Introduction
Climate is changing and extreme temperature and precipitation events are
expected to intensify over the coming century (IPCC, 2013a), presenting
immense challenges for immobile, long-lived organisms such as trees.
Rates of forest tree mortality are already increasing worldwide as a
result of shifting drought regimes, extreme temperatures, and pest
outbreaks associated with global change (Allen et al., 2010; Anderegg et
al., 2015; Anderegg et al., 2016; McDowell et al., 2018). In response to
change, individual plants may physiologically adjust by plastically
altering traits in the short term (Anderson, Inouye, McKinney, Colautti,
& Mitchell-Olds, 2012; Des Marais, Hernandez, & Juenger, 2013;
Pritzkow, Williamson, Szota, Trouve, & Arndt, 2020; Valladares et al.,
2014; Valladares, Sanchez-Gomez, & Zavala, 2006; Wortemann et al.,
2011), while populations may undergo local adaptive evolution through
shifts in their genetic composition over longer time scales (Aitken,
Yeaman, Holliday, Wang, & Curtis-McLane, 2008; Davis, Shaw, &
Etterson, 2005). Thus, understanding the extent to which trees are able
to alter their traits in response to climatic change over short
(plastic) and long (evolutionary) timescales is critical to predicting
species’ survival.
One key trait that enables plants to tolerate stochastic environments is
the storage of nonstructural carbohydrates (NSCs). NSCs are the sugars
and starches produced via photosynthesis and stored in the parenchyma
cells of plants’ woody tissues for later use (Chapin III, Schulze, &
Mooney, 1990; Plavcová & Jansen, 2015). NSCs fuel photosynthesis at
night, leaf-out in spring, and are also thought to serve as a back-up
osmolytic or energetic reserve during periods of environmental extremes,
like drought or freezing temperatures (Adams et al., 2017; Chapin III et
al., 1990; H. Hartmann & Trumbore, 2016; McDowell et al., 2008;
Sevanto, McDowell, Dickman, Pangle, & Pockman, 2014). Total NSC storage
concentrations are known to vary seasonally (eg. Furze et al., 2019),
across species (eg. Martínez-Vilalta et al., 2016), and within species
(eg. Blumstein et al., 2020) and this variation in NSC storage
concentrations has been linked to prolong survival under drought
(O’Brien, Reynolds, Ong, & Hector, 2017). However, what controls
variation in total NSC stores and how sensitive it is to environment is
still poorly understood. Thus, we are limited in our ability to predict
whether forest trees will be able to vary NSC stores in order to
tolerate or adaptively evolve in response to climate change.
Total NSC stores can be broken down into two categories: soluble sugars
and insoluble starches (Kaplan & Guy, 2004; Krasensky & Jonak, 2012;
Thalmann & Santelia, 2017). Sugars affect cells’ osmotic balances and
are readily accessible for metabolism as they are dissolved in solution,
while starches are considered the longer-term storage molecule as they
are insoluble and thus must be broken back down into sugar to be used by
the cell as an osmolyte or metabolite. Sugars can hydrolyze into and out
of starch form via a number of enzymatic pathways (Thalmann & Santelia,
2017). The rate of starch transition varies daily (A. Tixier, Orozco,
Roxas, Earles, & Zwieniecki, 2018), seasonally (Furze et al., 2019;
Martínez-Vilalta et al., 2016; Richardson et al., 2013; Wurth,
Pelaez-Riedl, Wright, & Korner, 2005), by plant tissue (Furze et al.,
2019; Martínez-Vilalta et al., 2016), and in response to stress, such as
water deficit (Adams et al., 2017; Landhäusser & Lieffers, 2011;
Mitchell et al., 2013; Sevanto et al., 2014), high salinity (Chen, Chen,
& Wang, 2007; Goyal, 2007; Kanai et al., 2007; Kempa, Krasensky, Dal
Santo, Kopka, & Jonak, 2008; Ma et al., 2013), or extreme temperatures
(Hoermiller et al., 2017; Kaplan & Guy, 2005; Nagao, Minami, Arakawa,
Fujikawa, & Takezawa, 2005; Vasseur, Pantin, & Vile, 2011). Under such
environmental stressors, starch is degraded, leading to a subsequent
rise in soluble sugars in stressed tissues (Thalmann & Santelia, 2017)
and an increased ability to withstand the applied stress. Thus, a
plant’s ability to convert NSC stores from starch to sugar, and back
again, is an important consideration for survival under climate change.
While variation in starch degradation rates in response to environment
is well document, it is not yet understood if individuals exhibit
differential sensitivities to environment, indicating a potential for
adaptive evolution in response to increased stress.
Given that both the total amount of NSC stores a plant holds and
the proportion of those stores reserved in starch can play
crucial roles in woody plant survival under environmentally induced
stress, measuring the degree of heritable genetic and plastic variation
in these traits will be critical for predicting tree species’
persistence under climate change. Transplant experiments across multiple
sites with replicate clones or related individuals can be used to
disentangle sources of variation (Josephs, Berg, Ross-Ibarra, & Coop,
2019; Nuismer & Gandon, 2008). Genetic variation in these designs is
measured within an environment across unique genotypes, while plastic
variation is measured across environments within unique genotypes. In
practice, because measuring plastic variation results in the additional
capture of genetic variation by virtue of gathering data from multiple
common environments, it is often broken down into three components;
variation within a garden attributed to genetic differences (G),
variation between gardens attributed to environmental plasticity
(E), and the interaction between genotype and environment (GxE) (ie.
some genotypes can be more plastic than others). While there is strong
evidence that heritable genetic variation is responsible for some of the
variation in NSCs in some plants such as Populus trichocarpa(Blumstein et al., 2020) and Pinus sylvestris (Oleksyn,
Zytkowiak, Karolewski, Reich, & Tjoelker, 1999), to our knowledge, no
study to date has quantified the extent of plastic variation in NSCs.
Furthermore, no study has examined genetic or plastic variation in the
proportion of NSC that is kept in starch versus sugar, a potentially
critical aspect of plant response to stress.
By partitioning the variation in NSCs we can not only predict the
potential for tress to respond to increased prevalence of stress, we can
also begin to understand if trees are already locally adapted to
variation in environmental stress across their ranges. If greater total
NSC storage and more rapid transition between starch and sugar storage
can increase survival in stressful events, we predict genetic variation
in these traits to reflect geographic variation in stress. By
controlling for neutral population genetic variation across the range of
a tree species (e.g. Fst) we can determine the extent to which
traits show genetic differentiation (e.g. Qst) reflective of local
adaption.
Here, we measure genetic and plastic variation in total NSC
storage and the proportion of NSC stores held in insoluble starch
versus soluble sugars. To do so, we utilized two Department of Energy
(DOE) common gardens of black cottonwood (Populus trichocarpa )
growing in the western United States. Each garden contains clonally
replicated genotypes from multiple populations across the species range.
We extracted sugars and starches from branch woody tissue of trees grown
in two common gardens located at similar latitudes, but spanning a
continental to coastal environmental cline (Figures 1 & 2A). Our
objective is to understand the acclimatory and evolutionary potential of
black cottonwood trees under future climate change. We accomplish this
by parsing the amount of phenotypic variation attributable to genetic
variation (G), environmental plasticity (E), and genotype-by-environment
interactions (GxE) for both the total concentration of NSC stores, as
well as the proportion of total stores in insoluble starch. In addition,
we search for signatures of current local adaptation in both traits
across the environmental gradient of source populations.