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ECOPHYSIOLOGY, STRESS AND ADAPTATION
Warming impacts on photosynthetic processes in dominant
plant species in a subtropical forest
Michael J. Aspinwall
1,2
| Jeff Chieppa
1,2
| Eve Gray
1
| Morgan Golden-Ebanks
1
|
Lynsae Davidson
1
1
Department of Biology, University of North
Florida, Jacksonville, Florida, USA
2
School of Forestry and Wildlife Sciences,
Auburn University, Auburn, Alabama, USA
Correspondence
Michael J. Aspinwall, School of Forestry and
Wildlife Sciences, Auburn University,
602 Duncan Drive, Auburn, AL 36849, USA.
Email: aspinwall@auburn.edu
Funding information
National Institute of Food and Agriculture,
Grant/Award Numbers: 1025522,
2019-67013-29161
Edited by I. Ensminger
Abstract
Climate warming could shift some subtropical regions to a tropical climate in the next
30 years. Yet, climate warming impacts on subtropical species and ecosystems
remain unclear. We conducted a passive warming experiment in a subtropical forest
in Florida, USA, to determine warming impacts on four species differing in their cli-
matic distribution, growth form, and functional type: Serenoa repens (palm), Andro-
pogon glomeratus (C
4
grass), Pinus palustris (needled evergreen tree), and Quercus
laevis (broadleaved deciduous tree). We hypothesized that warming would have
neutral-positive effects on photosynthetic processes in monocot species with
warmer climatic distributions or adaptations to warmer temperatures, but negative
effects on photosynthesis in tree species. We also hypothesized that periods of low
soil moisture would alter photosynthetic responses to warming. In both monocot
species, warming had no significant effect on net photosynthesis (A) or stomatal con-
ductance (g
s
) measured at prevailing temperatures, or photosynthetic capacity mea-
sured at a common temperature. In P.palustris, warming reduced A(15%) and g
s
(28%), and caused small reductions in Rubisco carboxylation and RuBP regenera-
tion. Warming had little effect on photosynthetic processes in Q.laevis. Interestingly,
A.glomeratus showed little sensitivity to reduced soil moisture, and all C
3
species
reduced Aand g
s
as soil moisture declined and did so consistently across temperature
treatments. In subtropical forests of the southeastern US, we conclude that climate
warming may have neutral or slightly positive effects on the performance of grasses
and broadleaved species but negative effects on P.palustris seedlings, foreshadowing
possible changes in community and ecosystem properties.
1|INTRODUCTION
Global mean surface temperatures increased 0.85C between 1880
and 2012 (IPCC, 2013), contributing to changes in species distribu-
tions, shifts in plant community composition, and alterations of eco-
system structure and function (Bertrand et al., 2011; Elmendorf
et al., 2015; Hughes, 2000; Kelly & Goulden, 2008; Lenoir
et al., 2008). Further warming of 2–4C is expected by the middle of
the century (IPCC, 2013). Warming impacts on many species and
communities remain difficult to predict, and may depend upon
variation in life-history traits, local adaptation, and physiological toler-
ances of component species (Li et al., 2016; Shi et al., 2015). Further-
more, the magnitude of warming, ecosystem structure, and a host of
biotic and abiotic factors can modify community and ecosystem
responses to warming (Coomes et al., 2014; Tingstad et al., 2015; van
Bogaert et al., 2011). New experiments are needed to provide insight
into warming impacts on plant species, communities, and ecosystems
in understudied regions and potential modifying factors.
Ecotones are boundary areas where vegetation types change or
species with different climatic distributions overlap. Experiments and
Received: 24 August 2021 Accepted: 20 February 2022
DOI: 10.1111/ppl.13654
Physiologia Plantarum
Physiologia Plantarum. 2022;174:e13654. wileyonlinelibrary.com/journal/ppl © 2022 Scandinavian Plant Physiology Society. 1of17
https://doi.org/10.1111/ppl.13654
observational studies in ecotones help reveal differential responses to
warming among component species and may help predict changes in
community and ecosystem attributes with further warming
(Cavanaugh et al., 2014; Harsch et al., 2009; Løkken et al., 2020).
Most studies have focused on elevational transects or high latitude
(boreal, tundra) ecotones; areas where vegetation transitions are
especially clear, rates of warming are higher, and ecosystem responses
to warming are more pronounced (Carroll et al., 2017; Pretzsch
et al., 2020; Reich et al., 2015). There have been relatively few
warming experiments in ecosystems located in transitional areas
between temperate and tropical climates. This is notable given that
some of the most productive and species-rich ecosystems on the
planet occur in these regions (Kreft & Jetz, 2007; Running
et al., 2004). Although rates of warming are generally slower in these
regions compared to higher latitude regions, small temperature
changes could still have significant impacts. Modest warming could
reduce the performance of species growing close to their warmest
geographic or physiological limit (Drake et al., 2015; Wertin
et al., 2011), improve the performance of species growing below their
temperature optimum (Kauppi et al., 2014), and promote the influx of
species from tropical regions resulting in community “thermophilization”
(Feely et al., 2020).
Changes in water availability (i.e. soil moisture) could enhance,
diminish, or possibly reverse positive effects of climate warming on
plant function and productivity. Warmer temperatures could also
increase evapotranspiration and reduce soil water availability (Xu
et al., 2013, but see Zavaleta et al., 2013), resulting in diminished or
negative responses to climate warming. However, direct tests of cli-
mate warming effects along temporal or spatial soil moisture gradients
are rare. According to the “law of the minimum”and multiple limita-
tion theory (Bloom et al., 1985), warming may alleviate the tempera-
ture limitation of enzyme activity when temperatures are below the
species optimum, resulting in higher photosynthetic rates and greater
carbon available for growth, respiration, and defense. Warming should
alleviate low-temperature limitations until water becomes limiting,
triggering stomatal closure and limitation of CO
2
for photosynthesis.
Indeed, there is evidence that low soil moisture diminishes the posi-
tive effects of climate warming on photosynthesis and productivity in
high latitude tree species growing in cold, seasonal climates
(D'Orangeville et al., 2018; Reich et al., 2018; Zhang et al., 2020).
Whether similar patterns exist in warmer and less seasonal lower lati-
tude ecosystems is unclear (Carter et al., 2020). In these ecosystems,
species may experience low-temperature limitations less frequently,
and warming might reduce carbon uptake and productivity of some
species when temperatures are high, even if water is not limiting.
Many plants acclimate to warmer growth temperatures through a
series of adjustments, including an increase in the short-term temper-
ature optimum of photosynthesis and reduced rates of Rubisco car-
boxylation (V
cmax
) or RuBP regeneration (J
max
) at a common
measurement temperature (Gunderson et al., 2010; Kumarathunge
et al., 2019; Sendall et al., 2015). But these adjustments vary among
species and environments and have variable impacts on realized
photosynthesis at warmer growth temperatures (see Way & Yamori,
2014). In some species and environments, a “detractive adjustment”
occurs when thermal acclimation results in lower net photosynthesis
(A) under warmer temperatures. In other cases, a “constructive adjust-
ment”occurs when thermal acclimation results in higher Aunder
warmer temperatures. Additional studies that investigate thermal
acclimation responses and impacts on realized Awould improve our
understanding of the consequences of photosynthetic temperature
acclimation (Carter et al., 2021). More broadly, there is a continued
need to examine physiological responses to warming in different plant
functional types and species in underrepresented regions. For exam-
ple, there is uncertainty about whether C
3
and C
4
species differ in
their capacity to acclimate to temperature changes (Smith &
Dukes, 2017; Yamori et al., 2014), and the extent to which species
representing different functional types and growth forms growing
together in a community differ in their response to warmer tempera-
tures remains understudied (de Valpine & Harte, 2001; Hoeppner &
Dukes, 2012; Volder et al., 2013; Wertin et al., 2015). Addressing
these uncertainties is important given that community and ecosystem
responses to warming will depend on the aggregate response of dif-
ferent functional types and individual species.
The southeastern US lies at the boundary of temperate and tropi-
cal climates and is classified as “warm temperate”or “humid subtropi-
cal”(Beck et al., 2018). The region contains the highest diversity of
plant species in North America (Keil & Chase, 2019; Weakley
et al., 2020), and warm-wet growing seasons contribute to higher
rates of primary production than many regions in the US (Turner
et al., 1995). Mean annual air temperatures in the region have
increased by approximately 1C since 1970 (Karl et al., 2009). This
rate of warming is low compared to higher latitude regions of North
America (IPCC, 2013). However, average temperatures in the region
are expected to increase by 1–3C over the next 30–50 years with
higher minimum temperatures and more extremely hot days (Carter
et al., 2018). Although there are signs that warming is driving
“tropicalization”in coastal wetland communities in the southeastern
US (Cavanaugh et al., 2019), warming impacts on terrestrial ecosys-
tems in the region remains uncertain.
The objective of this study was to determine the effects of exper-
imental warming on leaf physiology and biomass production in sub-
tropical forest species and the potential modifying effects of soil
moisture. To accomplish this, we grew seedlings of four species with
contrasting climatic distributions, growth forms, and functional strate-
gies (Andropogon glomeratus (C
4
grass), Serenoa repens (woody shrub
palm), Pinus palustris (needled evergreen tree), Quercus laevis
(broadleaved deciduous tree)) in a 1.5-year-long passive warming
experiment (mean daily air temperature +1.0C, mean daytime air
temperature +2.5C) in a subtropical forest in northeast Florida. We
repeatedly measured leaf gas-exchange at prevailing soil moisture and
leaf temperatures on seedlings of each species grown under ambient
and warm temperatures, and determined warming effects on photo-
synthetic biochemistry, total dry mass production, and biomass alloca-
tion after the experiment. Aggregated over time, we hypothesized
that warming would have negligible or positive effects on photosyn-
thesis and productivity in monocot species with warmer climatic
2of17 ASPINWALL ET AL.
Physiologia Plantarum
distributions or adaptations to warmer temperatures (A.glomeratus,S.
repens). Yet, warming would generally reduce photosynthesis and pro-
ductivity in tree species with more temperate distributions (P.pal-
ustris,Q.laevis). We also hypothesized that periods of low soil
moisture would negate or reverse the positive effects of warming on
photosynthesis in warm-adapted species and exacerbate the negative
effects of warming on photosynthesis in tree species.
2|MATERIALS AND METHODS
2.1 |Study site, experimental design, and species
This study took place on the University of North Florida campus in
Jacksonville, FL (30.2619N, 81.5165W). The mean annual tempera-
ture (1948–2012) is 20.4C. The highest mean daily maximum tem-
perature (August) is 29.4C, and the lowest mean daily minimum
temperature (January) is 6.7C (Western Regional Climate Center,
2020, Cooperative Climatological Data Summaries. Retrieved from
https://wrcc.dri.edu/cgi-bin/cliMAIN.pl?fl4358). Historically, night-
time minimum temperatures fall below 0C roughly 10–15 days per
year (1948–2012), but freezing temperatures are increasingly rare
(Cavanaugh et al., 2014). The mean annual precipitation is 1332 mm.
Rain predominantly falls during the summer wet season spanning May
to September/October. The end of the dry season (October–May)
coincides with the onset of high temperatures. The experimental site
contained a mixed pine-oak overstory and an understory of
broadleaved shrubs and grasses typical of a xeric longleaf pine ecosys-
tem (Peet & Allard, 1993). The site was last burned 5–10 years before
the experiment, although the year and season of burning are
unknown, as is the average fire interval. Soils at the site are Leon fine
sand (Sandy, siliceous, thermic Aeric Alaquods), characterized by deep
sand, low organic matter content and fertility, poor drainage, and slow
permeability.
The study was comprised of six blocks, each containing two
1.5 1.5 m plots spaced 0.75 m apart. Blocks were randomly posi-
tioned in high-light forest gaps within a one-hectare area. Plots within
each block were randomly assigned to one of two treatments: ambi-
ent or warmed. The warmed plots were enclosed in 1.5 1.5 1.5 m
chambers, framed with 2.5 cm diameter white PVC, and wrapped in
6 mil polyethylene greenhouse film [modified after Charles and
Dukes (2009)]. Passive warming chambers were chosen because they
require no electricity, are inexpensive, semi-permanent, and easy to
deploy at difficult to access field sites. They also provide significant
warming by trapping radiation. However, as discussed elsewhere
(e.g. Marion et al., 1997), passive warming chambers have limitations,
including modifications of the light environment, lack of night-time
warming, and reduced wind speed. We endeavored to mitigate these
limitations. The polyethylene film transmits full-spectrum ultraviolet
light and approximately 90% of photosynthetically active radiation. A
circular (0.75 m diameter) opening was cut into the chamber roof to
allow for air circulation (mixing) and natural rainfall. Small slits were
cut in the remaining roof area to facilitate additional air movement
and rainwater infiltration into the plots. The chambers “trap”radiation
causing the air temperature to increase. Although minimal warming
occurs during cloudy weather or at night, the chambers warm air tem-
peratures rather than canopy surface temperatures (as occurs with
infrared heaters), and do not inhibit dew formation (Feng et al., 2021).
Control plots were left uncovered. We did not include additional con-
trol plots with PVC frames (no polyethylene film) to determine the
influence of the PVC frame. We expected that the narrow, white-
colored PVC frames would have little influence on the light environ-
ment in the control plot, and the addition of these plots would require
a 50% increase in the number of sensors and measurements, which
was deemed unfeasible. Air temperature (T
air
) and relative humidity
(RH) were measured every 15 min in the center of each treatment
plot (0.8 m above the ground) using a shielded air temperature/RH
sensor (Model US23 Pro v2, HOBO Instruments Inc.) attached to a
wooden post.
Chambers effectively increased T
air
. Over the course of the exper-
iment, mean daily (24 h) T
air
in the warmed treatment was on average
1.0C higher than mean daily T
air
in the ambient treatment (ambient
mean daily T
air
=20.8 ± 5.9 [standard deviation]C, warmed mean
daily T
air
=21.8 ± 6.2C). Mean daytime (08:00–18:00 h) T
air
in the
warmed treatment was on average 2.5C higher than mean daytime
T
air
in the ambient treatment (Figure 1). Warming of this magnitude
(1–3C) is expected by 2050 throughout the southeast US (Carter
et al., 2018). Mean daytime RH in the ambient treatment was 5.3%
higher than mean daytime RH in the warmed treatment (Figure 1).
Mean daytime VPD in the warmed treatment was on average
0.54 kPa higher than mean daytime VPD in the ambient treatment
(Figure 1). Volumetric soil water content over 0–15 depth (VWC, m
3
m
3
or %) was measured in each quadrant of each treatment plot
every 2–3 weeks with a handheld time domain reflectometer (TDR)
probe (HydroSense II, Campbell Scientific, Logan, UT). VWC was mea-
sured on leaf gas-exchange measurement dates (see below for details)
as well as several timepoints between leaf gas-exchange measure-
ments. Over time VWC ranged from 10.9 to 0.1%. These VWC values
range from below the permanent wilting point (approximately
1.5 MPa) to near field capacity, for sandy soil. No obvious wilting
occurred at very low VWC. VWC did not differ between ambient and
warmed treatments (Figure 1, P=0.75, mean VWC =5.5 ± 2.1%).
Four species were chosen for this experiment: Andropogon glo-
meratus (bushy bluestem), Serenoa repens (saw palmetto), Pinus pal-
ustris (longleaf pine), and Quercus laevis (turkey oak). These species are
common at the site and represent the major growth forms and func-
tional types in forests of the southeastern US Andropogon glomeratus
is a C
4
NADP-ME type perennial bunchgrass (herbaceous monocot)
with a distribution spanning the Atlantic and Gulf Coastal Plains from
Texas to North Carolina, and all of Florida (latitude range: 25.2–
38.0N, longitude range: 75.5–98.0W). Serenoa repens is a low-
growing evergreen shrub palm (woody monocot) native to all of Flor-
ida and warm coastal areas of Louisiana, Mississippi, Georgia, and
South Carolina (latitude range: 25.2–32.4N, longitude range: 80.5–
89.6W). Pinus palustris is a needled evergreen tree with a natural dis-
tribution that spans the Atlantic and Gulf Coastal Plains of the
ASPINWALL ET AL.3of17
Physiologia Plantarum
southeastern US, from central Florida to east Texas and north
into North Carolina (latitude range: 26.8–36.6N, longitude range:
77.0–95.2W). The species occurs on inland montane and piedmont
sites, xeric sandhill sites, and poorly drained coastal flatwoods.
FIGURE 1 (a) Temporal variation in mean daytime air temperature (T
air
), (c) mean daytime relative humidity (RH), (e) mean daytime vapor
pressure deficit (VPD), and (g) mean volumetric soil water content (VWC). Ambient (blue) and warmed (red) treatment data are shown separately
in each panel. Panels (b, d, f, h) show the overall distribution (percentage) of T
air
, RH, VPD, and VWC data in each treatment, as well as the mean
and median values
4of17 ASPINWALL ET AL.
Physiologia Plantarum
Quercus laevis is a broadleaved deciduous tree species with a distribu-
tion from central Florida to southeastern Louisiana and north into
southeast Virginia (latitude range: 26.8–35.8N, longitude range:
77.0–89.6W). The species is limited to dry pinelands and sandy
ridges within its range.
One similarly sized seedling (or rhizome cluster in the case of A.
glomeratus) of each species was randomly transplanted into one quad-
rant of each treatment plot on February 21, 2019. Seedlings of S.
repens and rhizome clusters of A.glomeratus were collected from loca-
tions within the forest. At transplanting, S.repens seedlings contained
an average of four leaves with average petiole lengths and leaf widths
of 16.7 ± 2.9 and 34.0 ± 4.9 cm, respectively. Average petiole length
and leaf width did not differ between treatments (P=0.22 and
P=0.34, respectively). A.glomeratus rhizome clusters averaged 4.9
± 1.2 (standard deviation) cm in diameter at planting and did not differ
between treatments (P=0.59). Natural regeneration of P.palustris
and Q.laevis was very limited so seedlings of these species were sou-
rced from local nurseries. Containerized (164 ml Ray Leach “Cone-
tainer”) seedlings of P.palustris were sourced from the Florida Forest
Service nursery. These seedlings were produced from seed collected
throughout north and central Florida. Containerized (1 gallon) seed-
lings of Q.laevis were sourced from a local nursery in northeast Flor-
ida (Madison County). These seedlings were produced from seed
collected from naturally regenerated mature trees in the same county.
At planting, basal diameter (5 cm height) and stem length of Q.laevis
seedlings averaged 6.3 ± 2.5 mm and 47.1 ± 7.2 cm, respectively, and
did not differ between treatments (P=0.93, P=0.91).
2.2 |Leaf-level physiology
In situ rates of leaf, gas-exchange were measured monthly or semi-
monthly between May 2019 and July 2020 to determine the effects
of experimental warming on leaf physiology of each species. The num-
ber of measurement dates varied among species depending on the
availability of mature, fully expanded green (non-senescent) leaves/
needles (A.glomeratus and P.palustris =11 measurement dates, S.
repens =12 measurement dates, Quercus laevis =9 measurement
dates). On each date, two portable cross-calibrated photosynthesis
systems (LI-6800, LiCor., Inc.) were used to record steady-state mea-
surements of light-saturated net photosynthesis (A
sat
,μmol m
2
s
1
)
and stomatal conductance to water vapor (g
s
, mol m
2
s
1
) at a cham-
ber reference [CO
2
] of 420 μmol mol
1
. The ratio of A
sat
/g
s
was calcu-
lated as a measure of intrinsic water use efficiency (iWUE, μmol
mol
1
). Leaves typically reached steady-state within 7–10 min of
being enclosed in the cuvette. Photosynthesis systems were randomly
assigned to temperature treatments on each measurement date. Light
intensity within the leaf chamber was maintained at
1800 μmol m
2
s
1
photosynthetic photon flux density (PPFD) using
the red/blue LED light source. Flow rate varied between 500 and
600 μmol s
1
. Water vapor inside the leaf chamber was not scrubbed
so that RH inside the cuvette approximated ambient conditions. On
each measurement date, the LI-6800 temperature exchanger was set
to the prevailing ambient temperatures inside the ambient and
warmed treatment plots. Leaf temperature (T
leaf
)wasmeasured
with the built-in leaf temperature thermocouple. Measurements
occurred between 10:00 and 14:00 local time and were made on
1–3 recently mature, fully expanded, upper canopy leaves (A.glo-
meratus,S.repens,Q.laevis) or needle fascicles (P.palustris,three
needles per fascicles) per plant. Leaves of A.glomeratus and needles
of P.palustris were placed side by side (non-overlapping) within the
cuvette. The one-sided surface area (cm
2
) of leaves/needles within
the chamber was determined by measuring the length and width of
leaves/needles inside the chamber with a ruler. Leaf gas-exchange
data were then back-corrected using the corrected leaf area
estimate.
On the final date (July 6, 2020), we measured the CO
2
response
of leaf-level net photosynthesis (A-C
i
) on similar leaves/needles of all
plants. Light conditions within the cuvette were controlled at a PPFD
of 1800 μmol m
2
s
1
and all measurements were made at a common
aT
leaf
of 33 ± 1.0 (standard deviation)C. This was done to separate
warming effects on photosynthetic biochemistry from the effect of
measurement temperature. Each A–C
i
curve began with a steady-state
measurement of A
sat
and g
s
at a chamber reference [CO
2
]of
420 μmol mol
1
. For the C
3
plant species (S.repens,P.palustris,Q.
laevis), A–C
i
curves were constructed by measuring A
sat
and C
i
at a
series of reference [CO
2
]: 300, 250, 100, 50, 0, 420, 650, 800, 1200,
and 1500 μmol mol
1
. Each A–C
i
curve was parameterized using the
Farquhar model of C
3
photosynthesis (Farquhar et al., 1980). The
model estimates the maximum rate of Rubisco carboxylation (V
cmax
;
μmol m
2
s
1
) and the rate of electron transport for RuBP regenera-
tion (J
max
;μmol m
2
s
1
). Importantly, we did not measure mesophyll
conductance and rely on the simplifying assumption that C
i
equals the
[CO
2
] in the chloroplasts (as in Farquhar et al., 1980). Therefore, our
estimates of V
cmax
and J
max
are “apparent”rates that reflect both bio-
chemical limitations of photosynthesis and mesophyll conductance
(e.g. Salmon et al., 2020). The model was fit using nonlinear least
squared parameter estimation in SAS v9.3 (PROC NLIN, SAS Institute
Inc., 2010). For A.glomeratus (C
4
species), A–C
i
curves were con-
structed by measuring A
sat
and C
i
at the following series of reference
[CO
2
]: 300, 150, 50, 100, 200, 300, 340, 380, 500, 800, 1000, and
1200 μmol mol
1
.A–C
i
curves for A.glomeratus were parameterized
using the equations of von Caemmerer (2000), with modified code
from Smith and Dukes (2017). We used nonlinear least squared
parameter estimation in R version 3.2.1 (R Core Team, 2013). The
maximum rate of phosphoenolpyruvate carboxylase (PEPc) carboxyla-
tion (V
pmax
,μmol m
2
s
1
) and V
cmax
were estimated from the CO
2
-
limited portion of each curve, and J
max
was estimated from the light-
limited portion of each curve. In A.glomeratus,V
pmax
,V
cmax
, and J
max
were also “apparent”rates since mesophyll conductance was not
measured and bundle sheath conductance was held constant at
0.003 mol m
2
s
1
bar
1
(Alonso-Cantabrana et al., 2018). If warming
resulted in thermal acclimation of leaf photosynthetic capacity, we
expected that V
cmax
,J
max
, and V
pmax
(for A.glomeratus) measured at a
common temperature would be lower in warm-grown plants of each
species.
ASPINWALL ET AL.5of17
Physiologia Plantarum
Because A–C
i
data were measured at relatively high temperatures
and vegetation models often require estimates of photosynthetic
parameters at 25C, we also calculated estimates of V
pmax
,V
cmax
, and
J
max
at 25C. For A.glomeratus, we estimated photosynthetic parame-
ters at 25C using a third-order polynomial function fit to the temper-
ature response data for C
4
plants shown in Smith and Dukes (2017).
For the C
3
species, we estimated photosynthetic parameters at 25C
using a peaked Arrhenius equation (see Medlyn et al., 2002) with
assumed activation energies and entropy terms of 58.9 and
0.629 kJ mol
1
, respectively, for V
cmax
and assumed activation ener-
gies and entropy terms of 29.7 and 0.632 kJ mol
1
, respectively, for
J
max
. Deactivation energies for V
cmax
and J
max
were held constant at
200 kJ mol
1
as in other studies (e.g. Vårhammar et al., 2015).
2.3 |Biomass harvest
All plants of each species were harvested on July 8, 2020, roughly
17 months, and two growing seasons after the start of the experi-
ment. For each A.glomeratus plant, the entire aboveground portion
was cut at 5 cm above the soil surface and transported to the lab in a
paper bag. In the field, a 0.064 m
3
(40 40 40 cm) volume of soil
centered on the cluster of cut tillers was excavated and the root sys-
tem was washed free of soil. A random subsample of approximately
20 leaves was collected from each plant and subsample fresh mass
was determined. The area (cm
2
) of the subsampled leaves was deter-
mined using a portable leaf area meter (LI-3000C, LiCor., Inc.), and the
subsampled leaves were dried at 70C for 3 days. The resolution of
the portable leaf area meter is 1 mm
2
, which may lead to inaccurate
estimates of leaf area, especially for narrow needles of P.palustris.
However, we determined leaf area of plants in both treatments in the
same way such that assessment of treatment effects are still valid.
Leaf dry mass per unit area (LMA, g m
2
) was estimated by dividing
subsample leaf dry mass by leaf area. Remaining leaf/tiller and root
material were dried at 70C for 7 days. Leaf subsample dry mass was
added to the remaining leaf/tiller dry mass to estimate total above-
ground dry mass while total belowground dry mass was the sum of
root dry mass. Aboveground biomass was also harvested after the
first growing season (November 25, 2019) following the same
protocol.
Serenoa repens and P.palustris required a different protocol since
both species produce underground stems. For each plant of these
species, the entire plant and soil volume (0.064 m
3
) were excavated
together and transported to the lab in a plastic bag. In the lab, root
and stem biomass were separated, washed free of soil, and dried at
70C for 7 days to determine stem and root dry mass. Palm leaf lam-
ina and pine needles were immediately separated from each stem and
weighed to determine total leaf fresh mass. Palm petioles were dried
and weighed separately. A small subsample of palm leaf lamina or pine
needles (15–30 needles per seedling) were collected from each plant
and subsample fresh mass was determined. Lamina/needle subsample
area, subsample dry mass, and LMA were determined as described
above. Total leaf lamina or needle dry mass was estimated by
multiplying the subsample dry matter content (dry mass/fresh mass)
by total leaf fresh mass. Total leaf area (m
2
) was estimated by multi-
plying the subsample area to dry mass ratio by the estimate of leaf
lamina or needle dry mass. Total leaf mass of S.repens was estimated
by summing petiole dry mass, leaf lamina dry mass, and leaf lamina
subsample dry mass. For consistency, total leaf and stem dry mass
were summed to estimate aboveground dry mass while belowground
dry mass was the sum of root dry mass.
For Q.laevis, the entire aboveground portion of each seedling
was cut at the soil surface and transported to the lab where leaf and
stem material were separated. In the field, a 0.064 m
3
volume of soil
centered on the cut stem was excavated and the entire root system
was washed free of soil. All Q.laevis seedlings produced a clearly visi-
ble lignotuber which was considered part of the belowground root
system. Total leaf fresh mass was determined and a subsample of
approximately 10 leaves (approximately 20% of leaf fresh mass) were
collected from each seedling. Leaf subsample area, subsample dry
mass, LMA, total dry mass, and total leaf area were determined as
described above. Stem and root material were dried at 70C for
7 days. Total leaf and stem dry mass were summed to estimate above-
ground dry mass while belowground dry mass was the sum of root
dry mass. For all species, allocation to different biomass pools, relative
to total dry mass, was determined for each plant by calculating leaf
mass fraction (LMF, g g
1
), stem mass fraction (SMF, main stem +bra-
nches, g g
1
), aboveground (leaf +tiller/stem) mass fraction (AGMF, g
g
1
), and root mass fraction (RMF, g g
1
). Leaf area ratio (LAR, cm
2
g
1
) was also calculated for S.repens,P.palustris, and Q.laevis as the
ratio of total leaf area to total dry mass.
2.4 |Statistical analysis
All statistical analyses were performed in SAS v9.3 (SAS Institute Inc.,
2010). We separated species in all analyses due to considerable differ-
ences in plant size, morphology, growth pattern, and physiology
(e.g. tree vs. grass/shrub, C
3
vs. C
4
). Mixed-effect models (PROC
MIXED) were used to test the fixed effects of time (measurement
date), temperature treatment (ambient, warmed), and their interaction
on A
sat
,g
s
, and iWUE. Because leaf gas-exchange was measured
repeatedly over time on the same plants, we fit a random intercept
term for the effect of plant within temperature treatment. When tem-
perature treatment or date temperature treatment interactions
were statistically significant (P< 0.05) or marginally significant
(P< 0.10) we carried out pairwise comparison of treatment means,
overall or at individual measurement dates. Mixed-effect models were
also used to test the fixed effects of temperature treatment on photo-
synthetic parameters (V
pmax
,V
cmax
,J
max
at 33C), and seedling bio-
mass production and allocation (e.g. SMF, RMF, LAR), with block
considered a random effect.
For each species, analysis of covariance was used to deter-
mine whether temperature treatment effects on leaf physiology
(A
sat
,g
s
, and iWUE) were dependent upon soil moisture. In this
model, temperature treatment was treated as a factor and VWC a
6of17 ASPINWALL ET AL.
Physiologia Plantarum
covariate (continuous variable). A significant (P<0.05)or margin-
ally significant (P< 0.10) interaction between treatment and VWC
indicated that temperature treatment affected the relationship
betweenleafphysiologyandVWC,resultinginanequationwith
different slope parameters for each treatment. If treatment and
VWC were both significant (P> 0.10) equations with different
intercepts for each treatment, but a common slope, were fit to the
data. If only VWC was significant, one equation describing the
relationship between VWC and leaf physiology was fit to data
from both treatments. The same approach was used to test
whether the long-term temperature response of A
sat
differed
between temperature treatments.
FIGURE 2 Mean (± standard error, n=6) values for in situ (instantaneous) leaf temperature (T
leaf
), light-saturated net photosynthesis (A
sat
),
stomatal conductance to water vapor (g
s
), and intrinsic water-use efficiency (iWUE =A
sat
/g
s
) over time in four species grown under ambient
conditions and experimental warming in a subtropical forest in Northeast Florida. Andropogon glomeratus is a perennial C
4
grass (monocot).
Serenoa repens is a small woody palm (monocot) species. Pinus palustris is a coniferous tree. Quercus laevis is a broadleaved deciduous tree species.
When gaps exist in the data, leaves were brown or senesced. Inset figures show the overall mean (aggregated) values for T
leaf
,A
sat
,g
s
, and iWUE
under ambient and warmed conditions for each species
ASPINWALL ET AL.7of17
Physiologia Plantarum
3|RESULTS
3.1 |Leaf physiology
Over time and across species and treatments, in situ leaf gas-
exchange measurements occurred at leaf temperatures (T
leaf
) between
18 and 37C (Figure 2). Across all data, soil VWC tended to be low
when T
leaf
was high (Figure S1). On average, T
leaf
during leaf-gas
exchange measurements was 2.7, 2.6, 3.1, and 2.4C higher in
warmed than control treatment plants of A.glomeratus,S.repens,P.
palustris, and Q.laevis, respectively (Figure 2).
In A.glomeratus, warming effects on in situ rates of A
sat
,g
s
, and
iWUE measured at prevailing T
leaf
varied over time (date treatment
interaction, Table 1). Post-hoc comparisons indicated that warming
increased A
sat
in July (+30%) and November (+130%) 2019, but
reduced A
sat
in March 2020 (47%; Figure 2E). Warming also
increased g
s
in October 2019 (+37%) and February 2020 (+51%) but
reduced g
s
in March 2020 (19%). Warming reduced iWUE in
October 2019 (39%) and March 2020 (37%) but increased iWUE
in November 2019 (+138%). Aggregated over time, warming resulted
in small (nonsignificant) increases in A
sat
,g
s
, and iWUE in A.glomeratus
(Table 1, Figure 2).
In S.repens,A
sat
,g
s
, and iWUE varied over time, and warming
resulted in small but nonsignificant increases in A
sat
(+15%) and g
s
(+25%, Table 1, Figure 2). Warming resulted in a marginally significant
reduction in iWUE in S.repens (14%, Table 1, Figure 2N).
Averaged over time, warming caused a significant reduction in
A
sat
and g
s
in P.palustris (15 and 28%, respectively, Figure 2). A
marginally significant date warming interaction occurred for A
sat
(Table 1), caused by a negative effect of warming during August and
September 2019 (47%), when prevailing temperatures were high,
and a positive effect of warming in January 2020 (+39%) when preva-
iling temperatures were low. iWUE varied over time but was not
affected by warming (Table 1, Figure 2O).
TABLE 1 Analysis of variance of
measurement date, temperature
treatment, and date temperature
treatment effects on leaf-level
physiological variables in four species
growing in a subtropical forest
Species Variable
Date Treatment Date treatment
df F df F df F
Andropogon glomeratus A
sat
10.85 14.0*** 1.10 0.2 10.85 4.6***
g
s
10.85 6.9*** 1.10 0.3 10.85 3.9***
iWUE 10.85 14.5*** 1.10 0.5 10.85 2.4*
V
pmax
1.10 0.2
V
cmax
1.10 0.1
J
max
1.10 0.4
J
max
/V
cmax
1.10 0.8
Serenoa repens A
sat
11.96 8.0*** 1.10 2.1 11.96 1.6
g
s
11.96 12.2*** 1.10 3.3 11.96 1.2
iWUE 11.96 7.1*** 1.10 4.5
†
11.96 1.5
V
cmax
1.10 1.9
J
max
1.10 2.1
J
max
/V
cmax
1.10 0.2
Pinus palustris A
sat
10.76 7.4*** 1.10 6.5* 10.76 1.9†
g
s
10.76 9.2*** 1.10 8.4* 10.76 1.2
iWUE 10.76 2.4* 1.10 2.9 10.76 1.4
V
cmax
1.10 0.9
J
max
1.10 1.1
J
max
/V
cmax
1.10 0.6
Quercus laevis A
sat
8.69 4.0** 1.10 1 8.69 0.6
g
s
8.69 3.3** 1.10 0.3 8.69 1.3
iWUE 8.69 6.2*** 1.10 0.4 8.69 2.7*
V
cmax
1.10 0.1
J
max
1.10 0.1
J
max
/V
cmax
1.10 0.1
Note: Numerator and denominator degree of freedom (df ) and Fvalues are presented for each trait and
experimental factor. Fvalues denoted with “***”,“**”,“*”, and “†”are significant at P< 0.001, P< 0.01,
P< 0.05, and P< 0.1, respectively. Variable descriptions: A
sat
, light-saturated net photosynthetic rate; g
s
,
stomatal conductance to water vapor; iWUE, intrinsic water use efficiency (A
sat
/g
s
). For all species, g
s
and
iWUE data were square-root transformed to fulfill assumptions of normality.
8of17 ASPINWALL ET AL.
Physiologia Plantarum
In Q.laevis,A
sat
,g
s
, and iWUE varied over time and warming cau-
sed slight (non-significant) increases in A
sat
and g
s
(Table 1, Figure 2).
Warming effects on iWUE varied over time (date treatment interac-
tion, Table 1). Post hoc comparisons showed that warming increased
iWUE in November 2019 (+129%) but had little effect otherwise
(Figure 2P). Overall, these results provide general support for the
expectation that warming would have negligible or positive effects on
photosynthesis in monocot species adapted to warmer temperatures.
However, we found mixed support for the expectation that warming
would reduce photosynthesis in tree species with more temperate dis-
tributions given that P.palustris showed reduced photosynthesis with
warming but Q.laevis did not.
In general, warming had little effect on photosynthetic biochemis-
try (Table 1, Figure 3). In A.glomeratus, estimates of V
pmax
,V
cmax
, and
J
max
(measured at 33C) were similar in both temperature treatments
(Figure 3) and averaged 332.1 ± 21.5, 21.2 ± 1.4, and 136.5
± 9.1 μmol m
2
s
1
, respectively. In Q.laevis, estimates of V
cmax
and
J
max
were also similar between ambient and warmed treatments
(Figure 3), and averaged 114.9 ± 8.0 and 115.0 ± 7.6 μmol m
2
s
1
,
respectively. In S.repens and P.palustris, biochemical responses of
photosynthesis to warming tended to mirror average responses of A
sat
to warming. Serenoa repens showed small increases in V
cmax
and J
max
with warming (+32, +38%) while P.palustris showed small reductions
in V
cmax
and J
max
with warming (15, 19%). Nonetheless, warming
effects on photosynthetic parameters were not significant in any spe-
cies. The average J
max
/V
cmax
ratio (at 33C) in A.glomeratus was 6.45
± 0.2; substantially higher than J
max
/V
cmax
in the C
3
species in this
study (Figure 3). This is expected given that C
4
species are more com-
monly light-limited than CO
2
-limited at current atmospheric CO
2
and
tend to invest less nitrogen in Rubisco than C
3
species (Sage &
FIGURE 3 Mean (± standard error, n=6) values for biochemical parameters of photosynthesis in four species grown under ambient
conditions (blue bars) and experimental warming (red bars) in a subtropical forest in Northeast Florida. V
pmax
is the maximum rate of
phosphoenolpyruvate carboxylase (PEPc) carboxylation in C
4
plants (Andropogon glomeratus only). V
cmax
is the maximum rate of rubisco
carboxylation. J
max
is the maximum rate of electron transport for RuBP regeneration. All parameters were estimated at a common measurement
temperature (leaf temperature =33 ± 1.0 (standard deviation)C) in the field. For each species, the relative change in each parameter caused by
warming is shown above warmed treatment mean
ASPINWALL ET AL.9of17
Physiologia Plantarum
Kubien, 2007). Warming had no effect on J
max
/V
cmax
in any species
(Table 1, Figure 3). Analysis of photosynthetic parameters corrected
to 25C yielded similar results (Table S1).
In contrast to our expectation, we found that increasing or
decreasing soil moisture did not modify A
sat
and g
s
responses to
warming in any species (Table 2, Figure 4). In other words, the slope
parameter describing the increase in A
sat
and g
s
with VWC did not dif-
fer between temperature treatments for any species. We note that
A
sat
and g
s
showed no relationship with VWC in A.glomeratus,
reflecting high tolerance of reduced soil moisture (Figure 4). Relation-
ships between A
sat
and VWC and g
s
and VWC also appeared to be
similar across all C
3
species (Figure 4). Indeed, additional ANCOVA
tests revealed that the slope of the A
sat
versus VWC and g
s
versus
VWC relationships did not differ between C
3
species (P=0.94,
P=0.92). Across C
3
species, the relationship between individual mea-
sures of A
sat
and VWC could be described by the linear equation
y=4.465 +61.2VWC (r
2
=0.12, P< 0.0001). The relationship
between individual measures of g
s
and VWC could be described by
the linear equation y=0.0379 +1.13VWC (r
2
=0.15, P< 0.0001). In
A.glomeratus and Q.laevis, iWUE increased as VWC declined, and did
so consistently across temperature treatments (Table 2, Figure 4). In S.
repens, warming did not affect the slope of the iWUE versus VWC
relationship but reduced the intercept parameter, indicating lower
iWUE at a given VWC (Table 2, Figure 4). iWUE was not associated
with VWC in P.palustris (Figure 4).
For each species, we also examined the long-term (realized) tem-
perature response of A
sat
under ambient and warmed temperatures. A
second-order polynomial equation provided an adequate fit to the
temperature response for all species and treatments. In all species, the
intercept, slope, and quadratic terms describing the relationship
between A
sat
and T
leaf
were similar between treatments (all P> 0.10).
Thus, a single equation was used to describe the long-term
temperature response of each species (Figure 5). From the fitted
equations, the estimated temperature optima (T
opt
)ofA
sat
was highest
in A.glomeratus (36.5C) and lowest in P.palustris (23.1C, Figure 5).
The long-term estimated T
opt
of A
sat
was 31.6CinQ.laevis (Figure 5).
The estimated T
opt
of A
sat
in S.repens was 26.5C, but this estimate
should be interpreted cautiously given that A
sat
showed a very broad
temperature response with high rates of A
sat
above the apparent T
opt
(Figure 5).
3.2 |Biomass production and allocation
Warming resulted in small increases in total biomass production in A.
glomeratus (+22%), S.repens (+7%), and Q.laevis (+11%), but small
decreases in total biomass production in P.palustris (14%). This pat-
tern was similar for leaf, stem, and root biomass, as well as total leaf
area. In no cases were the effects of warming on biomass production
or leaf area significant (Table 3). In general, warming had no significant
effect on biomass allocation in any species. However, for Q.laevis,
warming resulted in marginally significant increases in LAR (+42%)
and LMF (+38%), indicating a shift in allocation to leaves relative to
stems and roots. For S.repens, warming resulted in a marginally signifi-
cant increase in LMA (+15%).
4|DISCUSSION
We carried out a warming experiment in a subtropical forest in north-
east Florida and assessed the impacts of warming on the physiology
and productivity of dominant species with different climatic distribu-
tions, growth forms, and functional strategies. We expected that
warming would have neutral or positive effects on photosynthesis in
TABLE 2 Results of analysis of covariance (ANCOVA) testing whether the relatoinship between volumetric soil water content (VWC) and leaf
gas-exchange variables is dependent upon temperature treatment (ambient, warmed)
Species Variable n
VWC Treatment VWC treatment
F P>F F P>F F P>F
Andropogon glomeratus A
sat
117 0.60 0.44 0.02 0.89 0.32 0.57
g
s
117 0.63 0.43 0.09 0.76 0.70 0.40
iWUE 117 6.6 0.01 0.01 0.96 0.04 0.84
Serenoa repens A
sat
130 26.0 <0.0001 0.01 0.94 0.70 0.41
g
s
130 25.9 <0.0001 0.03 0.86 1.07 0.30
iWUE 130 15.7 0.0001 4.0 0.05 1.35 0.25
Pinus palustris A
sat
108 20.1 <0.0001 0.83 0.37 0.12 0.73
g
s
108 20.3 <0.0001 0.81 0.37 0.01 0.97
iWUE 108 0.56 0.45 1.84 0.18 0.55 0.46
Quercus laevis A
sat
97 9.5 <0.01 0.07 0.79 0.44 0.51
g
s
97 15.6 0.0002 0.07 0.79 0.27 0.61
iWUE 97 7.7 0.007 1.00 0.32 1.92 0.17
Note: For each relationship (species, variable), the number of observations (n), model Fvalues, and associated probability (P) values are provided. Variable
descriptions: A
sat
, light-saturated net photosynthetic rate; g
s
, stomatal conductance to water vapor; iWUE, intrinsic water use efficiency (A
sat
/g
s
).
10 of 17 ASPINWALL ET AL.
Physiologia Plantarum
species with warmer climatic distributions or physiological adaptations
to warmer temperatures (A.glomeratus,S.repens), but negative effects
on photosynthesis in tree species with more temperate distributions
(P.palustris,Q.laevis). Our expectations were partially supported.
Aggregated over time, the effects of warming on A
sat
(and g
s
) were
not significant but slightly positive in A.glomeratus and S.repens.
Moreover, averaged over time, warming caused a significant reduction
in A
sat
and g
s
in P.palustris. However, in contrast to our expectation,
warming caused a small (nonsignificant) increase in A
sat
in Q.laevis.
We also expected that low soil moisture would negate or reverse the
positive effects of warming on photosynthesis in warm-adapted spe-
cies and exacerbate the negative effects of warming on photosynthe-
sis in tree species. In contrast, we found that A.glomeratus showed
little sensitivity to reduced VWC, and all three C
3
species reduced A
sat
and g
s
as VWC declined and did so consistently across treatments.
We conclude that P.palustris seedlings growing in canopy gaps may
be vulnerable to climate warming, but warming might have neutral, or
slightly positive, impacts on photosynthesis and productivity of domi-
nant monocot species (A.glomeratus and S.repens) as well as Q.laevis.
4.1 |Species responses to warming
Species' photosynthetic responses to warming should be viewed in
relation to (1) the temperature response of photosynthesis, (2) changes
in photosynthetic capacity in response to warming, and (3) biomass
responses to warming. Combined, these responses provide insight into
potential mechanisms and consequences of species photosynthetic
FIGURE 4 The relationship between volumetric soil water content (VWC) and instantaneous measures of leaf net photosynthesis (A
sat
),
stomatal conductance to water vapor (g
s
), and intrinsic water-use efficiency (iWUE =A
sat
/g
s
) in four species grown under ambient conditions
(blue symbols) and experimental warming (red symbols) in a subtropical forest in Northeast Florida. When experimental warming altered the
relationship (intercept, slope) between VWC and gas-exchange variables, separate lines (blue, red) were fit to the data for ambient and warmed
treatments. When experimental warming did not influence the relationship between VWC and leaf gas-exchange, we fit a single equation (one
line) across data for both treatments. Intercept and slope estimates are provided in each figure panel when the relationship between VWC and
gas-exchange parameters were significant (P< 0.10). Where appropriate, model coefficients of determination (r
2
) are provided
ASPINWALL ET AL.11 of 17
Physiologia Plantarum
responses warming. In A.glomeratus, the average T
leaf
under ambient
conditions was below the estimated long-term T
opt
of A
sat
(36.5C), such
that warming caused a slight increase in average A
sat
aggregated over
time. The high T
opt
of A
sat
in A.glomeratus is expected of C
4
plants which
are well-adapted to higher temperatures,andoftenoccurinwarmerhab-
itats (Sage & Kubien, 2007; Yamori et al., 2014). We note that A
sat
showed a significant date treatment interaction in A.glomeratus driven
by positive effects of warming on A
sat
in mid-summer and autumn, and
negative effects in early spring. Importantly, we observed no warming
effects on estimated rates of V
pmax
,V
cmax
,orJ
max
. Thus, little adjustment
in photosynthetic capacity occurred in A.glomeratus, which contributed
to little change in A
sat
with warming. It has been hypothesized that C
4
species are less capable of thermal acclimation than C
3
species (Yamori
et al., 2014). However, C
4
plants sometimes show equivalent acclimation
responses to warming (Sturchio et al., 2021; Yamori et al., 2014). Our
results are similar to those of Dwyer et al. (2007) who found slight
FIGURE 5 The realized long-term relationship between leaf-level light saturated net photosynthesis (A
sat
) and leaf temperature (T
leaf
) in four
species grown under ambient conditions (blue symbols) and experimental warming (red symbols) in a subtropical forest in Northeast Florida.
Second order polynomial equations were used to describe the relationship between A
sat
and T
leaf
in each species. In all cases, the relationship
between A
sat
and T
leaf
was consistent between treatments. Model coefficients of determination (r
2
) and significance (Pvalues) are provided
TABLE 3 Mean values (± standard error, n=6) for biomass production and allocation in four plant species grown under ambient conditions
and experimental warming in a subtropical forest in Northeast Florida
Variable
Andropogon glomeratus Serenoa repens Pinus palustris Quercus laevis
Ambient Warmed Ambient Warmed Ambient Warmed Ambient Warmed
Leaf dry mass (g) ––10.2 ± 1.9 12.0 ± 2.7 7.3 ± 2.1 6.8 ± 2.3 6.8 ± 1.5 10.5 ± 2.8
Stem dry mass
(g)
––32.1 ± 6.5 34.8 ± 8.7 3.0 ± 0.6 2.2 ± 0.7 11.2 ± 1.3 13.3 ± 3.0
AG dry mass (g) 19.4 ± 3.5 21.2 ± 7.6 42.3 ± 8.0 46.8 ± 8.9 10.3 ± 2.6 9.0 ± 2.8 18.0 ± 2.4 23.8 ± 4.7
AG
b
dry mass (g) 62.6 ± 7.3 75.7 ± 15.4 ––––––
Root dry mass
(g)
4.21 ± 0.35 7.61 ± 3.12 11.7 ± 1.2 10.8 ± 0.5 7.6 ± 1.9 6.4 ± 2.4 62.6 ± 11.2 65.6 ± 12.6
Total dry mass
(g)
23.6 ± 3.5 28.8 ± 10.6 54.0 ± 8.8 57.6 ± 9.2 17.9 ± 4.5 15.4 ± 5.1 80.6 ± 13.4 89.4 ± 17.0
Total leaf area
(cm
2
)
––591 ± 103 613 ± 131 481 ± 126 451 ± 159 653 ± 137 1044 ± 291
LAR (cm
2
g
1
)––11.0 ± 1.1 10.8 ± 2.4 26.2 ± 2.0 28.4 ± 1.0 7.7 ± 0.9 10.9 ± 1.4
a
LMA (g m
2
) 116.0 ± 7.7 115.3 ± 10.0 141.3 ± 5.2 161.8 ± 9.3
a
147.5 ± 8.0 154.0 ± 10.5 103.2 ± 2.5 103.3 ± 3.4
LMF (g g
1
)––0.19 ± 0.02 0.21 ± 0.05 0.39 ± 0.04 0.43 ± 0.02 0.08 ± 0.01 0.11 ± 0.01
a
SMF (g g
1
)––0.59 ± 0.03 0.58 ± 0.05 0.18 ± 0.03 0.17 ± 0.03 0.16 ± 0.04 0.15 ± 0.03
AGMF (g g
1
) 0.79 ± 0.05 0.76 ± 0.02 0.77 ± 0.02 0.79 ± 0.03 0.57 ± 0.02 0.60 ± 0.04 0.24 ± 0.03 0.26 ± 0.02
RMF (g g
1
) 0.21 ± 0.05 0.24 ± 0.02 0.23 ± 0.02 0.21 ± 0.03 0.43 ± 0.02 0.40 ± 0.04 0.76 ± 0.03 0.74 ± 0.02
Note: Variable descriptions: AG dry mass, aboveground dry mass (leaf +stem/tiller dry mass); LAR, leaf area ratio (LA/Total DM); LMA, leaf dry mass per
unit area (leaf dry mass/total leaf area); LMF, leaf mass fraction (Leaf DM/Total DM); SMF, stem mass fraction (Stem DM +Branch DM)/Total DM); RMF,
root mass fraction (Root DM/Total DM).
a
Indicates that mean values of biomass traits differ between ambient and warmed treatments at P< 0.1.
b
Aboveground biomass measured in November 2019. All other variables were measured at the end of the experiment (July 2020).
12 of 17 ASPINWALL ET AL.
Physiologia Plantarum
positive effects of warming on photosynthetic rates of three C
4
species.
However, the slight increase in A
sat
with warming we observed in A.glo-
meratus was less than the average increase in A
sat
with warming aver-
aged across a broader range of C
4
species (Liang et al., 2013). We also
found that warming caused a small increase in total biomass production;
although biomass production in both treatments was much lower in the
second year which could be a legacy of biomass clipping following the
first year. Other studies in a related species (A.gerardii)alsoobserved
slightly positive effects of experimental warming on biomass production
(e.g. Sherry et al., 2008).
The physiology of S.repens has not been widely studied, despite
being common and ecologically important in the southeastern US. We
expected this woody palm species would respond positively to
warming given its occurrence in the warmest habitats in the south-
eastern US, including tropical south Florida. This species showed a
very broad photosynthetic temperature response with relatively high
rates of A
sat
above the estimated T
opt
. Aggregated over time, warming
had no significant effect on A
sat
and g
s
although rates increased mod-
estly under warming. We also observed small increases in V
cmax
and
J
max
(at common measurement temperature) with warming which
could represent a “constructive adjustment”of photosynthetic capac-
ity that contributed to small increases in A
sat
under warming (Way &
Yamori, 2014). Interestingly, we found that whole-plant LMA mea-
sured at the end of the experiment was higher in warmed S.repens
seedlings than ambient seedlings, indicating increased leaf thickness
or density with warming. In contrast, most studies find reductions in
LMA with increasing temperature (Poorter et al., 2009). Increased
LMA with warming could indicate greater investment in structural
components than enzymatic components (Mediavilla et al., 2008;
Poorter & Villar, 1997). However, there is evidence that leaf N per
unit area (N
a
) increases with leaf thickness and density
(e.g. Niinemets, 1999), and V
cmax
generally increases with increasing
N
a
(Walker et al., 2014; Wilson et al., 2000). Although we did not
measure leaf N concentrations and were unable to examine relation-
ships between LMA and N
a
, increased LMA with warming could partly
explain increased V
cmax
. Despite slight increases in photosynthetic
capacity and A
sat
with warming, we found no change in biomass pro-
duction with warming in S.repens. More replicates may be needed in
future studies to determine the robustness of species productivity
responses to warming. Even so, we conclude that warming might have
neutral to slightly positive effects on S.repens in north Florida. This
conclusion fits with the prediction that S.repens habitat will increase
slightly with warming (Butler & Larson, 2020).
Aggregated over time, we expected that warming would reduce
A
sat
in P.palustris and Q.laevis given that north Florida is near the
southern range limit of both species. Previous studies have also found
negative effects of climate warming on photosynthesis and perfor-
mance of southeastern Quercus species at their southern range limit
(Wertin et al., 2011). As expected, warming caused a significant reduc-
tion in A
sat
in P.palustris. The negative effect of warming on A
sat
can
be partly explained by reduced g
s
(increased stomatal limitation), and
the observation that the average T
leaf
under ambient conditions was
above the long-term T
opt
of A
sat
(23.1C), such that warming caused a
further reduction in A
sat
. This T
opt
estimate is lower than the T
opt
of
photosynthesis in studies with mature P.palustris and Pinus elliotti
(a related sympatric species) at similar sites (Powell et al., 2008;
Teskey et al., 1994). There may be differences in the temperature
response of A
sat
between P.palustris seedlings and mature trees.
Seedling root systems are smaller and more shallow than mature tree
root systems, which could limit access to water on xeric sites and
increase stomatal limitation of A
sat
with increasing temperature (Lin
et al., 2012), although stomatal limitation of A
sat
is generally higher in
large trees than small trees (Drake et al., 2010). Also, foliar respiration
tends to be higher in seedlings than mature trees (Ryan et al., 1994),
and may represent an increasing proportion of gross photosynthesis
with increasing temperature (Way & Sage, 2008), which could result
in a lower T
opt
of A
sat
in seedlings than mature trees. Importantly, we
also observed reduced V
cmax
and J
max
with warming. Thus, reduced
A
sat
with warming in P.palustris appears to be related to stomatal and
biochemical limitations. While the reduction in V
cmax
and J
max
is sug-
gestive of thermal acclimation, it may represent a “detractive adjust-
ment”(Way & Yamori, 2014). This might also partly explain why P.
palustris seedlings tended to be smaller under warmer conditions.
Our results could have important implications for regeneration of
P.palustris; a foundational tree species in the southeastern US (van
Lear et al., 2005). It is well-established that P.palustris seedlings are
generally intolerant of shade (Boyer, 1979), yet there is also evidence
that seedlings growing in open conditions may be more vulnerable to
hot, dry conditions. Knapp et al. (2008) showed that P.palustris seed-
ling mortality increased in open conditions as soil temperature
increased and soil moisture decreased. Moreover, at xeric sites,
Loudermilk et al. (2016) found that P.palustris seedling survival
decreased in canopy gaps, and a moderate density of Q.laevis in the
mid-story had a facilitative effect on seedling survival and perfor-
mance. In this way, a healthy and functional cohort of Q.laevis may
improve P.palustris performance and survival (Johnson et al., 2021).
Nonetheless, the growth habit of P.palustris may partly explain its vul-
nerability to warmer temperatures at the seedling stage. The species
exists in the “grass-stage”for several years, where the stem remains
underground, and a thick arrangement of needles protects the apical
bud from fire. However, in the grass-stage, P.palustris foliage is often
in direct or close contact with soil where temperatures can exceed
50C (Loudermilk et al., 2016). Extreme temperatures could inhibit or
permanently damage photosynthetic machinery, increase respiratory
costs, and deplete carbon reserves (Aspinwall et al., 2019). Our results
suggest a potential narrowing of optimal conditions for P.palustris
regeneration with climate warming, particularly at xeric sites.
Although warming had no significant effect on A
sat
in Q.laevis,
A
sat
was slightly higher under warming, aggregated over time. The
slight increase in A
sat
could be partly attributed to the observation
that the average T
leaf
under ambient conditions was below the long-
term T
opt
of A
sat
. The T
opt
of A
sat
in Q.laevis is within the range of T
opt
observed in other warm-temperate oak species in the southeastern
US (Gunderson et al., 2010). Although A
sat
increased slightly with
warming, we found no change in V
cmax
and J
max
with warming. These
results differ slightly from studies across species that have observed
ASPINWALL ET AL.13 of 17
Physiologia Plantarum
reductions in V
cmax
,J
max
, or both parameters (measured at a common
temperature) with increasing growth temperature (Aspinwall
et al., 2016; Kattge & Knorr, 2007; Kumarathunge et al., 2019). How-
ever, our results are similar to studies that have found no reduction in
V
cmax
and J
max
with increasing growth temperature, coupled with the
relative stability of A
sat
across different growth temperatures (Ow
et al., 2010; Slot & Winter, 2017; Stefanski et al., 2020). We also
found that warming caused a slight increase in total biomass produc-
tion, mostly driven by increased leaf production. We emphasize that
warming of a larger magnitude may induce different responses in each
species. Studies that examine species responses to multiple tempera-
ture treatments are required to understand potential threshold
responses for species performance.
We acknowledge some unavoidable limitations and uncertainties
in our study. Passive warming chambers cause small reductions in light
intensity and reduce maximum wind speed, and do not cause warming
at night. Lower light intensity might limit daily whole-plant C uptake
and plant growth. However, P.palustris was the only species to show
lower growth with warming. We argue that small reductions in light
intensity were unlikely to cause reduced growth in P.palustris given
that previous studies have shown that larger reductions in light are
required to reduce P.palustris seedling growth (Jose et al., 2003;
McGuire et al., 2001; Palik et al., 1997). Lower wind speeds in the
chambers could also intensify warming effects by reducing air flow
and convective heat transfer. We added openings and slits to the top
of the chambers to mitigate this effect. Yet, lower wind speeds are
occurring alongside climate warming (McVicar et al., 2012; Zhang
et al., 2021), such that passive warming chambers could simulate both
aspects of climate change. Previous studies have shown that daytime
and nighttime warming treatments can have different impacts on
plant function and growth. In Populus deltoides, Turnbull et al. (2002)
found that daytime warming alone had little effect on leaf respiration
or A
sat
. However, combined night and daytime warming increased leaf
respiration and A
sat
. Increased A
sat
was caused by greater consump-
tion of carbohydrates by respiration during warmer nights, which
stimulated higher demand for carbohydrates and higher photosyn-
thetic capacity. The impacts of nighttime warming on plant growth
remain unclear. Theoretically, nighttime warming could increase main-
tenance costs and reduce growth, yet some studies show increased
growth with nighttime warming despite increased respiration
(Cheesman & Winter, 2013; Krause et al., 2013). Finally, our estimates
of warming impact on apparent rates of photosynthetic capacity could
be improved with measures of mesophyll conductance, which could
vary among species (e.g. Niinemets et al., 2009).
4.2 |Climate warming–soil moisture interaction
Soil moisture has been hypothesized to be an important modifier of
plant responses to warming. In cool climates, warming effects on A
sat
might remain positive until water limitations increase stomatal limita-
tion of A
sat
. Although there are exceptions (Moyes et al., 2013),
previous studies have generally found support for this hypothesis
(D'Orangeville et al., 2018; Lazarus et al., 2018; Reich et al., 2018).
We tested this hypothesis in a subtropical forest where temperature
limitation of A
sat
is probably less common and found no interaction
between soil moisture and temperature treatment. There are a few
possible explanations for this result. First, warming effects on leaf gas-
exchange were generally small and may not have been strong enough
to drive synergistic interactions with soil moisture, which may be the
dominant constraint on plant function at our xeric site. Indeed, all
three C
3
species reduced A
sat
and g
s
as VWC declined and did so
consistently across treatments. Second, unlike previous warming
studies that have shown reduced soil moisture under warming
(e.g. Liu et al., 2021; Reich et al., 2018), warming did not reduce
VWC such that plants in both treatments operated at similar VWC
onanygivenday.Thismayhave contributed to convergent
responses to VWC across treatments. Third, high temperature, high
VPD, and low VWC conditions tended to co-occur naturally over
seasons, which is typical for northern Florida (Figures 1 and S1). In
much of Florida, maximum daily T
air
and VPD increase sharply at
the end of the dry season (May) when soil moisture is generally
low. This means that ambient and warmed plants experienced the
driest conditions when prevailing T
air
and VPD are near their peak.
We conclude that soil moisture and warming are unlikely to have syner-
gistic effects in xeric forests in the southeastern US. However, additional
warming studies across different sites (upland, lowland), microhabitats, or
soil moisture treatments would help resolve the potential interactive
effects of warming and water availability on plant performance over
space and time.
Experimental warming studies have revealed that species adapta-
tion, functional type, and prevailing abiotic and biotic conditions may all
be important determinants of warming impacts on plant performance
and community and ecosystem properties (Dusenge et al., 2020; Reich
et al., 2018; Volder et al., 2013). In a subtropical forest in northeast Flor-
ida, we found that warming had neutral, or slightly positive effects on
seedling photosynthesis and productivity in warm-adapted monocot spe-
cies and a sympatric oak species. However, warming negatively affected
photosynthesis in P.palustris seedlings. Soil moisture did not interact
with temperature treatments. Our results highlight the importance of
species functional type and biogeography in influencing species physio-
logical responses to warming, especially in the southeastern US. Species
differences in warming responses observed here could foreshadow
changes in community and ecosystem properties. Nonetheless, future
experiments should focus on species responses to warming across a
broader range of abiotic and biotic conditions, as well as warming
impacts on species interactions.
ACKNOWLEDGMENTS
Michael J. Aspinwall and Jeff Chieppa were supported by the USDA-
NIFA award 2019-67013-29161, the University of North Florida, and
Auburn University. Additional support was provided by Hatch NIFA,
Grant/Award Number: 1025522. The authors thank Martina Faciane,
John Clarke, and Jake Tucker for technical assistance.
14 of 17 ASPINWALL ET AL.
Physiologia Plantarum
AUTHOR CONTRIBUTIONS
Michael J. Aspinwall and Lynsae Davidson conceived and designed
the experiment. Michael J. Aspinwall, Jeff Chieppa, Eve Gray, Morgan
Golden-Ebanks, and Lynsae Davidson collected data. Michael
J. Aspinwall led the data analysis and wrote the manuscript with input
from all authors.
DATA AVAILABILITY STATEMENT
Data are freely available here: Aspinwall, Michael (2021): Sawmill
Slough Raw data.zip. figshare. Dataset. https://doi.org/10.6084/m9.
figshare.15157707.v1.
ORCID
Michael J. Aspinwall https://orcid.org/0000-0003-0199-2972
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version
of the article at the publisher's website.
How to cite this article: Aspinwall, M.J., Chieppa, J., Gray, E.,
Golden-Ebanks, M. & Davidson, L. (2022) Warming impacts on
photosynthetic processes in dominant plant species in a
subtropical forest. Physiologia Plantarum, 174(2), e13654.
Available from: https://doi.org/10.1111/ppl.13654
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