassland
 functioning. With warming, C4 grasses are expected to increase relative to
 C3 grasses. But, alterations in the seasonal availability of water may
 also influence C3/C4 dynamics because of their distinct seasons of growth.
 C4: Mass of C4 perennial grasses in sample, in grams Forb: Mass of forbs
 in sample, in grams Woody: Mass of woody species in sample, in grams
 Annual: Mass of C3 annual grasses in sample, in grams Cactus: Percent
 cover of cactus in the sample Total_biomass: Total mass of entire sample,
 in grams ANPP_total: Aboveground net primary production in g m-2
 (total_biomass converted to an area) 3. Canopy greenness. Plot greenness
 from repeat digital photography, as calculated with the green chromatic
 coordinate (GCC), capturing the growing season phenology dynamics
 (2021-04-09 2021-09-23). File format: .csv Variables: Date: Date of
 measurement (dd-mm-yy) Plot: Plot number of sample (Plots 1-20) Trt:
 Treatment (AMB = Ambient, SEAS = Seasonally shifted treatment) Greenness:
 Average green chromatic coordinate of the pixels for each plot image 4.
 Soil moisture. Soil moisture measured integrated across the upper 20 cm
 from 2021- 04-10 2021-09-23. File format: .csv Variables: Date: Date of
 measurement (dd-mm-yy) Plot: Plot number of sample (Plots 1-20) Trt:
 Treatment (AMB = Ambient, SEAS = Seasonally shifted treatment) Season:
 Corresponds to the first or second half of the growing season (Pre = roofs
 in place; post = after roof removal) sm_20: soil moisture measured as the
 % volumetric water content integrated over the upper 20 cm of soil 5. Soil
 respiration. Soil CO2 efflux of the plot in mol CO2 m-2 s-1 from
 2021-05-05 2021-09- 23 File format: .csv Variables: Date: Date of
 measurement (dd-mm-yy) Plot: Plot number of sample (Plots 1-20) Trt:
 Treatment (AMB = Ambient, SEAS = Seasonally shifted treatment) Season:
 Corresponds to the first or second half of the growing season (Pre = roofs
 in place; post = after roof removal) Efflux: CO2 efflux (soil respiration)
 of the plot in mol CO2 m-2 s-1. 6. Soil temperature. Soil temperature to a
 depth of 10cm of the plot in degrees Celsius from 2021-05-07 2021-09-23
 File format: .csv Variables: Date: Date of measurement (dd-mm-yy) Plot:
 Plot number of sample (Plots 1-20) Trt: Treatment (AMB = Ambient, SEAS =
 Seasonally shifted treatment) Season: Corresponds to the first or second
 half of the growing season (Pre = roofs in place; post = after roof
 removal) Soil_temp: Measurement of soil temperature to a depth of 10 cm
 for each plot, in degrees Celsius 7. Water potential. Water potential in
 MPa measured throughout the growing season for a dominant C3 grass
 (Pascopryum smithii; 2021-05-27 2021-09-23) and the dominant C4 grass
 (Bouteloua gracilis; 2021-06-15 2021-09-23). File format: .csv Variables:
 Date: Date of measurement (dd-mm-yy) Plot: Plot number of sample (Plots
 1-20) Trt: Treatment (AMB = Ambient, SEAS = Seasonally shifted treatment)
 Season: Corresponds to the first or second half of the growing season (Pre
 = roofs in place; post = after roof removal) Species: 4-letter code for
 each plant species (PASM = Pascopryum smithii; BOGR = Bouteloua gracilis)
 Functional_Group: Functional group that the plant belongs to (C3 = C3
 perennial grass; C4 = C4 perennial grass) WP: Measured water potential in
 MPa 8. Species Composition. Visual estimate of the plot cover by species
 for all experimental plots, assessed in mid-August File format: .csv
 Variables: Species: Name of plant species present in the plot
 Functional_group: Functional group that the plant belongs to p_1 p_20:
 Correspond to plot where measurement was taken (ie. p_1 refers to plot 1)
 and the value represents the % cover of each species 9. Light
 transmission. Measurement of the ratio of light transmitted through the
 rainout shelters observed on three different sunny days. File format: .csv
 Variables: Date: Date of measurement (dd-mm-yy) Plot: Plot number of
 sample (Plots 1-20) Trt: Treatment (SEAS = Seasonally shifted treatment)
 Outside: PAR, radiation in the 400- to 700-nm waveband, representing the
 portion of the spectrum that plants use for photosynthesis, observed
 outside of the shelter Underneath: PAR, radiation in the 400- to 700-nm
 waveband, representing the portion of the spectrum that plants use for
 photosynthesis, observed underneath the shelter t: ratio of below-canopy
 PAR measurements to the most recent above-canopy PAR measurement,
 calculated automatically by the LP-80. 10. Photosynthesis. Photosythesis
 measurements taken with a LI-6400 (LiCor., Inc, Lincoln NE, USA) in June
 prior to roof removal and in July post roof removal for a dominant C3
 grass and the dominant C4 grass. File format: .csv Variables: HHMMSS:
 Realtime clock FTime: Time since logging started Photo: Photosynthetic
 rate (mol CO2 m-2 s-1) Cond: Stomatal conductance mol m-2 s-1 Ci:
 intercellular CO2 concentration, mol CO2 mol air-1 Trmmol: Transpiration
 in mmol m-2s-1 VpdL: Vapor pressure deficit based on leaf temp in kPa
 Area: In-chamber leaf area cm-2 StmRat: Stomatal ratio estimate BLCond:
 Total boundary layer conductance for the leaf in mol m-2 s-1 Tair: Chamber
 air temperature in degrees Celsius Tleaf: Leaf thermocouple in degress
 Celsius Tblk: IRGA Block Temp C CO2R: Reference cell CO2 concentration in
 mol mol-1 CO2S: Sample cell CO2 concentration in mol mol-1 H2OR: Reference
 cell H2O concentration in mmol mol-1 H2OS: Sample cell H2O concentration
 in mmol mol-1 RH_R: Reference relative humidity % RH_S: Sample relative
 humidity % Flow: Flow rate into chamber in mol mol-1 PARi: In-chamber PAR
 mol m-2 s-1 PARo: External PAR mol m-2 s-1 Press: Atmospheric pressure in
 kPa CsMch: Sample CO2 offset mol mol-1 HsMch: Sample H2O offset mmol mol-1
 StableF: Stability status as a decimal value Status: Primary numerical
 status information on the state of the system KEEP: Y means included in
 analyses; N is excluded Date: Date of measurement (mm/dd/yy) Plot: Plot
 number of sample (Plots 1-20) Trt: Treatment (AMB = Ambient, SEAS =
 Seasonally shifted treatment) C: value 3 correspond to C3 (Pascopryum
 smithii) and 4 corresponds C4 (Bouteloua gracilis) plant species
 Experimental Design Before the 2021 growing season, we established twenty
 1 m2 plots (n=10 per treatment). Plots were separated by at least 3 m, and
 aluminum flashing was installed (10 cm belowground and 5 cm aboveground)
 20 cm outside of the plot perimeter to reduce surface and shallow soil
 water movement into and out of each plot. Rainout shelter roofs (2.44 m
 3.05 m made of clear corrugated polycarbonate, Suntuf, Palram Americas)
 that were larger than the 1 m2 plots were then placed over ten of the
 plots. Roofs were initially installed 80 cm above the ground at a slight
 angle to allow water to drain away from the plot; later in the season, the
 shelters were raised to 100 cm. Although previous work has demonstrated
 that these shelters have minimal influence on the microclimate (Loik et
 al. 2019; Post and Knapp 2020; Hoover et al. 2022), we monitored soil
 temperatures at 10 cm weekly and evaluated light transmission under the
 roofs using a 1-m linear quantum light sensor (Decagon AccuPAR, model
 LP-80). We altered the seasonal dynamics of soil water availability
 (seasonally shifted treatment, SEAS) by using the clear roofs to exclude
 all precipitation from April 10 June 30 (some minor blow-in of
 precipitation during storms was inevitable). We removed the shelters July
 1 and added the equivalent amount of water excluded in the spring to these
 plots in addition to the ambient precipitation received. The additional
 precipitation was applied manually throughout July September replicating
 the distribution of rainfall events from the spring (i.e., additions were
 similar in event frequency and magnitude, Table S1). This ensured that
 total growing season precipitation was similar for the SEAS and the
 ambient (AMB) treatment, which received natural precipitation for the
 entire growing season. Precipitation was recorded at a nearby NOAA weather
 station (Cheyenne Weather Forecast Office, (41.1516, -104.80622)).
 Treatments (n=10) were randomly assigned, and because there was some minor
 topographical variation in the landscape (10 plots were slightly uphill
 from the others), we assigned treatments within two blocks to control for
 any effects topography. Block effects were non-significant in our
 analyses, but after plants became active, we noted that the cover of C3
 and C4 functional groups varied widely among plots. Thus, we estimated
 total plant cover by species in each plot to account for this variation in
 our analyses. Measured Responses We measured soil moisture (volumetric
 water content, VWC) weekly in all plots throughout the experiment (April
 10 September 23) with a 20 cm handheld soil moisture time-domain
 reflectometry probe (Campbell-Scientific Hydrosense II). This instrument
 integrates soil moisture in the top 20 cm of soil where most of the root
 biomass in this grassland is located (Sun et al. 1997; Carillo et al.
 2014). To assess treatment effects on plant water status, we estimated
 mid-day (12:00 - 14:00hr MST) leaf water potential with a Scholander
 pressure chamber (PMS instruments) for a dominant C3 grasses, Pascopyrum
 smithii, and the dominant C4 grass, Bouteloua gracilis. Fully expanded,
 mature canopy leaves (1-2 leaves per plot, n=6 per treatment) were
 collected each week. Because the C3 and C4 grasses become active at
 different times of the growing season, we measured P. smithii water status
 from May 27 Sept. 16 and B. gracilis from June 15 - Sept. 16. To evaluate
 how differences in the seasonal availability of water influenced C3 and C4
 dynamics and ecosystem function, we measured canopy greenness and soil CO2
 efflux weekly, photosynthetic rates for the primary C3 and C4 species in
 June and July, and ANPP at the end of the growing season (September).
 Canopy greenness, measured to assess canopy-scale phenological responses
 and to serve as a proxy for potential ecosystem carbon uptake, was
 estimated with repeat digital photography (following the methods of Post
 and Knapp 2020, Hoover et al. 2022). Briefly, an iPhone camera was
 positioned directly above a marked 50 cm x 50 cm frame in a corner of each
 plot, and each photograph was then cropped to contain only the interior
 area of the frame. These cropped photos were processed using the R package
 EBImage (Pau et al. 2010) to calculate the average green chromatic
 coordinate (GCC) index (Filippa et al. 2016). The GCC index accounts for
 variation in pixel brightness (Filippa et al. 2016), thus avoiding
 background light levels and potential infrastructure impacts. Soil
 respiration was measured weekly (May 5 Sept. 23) to quantify how the
 treatments influenced this important carbon flux (Hashimoto et al. 2015).
 Permanent PVC collars (10 cm in diameter, n=6 per treatment) were
 installed in locations between grasses at the end of April (2.4 cm
 belowground and 2 cm aboveground), and all vegetation within the collars
 was removed (clipped at the base). Before each measurement, any new
 vegetation growth was also gently removed. Soil respiration was measured
 using a 6400-09 soil flux chamber attached to an LI-6400XT (LiCor., Inc,
 Lincoln NE, USA). Measurements were taken mid-day (between 8:30hr 12:30hr
 MST) at ambient CO2 concentration, humidity, and temperature. Leaf
 gas-exchange was measured (June 23-24) prior to roofs coming off and after
 the roofs were removed (July 24-25). On each date, a portable
 photosynthesis system (LI-6400, LiCor., Inc, Lincoln NE, USA) was used to
 measure the CO2 uptake (net photosynthesis, or A) on 12 fully expanded
 mature upper canopy leaves for both C3 (P. smithii) and C4 (B. gracilis)
 individuals in each treatment. The LI-6400 was fitted with a 32 cm cuvette
 head and a red-blue LED light source. For all measurements, flow rate was
 held constant at 600 mol s-1. The temperature exchanger was set to an
 average midday summer temperature of 30 C. Leaf temperature (Tleaf) was
 measured with a thermocouple and averaged 31 1.7 C (standard deviation)
 across all measurement dates. Relative humidity conditions in the chamber
 were controlled near ambient levels but did vary slightly depending upon
 water vapor fluxes from the leaf. Photosynthetic photon flux density in
 the chamber was set at 1800 mol m-2 s-1, approximating full-sun conditions
 to measure light-saturated net photosynthesis (Asat) and stomatal
 conductance to water vapor (gs, Fig. S1) at a chamber reference [CO2] of
 420 mol mol-1. All measurements occurred between 10:00hr and 15:00hr MST
 Finally, ANPP was estimated near the end of the growing season (Sept. 1-2)
 in all plots as plants began to senesce. For each plot, all aboveground
 vegetation within two 0.1 m2 subplots was harvested to ground height,
 sorted by functional group (C3 grass, C4 grass, forb, woody, or annual
 grass), and then dried at 60 C for 48 hours before being weighed to the
 nearest 0.01g. Previous years growth was easily distinguished from current
 year growth and was not included.