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Functional Ecology. 2024;00:1–13.
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1wileyonlinelibrary.com/journal/fec
Received: 12 April 2024
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Accepted: 29 September 2024
DOI : 10.1111/136 5-243 5.14 677
RESEARCH ARTICLE
Phenology- Trait Relationships Across Different Scales and Organisational Levels
New opportunities for grassland species in warming temperate
winters
F. Curtis Lubbe1 | Andrea Kučerová1 | Martin Bitomský1 | Jitka Klimešová1,2
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2024 The Author(s). Functional Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.
1Institute of Botany of the Czech Academy
of Sciences, Třeboň, Czech Republic
2Department of Botany, Faculty of
Science, Charles University, Praha 2,
Czech Republic
Correspondence
F. Curtis Lubbe
Email: curtis.lubbe@ibot.cas.cz
Funding information
Akademie Věd České Republiky; Grantová
Agentura České Republiky, Grant/Award
Number: 22- 10897S and RVO 67985939
Handling Editor: Sabrina Träger
Abstract
1. Temperate winters are getting warmer, the length of the growing season is in-
creasing and mid- winter fluctuations of warm and freezing temperatures are
more frequent. Although typically winter dormant, some herbaceous perennials
can maintain or grow green leaves during the winter and more species may do so
as winters become warmer. For wintergreen leaves to provide an advantage, they
must be able to photosynthesize during the warm spells but also withstand freez-
ing temperatures and fluctuations between the two conditions.
2. To understand the leaf traits of wintergreen herbs, we studied three widely
distributed perennial species: Bellis perennis, Plantago lanceolata and Trifolium
repens. For contrast between winter and growing season leaves, we measured
three common leaf economic traits (LDMC, SLA and leaf N content). To assess
freezing tolerance, we measured the ice nucleation temperature and LT50 after
frost exposure in winter leaves. To confirm photosynthetic ability, we measured
photosynthetic efficiency (Fv/Fm value) in winter and late spring. Additionally,
we surveyed the green and dead leaf coverage for herbaceous species in a lawn
community from winter to early summer (four surveys in total). We used our ob-
servations to determine phenological categories and compared them to historical
observations of wintergreenness.
3. All three species had denser leaves with lower nitrogen content in winter than in
the growing season and relatively high tolerance of freezing temperatures (aver-
age of −8°C ice nucleation temperature and −15°C LT50). Both winter and grow-
ing season leaves were capable of photosynthesis, but the efficiency was greater
for growing season leaves. In the community survey, a majority of the plant spe-
cies had green leaves in winter, and many were without dead winter leaves. Many
wintergreen herbs had previously been labelled ‘summer green’.
4. With these initial investigations into the traits and phenology of wintergreen
herbs, we confirm that wintergreen leaves have different traits from typical grow-
ing season leaves and are capable of both photosynthesis and freezing tolerance.
Wintergreen herbs have a broad range of phenological strategies and differ from
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LUBBE et al .
1 | INTRODUC TION
Climate change has already greatly altered weather conditions across
the globe and these changes will continue to shift temperature av-
erages and increase fluctuations of both temperature and precip-
itation (IPCC, 2023). These changes occur across all seasons, and
the effect of warming may be greater during the winter than during
summer in some regions (Xia et al., 20 14). Winter warming varies
across latitudes (Henry, 2008) but we can expect stronger effects
in regions that experience freezing temperatures and snow cover
during winter where increasing temperatures might cause extended
growing seasons (characterized by photosynthetic activity and not
necessarily active growth), decreases in number of days with freez-
ing temperatures and loss of snow cover (Kreyling & Henry, 2011;
Sanders- DeMott & Templer, 2017 ). Longer winter periods with soil
and air temperatures above the freezing point may allow the per-
sistence or growth and development of aboveground organs such as
leaves, stems or even flowers in some ‘phenologically plastic’/non-
fully winter dormant plant species (Inouye, 2008). Although warmer,
these changing winters may still have periods of freezing tempera-
tures and vulnerable plant tissues may freeze without a layer of
protective snow cover (Henry, 2007). Thus, during warmer winters,
plants may have an opportunity to produce carbohydrates but also
have the challenge to avoid damage (Grossman, 2023).
Herbaceous plants in cold temperate climates generally lose all
aboveground structures during the winter to avoid damage from
freezing temperatures. However, the depth of winter dormancy
varies greatly across species (Gillespie & Volaire, 2017) and many
herbs may have green leaves during the winter (Beatley, 1956;
Geißelbrecht- Taferner et al., 1997). Some herbaceous species are
only green during the winter, taking advantage of the lack of canopy
cover, similar to spring ephemeral species (Damascos & Prado, 2001;
Tissue et al., 1995). Other species may either retain their summer
leaves into the winter or grow a separate group of leaves specifically
for the winter (Beatley, 1956; Minoletti & Boerner, 1993). The term
wintergreen can sometimes be used to describe the species that are
only green during winter, in contrast to the terms evergreen or per-
sistent green for herbaceous plants with green leaves in winter in ad-
dition to the other typical growing seasons (Klotz et al., 2002; Yoshie
& Yoshida, 1989). Here we will use the term wintergreen to indicate
the presence of green leaves regardless of summer- greenness or
time of their formation because many species may form separate
winter and summer leaves, instead of leaves that persist all year, as
is often implied by the term evergreen (Demascos & Prado, 2001;
Minoletti & Boerner, 1993). Although the ecology and economy of
wintergreen herbs is little understood, some forest species have
been examined. The wintergreen species of forests are generally
short and with few relatively thick basal leaves to avoid the flow
of cold air (Givnish, 1982) but less is known regarding leaf traits of
wintergreen plants across different habitats.
Wintergreen leaves may differ in their traits in comparison to
leaves that are only green during spring and summer, including
qualities that may be gained by winter- hardening and potentially
increased freezing tolerance after previous exposure to cold condi-
tions (Alves de Freitas Guedes et al., 2019). Growing season leaves,
with warmer conditions, may be relatively thin and large to maxi-
mize photosynthetic area and output (frequently referred to as a fast
economic strategy; Wright et al., 2004) whereas winter leaves must
survive harsher conditions and thus may be smaller and tougher,
with stronger cell walls which takes resources away from maximiz-
ing photosynthetic area (a comparatively conser vative and slow
economic strategy; Reich, 2014). Freezing tolerance and growth re-
sponse after freezing temperature exposure are generally studied
during the beginning and end of the growing season, and thus little
is known about the response of the winter leaves of herbs (Volaire
et al., 2023). Winter- adapted leaves may be denser, have smaller
cells and higher concentration of nitrogenous compounds and car-
bohydrates that may be part of a physiological frost stress reduction
strategy (Poorter et al., 2009; Valluru et al., 2008). Some winter-
green plants are often still capable of photosynthesis from beneath
the snow and may be vulnerable to the loss of consistent snow cover
(Starr & Oberbauer, 2003).
Warming has already contributed to phenological shifts
for plants across the globe and will continue to do so (Menzel
et al., 2020; Piao et al., 2019), but most of these records are for
the beginning and the end of the growing season. To understand if
herbs can take adv antage of warming winters in Centr al Europe (in
a typical temperate region where freezing temperatures are com-
mon in winter) via the presence of wintergreen leaves, we must
first know (i) if green leaves of herbs are present during winter,
(ii) if wintergreen leaves are physiologically functional and (iii) if
leaf traits differ between winter and growing season leaves to
best take advantage of either cold or growing season conditions.
Thus, we hypothesize that green winter leaves of herbs can pho-
tosynthesize as well as survive frost yet have more conservative
traits (are smaller and tougher) than typical growing season leaves
(late spring and summer). Additionally, we expect that there is an
increasing number of wintergreen herbaceous plant species in
observations 20 years ago. In many temperate regions, winter is becoming a new
growing season and thus should be included in phenological studies.
KEYWORDS
climate change, freezing tolerance, herbaceous perennial, leaf persistence, leaf traits,
overwintering, phenology, winter warm spell
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LUBBE et al.
Central Europe than previously reported. To test these hypothe-
se s, we mea sure d leaf trai t s , freez ing re sponse tr aits, ph otosy s tem
health and phenology for three common herbaceous species. We
also assessed the presence of species with green (photosynthet-
ically active) and brown (photosynthetically inactive and poten-
tially dead) leaves in winter, cold early spring, mid- spring and early
summer at a grassland site to examine winter leaf phenology, the
ex tent of win tergr e enne ss an d pla nt domi n ance in a co mmon law n.
We focused on herbaceous species of Central Europe, a region
likely to continue to lose winter snow cover and consistent freez-
ing (IPCC, 2023; Kreyling et al., 2019; Kreyling & Henry, 2011).
2 | METHODS
2.1 | Species and sites
Bellis perennis (Asteraceae), Plantago lanceolata (Plantaginaceae) and
Trifolium repens (Fabaceae) are perennial herb species, frequently
present in anthropogenic habitats such as managed grasslands
(e.g. lawns and meadows). All frequently have green leaves present
during the winter (Beatley, 1956; Klotz et al., 2002). Bellis perennis
and Plantago lanceolata grow from short and shallow rhizomes and
Trifolium repens grows from stolons that often have superficially bur-
ied increments (Klimešová et al., 2017).
Plants were collected or observed from two different lawns
in the South Bohemian region of the Czech Republic, either at
Lužnice (49°4.81855′ N, 14°45.38945′ E) or Třeboň (49°0.33763′ N,
14°46.40380′ E). These lawns are private property of the Institute
of Botany of the Czech Academy of Sciences and thus do not re-
quire permissions for fieldwork. Both sites were mowed three to
four times during the growing season and have similar species as-
semblages and environmental conditions. Mean annual air tempera-
ture was 7.4°C and mean annual precipitation 614 mm for the period
1977–2009 from the nearest meteorological station in a natural
sedge- grass marsh; the absolute minimum air temperature (−30.9°C)
was recorded in January 1985. The average number of arctic days
(days with a maximum temperature equal to or lower than −10°C)
decreased from 1.3 in the decade 1977–1986 to 0.1 in the decade
2000–2009 (Dušek et al., 2013).
Plants grow in diffe rent ways and at diff erent rates th roughou t
the year, making the defining of the growing season quite difficult
(Körner et al., 2023), especially regarding climate change (Sutton
et al., 2021). Here we describe the growing season as the tradi-
tional window of opportunity for growth (Körner et al., 2023), in
alignment with the meteorological seasons (Sutton et al., 2021),
such that winter occurs from 1 December to 28 February as op-
posed to later into March as according to the astronomical defi-
nition (NCEI, 2023). According to the meteorological definition,
March is a transitional state into true spring with greater vari-
ability and still frequent presence of freezing conditions (Hájková
et al., 2012). For this reason, we will generally refer to our mea-
sures as during either the cold season (from December to late
March) or the growing season (from late March to September). The
growing season term is meant to delineate the typical warmer and
more hospitable conditions for growth, photosynthetic activity
and general aboveground presence for herbs in the region (Körner
et al., 2023). When usin g the term winter, we mean specific ally the
meteorological definition above.
2.2 | Leaf and air temperatures
Leaf temperatures for the three studied plant species were meas-
ured in a lawn in Třeboň. Three specimens per species were se-
lected. Leaf temperatures were recorded by fine wire Cu- Co
thermocouples attached to the abaxial leaf surface (underside of
the leaf). Data were recorded with three 3- channel data loggers
(EMS Brno, Czech Republic) with a recording interval of 30 min
from 31 January to 28 February 2022 with increased frequency
(every 6 s) during a period with night frosts between 28 February
and 2 March 2022.
Additionally, a weather station was operated in Třeboň, very
close to the measured plants. The station was equipped with a set
of dataloggers with built- in sensors to record incident global radi-
ation (Rg), air temperature (T200) and relative air humidity (RH),
positioned on an aluminium pole at 200 cm height above ground.
The T200 and RH sensors and loggers were screened by a standard
white radiation shield. The sensors and dataloggers were supplied by
EMS Brno, Czech Republic (w w w . e m s b r n o . c z ). Measurements were
taken from 15 February 2022 to 2 March 2022, with a recording
interval of 1 h.
2.3 | Leaf traits
For assessment of trait differences between overwintering cold
season leaves and growing season leaves, we collected 10 indi-
viduals per species and one leaf per individual for specific leaf area
(SLA: leaf area divided by dry weight; cm2/g), leaf dr y matter con-
tent (LDMC: leaf dry weight divided by fresh weight), leaf water
content (LWC: leaf fresh weight minus dry weight then divided
by dry weight) and leaf area (cm2). Each leaf was weighed fresh,
scanned on an Epson V800 photo scanner, oven- dried at 60°C
for 48 h and weighed again. Additional leaves were collected from
each specimen and also dried and weighed to have adequate sam-
ple size for nitrogen analysis. Only Trifolium repens leaflets were
collected and not the petiole. Leaf samples were collected at the
end of freezing winter conditions and beginning of warm early
summer conditions (8 March and 2 May 2022, respectively). Cold
seasons leaves were those that were green and undamaged and
had been present during the winter, thus we did not select newly
formed leaves for these measurements. Growing season leaves
were collected after the formation and maturation of leaves pre-
sent and active for peak warmth and carbon acquisition of the full
growing season.
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LUBBE et al .
For nitrogen content, samples were mineralized with sulphuric
acid on a semi- micro scale. The organic forms of nitrogen were
converted by mineralization to ammonium ions (N- NH4
+), which
were determined spectrophotometrically after reaction of am-
monia gas with an acid–base indicator on an FIA flow analyser at
590 nm. Samples were pooled to reach an adequate weight for the
analysis, providing from four to five replicates for each species
and season.
2.4 | Plant cover survey and leaf phenology
observations
Green leaf and brown/dead leaf cover was assessed at species level
and surveyed during cold season conditions (15 February (winter)
and 17 March 2022 (cold early spring)) and growing season condi-
tions (13 April (warm spring) and 27 May 2022 (late spring/early
summer)) in 10 permanent plots (each 1 × 1 m, along 2 transects) at
the Lužnice site. Additionally, the plant cover survey was repeated
during the next two winters (February 2023 and 2024) in the same
plots. Species were classified as three different strategies based on
the presence or absence of green or brown leaves during the win-
ter survey (wintergreen: only green leaves present, partially win-
tergreen: green and brown leaves present, and not- wintergreen: no
green leaves present) and compared to leaf persistence observations
in the Bioflor trait database (Klotz et al., 2002; leaf persistence cat-
egories are given in Table S2).
2.5 | Freezing measurements
We assessed the freezing tolerance of leaves of Bellis perennis,
Plantago lanceolata and Trifolium repens: nucleation temperature and
LT50 temperature, corresponding to 50% injury of the tissues by
freezing. We only sampled and measured winter season leaves for
freezing tolerance because it is needed by cold season leaves for sur-
vival but would be otherwise superfluous for growing season leaves.
We sampled fully developed and undamaged leaves of six individu-
als of each species and placed them, along with an attached Cu–
Co thermocouple, in Peltier cooling chambers (ConBrio, Pardubice,
Czech Republic) at around 5°C for about 30 min, then lowered the
temperature at a constant rate of 5 K per hour (Sklenář, 2017). Leaf
temperature was recorded in 6- s intervals by dataloggers (EMS
Brno, Czech Republic). Nucleation temperature was indicated by a
sudden rise in leaf temperature. LT50 was estimated using the same
cooling rate in Peltier chambers. After about 30 min of exposure to a
target temperature, the temperature in a chamber was increased at
5 K per hour to 5°C. Freezing injury was estimated by the conductiv-
ity method (Prášil & Zámečník, 1998). The LT50 temperature, cor-
responding to 50% injury of the tissues by freezing, was estimated
by fitting the tissue injury data (calculated from the conductivity
measurement) to a sigmoid curve (Janáček & Prášil, 1991). For fur-
ther details, see Appendix S1.
2.6 | Health of photosynthetic systems
A portable chlorophyll fluorometer (Fluorpen FP100, PSI company,
Czech Republic) was used to measure an Fv/Fm value (ratio of vari-
able to maximum fluorescence). This value estimates the photosyn-
thetic efficiency of photosystem II in leaves in a dark- adapted state.
An Fv/Fm value in the range of 0.79–0.84 is the approximate optimal
value for many plant species, with lowered values indicating plant
stress (Maxwell & Johnson, 2000). We sampled 10 individuals of
Bellis perennis and Plantago lanceolata in Třeboň and Lužnice stations
on 1 March (during the end of cold conditions) and 3(6) May 2022
(during warm late spring/early summer), to compare the health of the
photosynthetic systems between the wintergreen and growing sea-
son leaves. No measurements were performed on Trifolium repens
because of the limited size of wintergreen leaflets.
2.7 | Replication statement
We wished to examine the traits of wintergreen herbs at multiple
scales, to better understand the intersection of leaf traits and toler-
ance and phenology. Economic, freezing and photosynthesis traits
were assessed for up to three species and two seasons (cold and
growing seasons; Table S1). We also conducted cover surveys of 10
plots, assessing both live and dead leaf cover at species level four
times in 1 year and then once each winter for another 2 years. Data
were analysed either based on species identity or phenological type
either through a single year (four surveys) or among winters (three
surveys).
2.8 | Statistical analysis
The comparison of leaf economic, freezing and photosystem traits
between cold and growing season leaves in the three model species
(Bellis perennis, Plantago lanceolata and Trifolium repens) was done
using factorial analysis of variance (ANOVA).
For the leaf cover survey, we worked with two forms of the
dataset: species- level (n = 2400) and plot- level (cover values ag-
gregated per plot, n = 40). For the species- level analysis, we used
generalized linear mixed models (GLMM; Ives & Helmus, 2011; Li
et al., 2020) with the Integrated Nested Laplace Approximation
(INLA; Rue et al., 2009) technique implemented in the pglmm
function (Li et al., 2020). We considered leaf cover as a response
variable, while we set months (February, March, April and May
as an ordinal variable), leaf type (green vs. dead) and phenology
type (based on survey observations: not- wintergreen vs. partially
wintergreen vs. wintergreen) as fixed effects. We also tested the
interaction between season and phenology type. Further, we set
three random- effect terms to account for variability in leaf cover
across species (random intercept, 1|Species) or plots (1|Plot), and
variability in leaf cover across seasons within species (random
slope, Month|Species). We used default INLA priors and assumed
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LUBBE et al.
zero- inflated Poisson error distribution to account for potential
excess zeros in the dataset generated by the sampling limitations
(e.g. some spatially aggregated species could have been absent
from a number of plots because of plot size and the spatial struc-
ture of plots in the transects, thus leading to more zeros in the
dataset).
To analyse seasonal dynamics of total green and dead leaf
cover and total leaf cover of wintergreen, partially wintergreen
and not- wintergreen species at the plot level, we fitted Markov
chain Monte Carlo (MCMC) GLMM using the MCMCglmm function
(Hadfield, 2010). We assumed normal error distribution and consid-
ered month as a fixed effect and plot identity as a random effect.
To achieve sufficient effective sample sizes (ESS) for posterior esti-
mates, we set 100,000 MCMC iterations (thinning interval = 5) and
discarded the first 10,000 as burn- in. We set a weakly informative
inverse- Wishart prior on the residual variance and the variance of
the random effects (V = 1, nu = 0.002). We visually checked for the
algorithm convergence.
To compare the three winters (leaf cover surveys in February
2022, 2023 and 2024), we performed non- metric multidimensional
scaling (NMDS) on the community data (species × site matrix) using
the metaMDS function (Oksanen et al., 2022). Data was standard-
ized (Wisconsin double standardization) and Bray–Curtis distance
was used as a measure of dissimilarity between plots (Oksanen
et al., 2022). We selected three dimensions (k = 3) to achieve suffi-
cient fit accuracy (stress = 0.13).
We did all analyses in R 4.2.1 (R Core Team, 2022) using the
MCMCglmm 2.34 (Hadfield, 2010), phyr 1.1.0 (Li et al., 2020) and
INLA 23.04.24 (Rue et al., 2009) packages to fit generalized linear
mixed models, vegan 2.6- 4 (Oksanen et al., 2022) to perform NMDS
and ggplot2 3.4.0 (Wickham, 2016) to draw graphics.
3 | RESULTS
3.1 | Leaf temperatures measured in situ
Mean January and February 2022 air temperatures were 1.2 and
3.3°C, respectively, slightly higher than the long- term mean air tem-
peratures for the period 1993–2022 (−1.1 and 0.1°C, respectively,
Czech Hydrometeorological Institute). Both January and February
2022 were almost without any snow cover (snow cover between
1 and 2 cm only for 4 days in January, Czech Hydrometeorological
Institute) and had frost nights with clear sky, especially during the
end of February and beginning of March. Leaf temperatures of all
three species dropped well below −5°C during clear nights in that
time (down to −7.0, −8.1 and −10.5°C in Bellis perennis, Plantago
lanceolata and Trifolium repens, respectively, Figure 1; Table S3).
The minimum and mean leaf temperatures of Trifolium repens were
slightly lower than in the other two species. Marked daily leaf tem-
perature amplitudes were measured in all studied species during
clear sky conditions reaching 26.5, 28.1 and 29.9°C for Bellis peren-
nis, Plantago lanceolata and Trifolium repens, respectively. The rate
of leaf temperature change around zero value was between 2.5 and
5.0°C per hour, closely tracking changes in global radiation (during
morning) and air temperature (during night) (Figure 1).
3.2 | Leaf traits
In the comparison of leaf traits between winter and early spring
(cold season) and mid- spring and early summer (growing season),
all species had winter leaves that had significantly greater LDMC,
lower leaf area, lower LWC, lower SLA and lower nitrogen con-
tent in winter leaves than growing season leaves (Table 1a). These
species all had a freezing strategy of tolerance of ice formation,
with ice nucleation temperature around −8°C for all species and
an LT50 around −24°C for Plantago lanceolata and Trifolium repens,
and −15°C for Bellis perennis (Table 1b). Leaves of Bellis perennis
and Plantago lanceolata were both capable of photosynthetic ac-
tivity during winter, although this was greater in the spring meas-
urements (Table 1c).
FIGURE 1 Air and leaf temperatures during 1–2 March 2022
with clear sky and pronounced night frost. (a) Daily courses of air
temperatures (T200) and global radiation (Rg) measured 2 m above
soil surface. (b) Mean leaf temperatures for the same day measured
on wintergreen leaves in a lawn, n = 3. Means and extreme values
of leaf temperatures from January 31 to 2 March 2022 are given in
Table S3.
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LUBBE et al .
TAB LE 1 Results of assessment of leaf economic, freezing and photosystem traits.
Season Cold Growing Cold Growing Cold Growing ANOVA results
Species Bellis perennis Plantago lanceolata Trifolium repens
Parameter F p
(a) Leaf economic traits
LDMC (g g−1 ) 0.33 ± 0.01 0.19 ± 0.02 0.39 ± 0.01 0.19 ± 0.01 0.40 ± 0.02 0.22 ± 0.01 274.6 <0.001
SLA (m2 kg−1) 120.34 ± 5.57 201.89 ± 13.55 100.50 ± 4.15 165.68 ± 7. 73 172.96 ± 6.06 283.75 ± 18.53 98.7 <0.001
Leaf N (%) 1.56 ± 0.02 1.74 ± 0.08 1.86 ± 0.16 2.65 ± 0.10 3.69 ± 0.08 4.77 ± 0.16 5 7. 5 <0.001
Leaf area (cm2) 1.09 ± 0.07 1.43 ± 0.07 1.44 ± 0.09 5.60 ± 0.38 0.84 ± 0.07 3.43 ± 0.15 8 7. 7 <0.001
LWC (g g−1) 2.08 ± 0.14 4.36 ± 0.33 1.57 ± 0.07 4.46 ± 0.33 1.53 ± 0.10 3.71 ± 0.26 166.6 <0.001
(b) Freezing traits
Ice nucleation
temperature
−7.7 ± 0.6 −7. 3 ± 0.5 −8.2 ± 0.1
LT5 0 −15. 3 −23.8 −25.0
Freezing strategy Tolerance of ice formation Tolerance of ice formation Tolerance of ice formation
(c) Photos ystem health
Fv/Fm
Lužnice site
0.735 ± 0.037 (n = 7) 0.815 ± 0.004 (n = 10) 0.639 ± 0.073 (n = 6) 0.823 ± 0.003
(n = 10)
23. 24 <0.001
Fv/Fm
Třeboň site
0.573 ± 0.039 (n = 9) 0.803 ± 0.0 06 (n = 10) 0.534 ± 0.017 (n = 8) 0.814 ± 0.006
(n = 10)
150.40 <0.001
Note: (a) Comparison of leaf economic traits (LDMC, SLA, leaf area, LWC and leaf N content) estimated for three wintergreen species during the cold season (winter) and growing season (late spring). Means
± SE intervals are shown, n = 10 for leaf area, LWC, LDMC and SLA and n = 5 for leaf N content. Statistical F and p values correspond to the main effect of season (df = 1). The results are based on factorial
ANOVA. (b) Ice nucleation temperature (means ± SE intervals), freezing tolerance (LT50) and freezing strategy estimated for leaves of three wintergreen species in late winter. (c) Fv/Fm values for two
wintergreen species measured in late winter and late spring in two study sites. Means ± SE intervals are shown. Small size of winter leaflets of Trifolium repens limited the measurement of Fv/Fm values.
Abbreviations: LDMC, leaf dry matter content; LWC, leaf water content; SLA, specific leaf area.
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LUBBE et al.
3.3 | Community survey
From the 2022 surveys, 26 out of 37 species had some winter
green leaves present, 15 species only had green leaves and 11
had a mixture of both green and brown (dead) leaves that var-
ied through the season based on the species. One species with
winter leaves was previously labelled as spring green and seven
were originally labelled summer green, one was a dominant spe-
cies during the winter (Luzula multiflora) and four of the species
were exclusively green (Table S2). Seven species previously la-
belled persistent green had both green and dead leaves present
during this survey and five additional species labelled persistent
green were only present and green during spring and summer, not
in winter of 2022.
When including all three winter surveys, 34 of the 37 species
had green leaves during at least one winter and 25 also had brown/
dead leaves present at least one winter (especially 2024, which had
23 such species in contrast to the 11 and 12 in 2022 and 2023, re-
spectively). Only Holcus lanatus, Stellaria graminea and Trifolium rep-
ens were present all three winters with only green leaves and only
Alchemilla glaucescens, Arrhenatherum elatius and Campanula patula
were absent all three winters (but present in spring and summer
2022). Eight species were consistent across all years in the presence
of both green and dead leaves.
The stand height in winter was generally 5–10 cm in all plots
during the years observed, corresponding to the presence of win-
tergreen herbs with low stature (e.g. Lysimachia nummularia) and
often forming basal leaf rosettes (e.g. Bellis perennis and Plantago
lanceolata) or graminoids with short leaves (e.g. Carex caryophyllea
and Luzula multiflora). In early summer, most wintergreen species
were taller (in the range of 10–30 cm) than in winter but usually they
formed a lower layer in the grassland community. In contrast, most
summer green species had different growth forms and reached up
to 50 cm in height (e.g. Arrhenatherum elatius, Phleum pratense and
Securigera varia).
In our plant community survey, we observed higher cover of
green than dead- leaf biomass in all studied months, except for
March (Figure 2a; Table S4C) when dead- leaf biomass peaked.
Compared to February, overall leaf cover was slightly lower in
March, but then progressively increased over the season (Figure 2a;
Table S4C). Partially wintergreen species (species with both green
and dead leaves present) tended to produce more green- leaf bio-
mass in all months, except for March (where similar cover was ob-
served for both partially and fully wintergreen species), compared
to wintergreen and not- wintergreen species (Figure 2b; Table S4C).
The GLMM model also revealed synergistic effects of season and
species phenology type (Table S4D). Negative interaction coeffi-
cient s for April and May with partially wintergreen and wintergreen
phenologies (Table S4D) suggest that the relative positive increase
in leaf cover is lower for these phenology types compared to not-
wintergreen species. Conversely, the positive interaction coeffi-
cients for March (Table S4D) indicates a stronger increase of cover
of partially wintergreen and wintergreen from February to March
compared to not- wintergreen species, which should be the case be-
cause not- wintergreen species produce no leaf biomass in March
by definition.
Sp ecies iden t ity was the la r gest so urce of var iation of le af cov er
from all random- effect terms (Table S4B). The variance attributed
to plots wa s negli gibl e (Table S4B). Som e por tion of variatio n in le af
cover was attributed to the random slope term (Month|Species,
Table S4D), suggesting species- specific seasonal dynamics in green
and dead leaf cover over the season. Examining the eight most
abundant species, we observed a few general patterns (Figure 3).
Green- leaf cover was typically the lowest in March, while dead-
leaf cover peaked in March in most species (Figure 3). Green- leaf
cover started to rapidly and monotonically increase from March
in most species (Figure 3). Finally, we observed a few exceptions:
green- leaf cover of Agrostis capillaris peaked in February then rap-
idly declined till March and after that stayed constant (Figure 3a).
In Ranunculus bulbosus green- leaf cover increased throughout the
season but peaked in April (Figure 3f). In Luzula multiflora, dead-
leaf biomass monotonically decreased over the season and in
Festuca rubra, dead- leaf biomass peaked in April and then rapidly
decreased.
FIGURE 2 Seasonal dynamics of
green- and dead- leaf cover aggregated
per plot for (a) total cover (lines represent
single plots) and (b) cover of species with
only green leaves present (wintergreen),
green and dead leaves present (partially
wintergreen) or no green leaves present
during winter. Dead- leaf biomass was,
by definition (for list of species and
phenology see supplemental material,
Table S2) observed only in partially
wintergreen species. Vertical lines
represent 95% Bayesian credible intervals.
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LUBBE et al .
3.4 | Among- winter plant cover comparison
The first two NMDS dimensions separated the plots from February
2024 from the plots sampled in February 2022 and 2023 (Figure 4a).
Because Februar y 2024 showed no deviation in terms of total green
(Figure 4c) or dead- leaf cover (Figure 4d), this separation was mostly
because of differences in species composition. In February 2024,
we observed relatively higher cover of both green and dead leaves
of Crepis biennis and Poa pratensis than in Februar y 2022 and 2023.
The second and third NMDS dimension separated the samples from
FIGURE 3 Seasonal dynamics in green
and dead leaf cumulative cover (across all
plots for each month) of eight dominant
species (a- h) in 2022 (2 = February,
3 = March, 4 = April and 5 = May). With
the exception of wintergreen Trifolium
repens, all other species are classified as
partially wintergreen (for list of species
and phenology see supplemental material,
Table S2). To fully demonstrate species-
specific strategies and trends, each
subplot has its own y- axis scale. Cover
includes all plants, including canopy and
understorey and can be over 100%.
FIGURE 4 Community- level
comparison between the three winters.
(a) First two dimensions of non- metric
multidimensional scaling (NMDS) based
on species community data in 10 plots
over three winters (February sampling in
2022, 2023 and 2024). (b) Visualization of
the second and third NMDS dimension.
(c) Green- leaf cover values across the
three winters per plot. (d) Dead- leaf cover
values across the three winters per plot.
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LUBBE et al.
February 2022 and 2023 (Figure 4b), which was most likely because
of the decline of green- leaf cover in the majority of plots in February
2023 (Figure 4c).
4 | DISCUSSION
The ther mal conditions during th e wi nt er of 2022 were slight ly warmer
than the historical temperature conditions of the area (the difference
between January and February 2022 mean air temperature was 2.3
and 3.2°C, respectively, compared with the means in 1993–2022,
Czech Hydrometeorological Institute). From our assessment of three
wintergreen herbaceous species, we supported our hypothesis that
winter leaf traits (green leaves present during winter regardless of time
of formation) differ in comparison to growing season leaves, specifi-
cally winter leaves had lower leaf area, LWC, SLA and content of ni-
trogen and higher LDMC than growing season leaves. Winter leaves
also had relatively high tolerance to freezing damage. Both Plantago
lanceolata and Bellis perennis had lower photosynthetic efficiency (as
Fv/Fm values) in cold season than in growing season leaves, indicating
some stress, but values were high enough for plants to be capable of
photosynthetic activity during the cold season. We have thus verified
that these three species have all requirements to take advantage of
warming winter conditions because (i) green leaves are present during
winter, (ii) winter green leaves are capable of photosynthesis and frost
tolerance and (iii) winter green leaves differ in their economic traits
compared to growing season leaves. From our assessment of a plant
lawn community, we identified several wintergreen species that were
previously reported as ‘summer green’, supporting our hypothesis that
there are more herbaceous species with green leaves present in winter
than previously reported. The presence and cover of green or dead
leaves also varied across years.
4.1 | Economic leaf traits
Winter and growing season leaves of all three species were distinctly
different depending on the season and all species had leaves during
the winter that were significantly smaller and denser but lower in
nitrogen than leaves during the growing season. This indicates more
investment into tissues for protection (including toughness and cell
wall reinforcement) as more conservative traits (Reich, 2014) than is
typical for those species in summer. This is in alignment with previous
findings that SLA and LDMC both vary through the growing season
(with cold spring and autumn and warm summer; Gast et al., 2020;
Römermann et al., 2016). In a study using summer green tree spe-
cies, Römermann et al. (2016) found an increase in LDMC through
the season with leaf longevity until a decline upon leaf senescence,
while Gast et al. (2020) found high spring and autumn LDMC com-
pared to summer values in evergreen trees, which aligns with the
higher winter LDMC of our wintergreen herbs. Our results may be
evidence of adaptation of winter leaves to survive both incidental
frosts and warm spells during the cold season. The trait values we
recorded may also mirror those for plant species from harsher envi-
ronments (e.g. cold or dry), that often have smaller and denser leaves
(i.e. lower SLA and higher LDMC) (Reich, 2014; Wright et al., 20 04).
Thus, the comparatively dense leaves of these wintergreen species
would also align to a conservative, slow economic strategy. Higher
leaf nitrogen content is generally a trait of more competitive plant
species that have fast leaf economic strategies (Reich, 2014), al-
though increased nitrogen content in winter leaves may also be an
adaptation to survive freezing temperatures and decrease damage
from radiation (Ensminger et al., 2006; Nishitani et al., 2020), yet
this was not found in this study. Some studies find a general cor-
relation between high nitrogen content and increased freezing dam-
age (Malyshev & Henry, 2012) which may align with our results of
lower nitrogen content in winter leaves with relatively high freezing
tolerance.
In contrast to woody deciduous or evergreen species, wherein
deciduous leaves are grown and shed for activity only during the
favourable seasons and evergreen species form leaves with lon-
gevity of 1 year or greater (Damascos & Prado, 2001; Minoletti &
Boerner, 1993), wintergreen herbaceous species may have different
seasonal cohorts of leaves allowing for both the growing and win-
ter seasons. Wintergreen herbs thus may have a competitive and
fast leaf economic strategy during the summer with fast growth and
high SLA and a slow strategy during the winter with smaller leaves
with high LDMC (Reich, 2014; Wright et al., 2004). In a compari-
son among leaf longevity strategies, evergreen species (longevity
greater than 1 year) had the densest leaves, deciduous species had
the least dense leaves and traditional wintergreen species (primary
growth and green leaf presence during winter) had intermediate
values (Demascos & Prado, 2001) perhaps to accommodate for the
harsh winter conditions but not to support increased longevity. This
variation in leaf traits and economic strategies also challenges the
idea of a single fast or slow strategy of plants for the full year and
instead indicates varying strategies that can be adapted to survive
a variety of conditions through the years (Volaire et al., 2023), in-
cluding as conditions change, such as through winter warming trends
(Grossman, 2023).
4.2 | Leaf physiology
The successful winter survival of leaves depends on their ability to
assimilate carbon and to resist, tolerate or avoid frost and photo-
oxidative stress (Malyshev & Henry, 2012; Slováková et al., 2011). All
three species used tolerance of ice formation and the temperatures
for the formation of ice (ice nucleation) and 50% mortality (LT50) of
winter leaves were relatively low and comparable to the frost tol-
erance of the cold- hardened growing- season leaves of some alpine
herbs (Sklenář, 2017). Our measures of frost tolerance for leaves of
Bellis perennis (during a relatively warm winter without protective
snow cover) was even higher (−15.3°C, Table 1) than previously re-
ported values for leaves of this species sampled below snow cover
(−11.5°C for fully developed and −13°C for young leaves; Sakai &
10
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LUBBE et al .
Larcher, 1987). The same study reported relatively high frost toler-
ance for some other wintergreen species in our study (e.g. −17 to
−27°C for Festuca rubra).
Freezing tolerance is one strategy used by plants to survive
freezing temperatures and provides greater resistance to, and safety
from, freezing damage than the strategy of frost avoidance via su-
percooling (Schulze et al., 2006). Plants can use many different types
of compounds to promote freezing tolerance, including accumula-
tion of water- soluble carbohydrates and nitrogenous compounds
such as proline (Koocheki & Seyyedi, 2019; Patton et al., 2007;
Valluru et al., 2008) or different types of lipids to protect tissues
against damage (Raju et al., 2018). However, we do not identify the
exact mechanisms here, which likely varies, especially among the
unrelated species in our field survey. There is little known about
the freezing traits of wintergreen herbs because leaf and freezing
traits have only occasionally been measured on them because the
focus has usually been on early- growing season frosts and spring
leaves. The three species measured for leaf traits here were capable
of maintaining and protecting their leaves through these stresses to
remain green all winter. This may also be valid for other wintergreen
species from our survey.
Plantago lanceolata and Bellis perennis winter leaves had lower
photosystem health than growing season leaves. We do not know
if this lower capability is because of differences in formation and
allocation between winter and spring leaves, or the effect of damage
from freezing temperatures or radiation (Ensminger et al., 2006). The
leaves of some wintergreen and spring ephemeral species have a de-
creased ability to alter their response to temperature (i.e. to increase
photosynthetic capacity with increasing temperature), compared
to deciduous or evergreen leaves (i.e. with multiple year lifespans)
(Tissue et al., 1995).
4.3 | Wintergreenness of herbs
Although it is taken for granted that herbs in regions with cold win-
ters undergo dormancy, incomplete dormancy (and thus greater
flexibility in phenology) is quite common (Schnablová et al., 2024).
Wintergreen herbs have repeatedly been documented in cold tem-
perate regions (Beatley, 1956; Jager & Werner, 2002; Yoshie &
Yoshida, 1989), but the number of wintergreen herb species may be
greater th an pre vio usly note d, esp ecially for re gio nal fl oras with win-
ter warming trends, such as Central Europe (Kreyling et al., 2019). We
observed a strong disparity between historical species observations
and phenological categories. From our survey, many species previ-
ously categorized as ‘summer green’ had wintergreen leaves and al-
most half of those species only had green leaves during the first year
of the survey; this increase in number of wintergreen plant species
may be in response to the growing trend in winter warming (Kreyling
et al., 2019; Kreyling & Henry, 2011). In contrast, many species pre-
viously recorded as ‘persistent green’ often had large percentages
of dead leaves, and the ratio of green to dead leaves varied across
the season and years, dependent on species (Figure 3). Additionally,
some ‘persistent green’ species had no wintergreen leaves present
and only started growing from early spring. The presence of green
leaves during winter is not just an acquisitive advantage but may also
be a cost. The abundance of brown leaves and lack of presence dur-
ing the winter for many species in this survey may be in response to
greater exposure to freezing temperatures and solar radiation with
a loss of snow cover and pronounced temperature fluctuations with
further climate change (Williams et al., 2015).
Many species at our study site were able to take advantage of
warm and snow- free spells to have green and potentially photosyn-
thetically active leaves. Most wintergreen herbs form leaf rosettes
or leafy shoots close to the soil surface and thus may profit from
the greater heat absorption by the soil surface and the decoupling
from the atmosphere, similarly to many alpine herbs (Körner, 2003;
Sklenář, 2017 ). Wintergreen herbs are relatively well documented
from forest habitats (Givnish, 1982) where they are partially pro-
tected by the tree layer and leaf litter and profit from the high
irradiance during early spring. Yet many herbs of open habitats
may also have green winter leaves (Doležal et al., 2019; Fischer
et al., 2023), an d with greater variet y in form and habit than among
forest herbs (Beatley, 1956). Wintergreen herbs are a phylogenet-
ically diverse group, and their phenological strategies are similarly
diverse with a wide range of variation in the time of formation and
loss of winter leaves (Beatley, 1956). The contrast between winter
and growing season leaves is also quite variable, with some species
forming relatively similar leaves in both seasons (such as Sedum
ternatum, Fragaria virginiana and Houstonia caerulea), or can be ve ry
different in their timing and appearance (such as some Viola spe-
cies; Beatley, 1956). It is unknown how many and what percentage
of species in other habitats undergo incomplete dormancy and
would be capable of such strategies. Our study site is under a rela-
tively high disturbance regime (a lawn mown approximately every
4 weeks), thus litter accumulation is relatively low and light avail-
ability may be less obstructed than in other grasslands that have
a consistent layer of dead leaves that could shade the compara-
tively smaller wintergreen plants. We also do not know to what
extent this community has changed over time if recent warming
has already caused the loss of the more vulnerable species in fa-
vour of species with more plastic phenology and greater freezing
tolerance.
The presence of green winter leaves is not a constant, as it is
for growing season leaves, the conditions of a single winter may be
inhospitable for leaf production or maintenance but acceptable the
ne xt. Les s tha n one - thir d (10/35 ) of spec ies ha d consi s tent pre sen ce/
absence observations for green and dead leaves across all 3 years.
Jus t as we saw variation in strategy across species (Figure 3), we saw
variation among years, as conditions may have promoted or discour-
aged green leaf production/persistence. Additionally, many species,
especially graminoids, may not only have green and brown leaves on
the same plant, but single leaves themselves may be partially dead
with more basal portions of the leaf still alive and active. This was
frequently observed in this survey and may be more common in lo-
cations with less litter removal.
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LUBBE et al.
4.4 | Future directions
Although we found many links between phenology, physiology, and
functional traits, there is still much to learn about these connec-
tions in the wintergreen strategies of herbs. Although we already
see great diversity among the plants in this study, plants may form
their wintergreen leaves any time before or during winter, and these
differences could be vital for better understanding the costs and
benefits of wintergreenness for different species. Freezing toler-
ance is important for the survival of wintergreen leaves, but this can
be achieved many different ways, including different water- soluble
carbohydrates, nitrogenous compounds or membrane lipids (Patton
et al., 2007; Raju et al., 2018; Valluru et al., 2008). Future studies
will need to identify what mechanisms are used, especially because
of the strong phylogenetic conservation of many water- soluble car-
bohydrates (Hendry, 1987; Lubbe et al., 2021; Van den Ende, 2013).
Winter survival of green leaves, and acquisition of carbon during
winter may be an advantage for spring and summer competition of
some plants, but for others it may simply prolong persistence within
the community when summer shading by competitors is very strong.
More complex study of phenology and ecophysiology will be neces-
sary to elucidate the role of wintergreenness for different herba-
ceous species and whole plant communities.
5 | CONCLUSIONS
Whether costs or benefits of wintergreen strategies will prevail de-
pends on temperature, precipitation, their fluctuations and inter-
actions during the winter, and plant responses to these conditions
which may result in shifts in species dominance in a community. We
do not know how many herbaceous plant species can take advantage
of warming winters. Winter survival strategies are complex and di-
verse, and future work will be needed to properly record and recog-
nize the different traits and phenology across species and between
winter and the traditional growing seasons. Additionally, the carbon
gain of plants and input into the soil as litter may be very different
fro m historical conditions, with the potential inclusion of new pe riods
of leaf gain and loss occurring through the winter. Altered compe-
tition and community composition can even scale up to ecosystem
level changes with altered regimes of carbon input and cycling.
AUTHOR CONTRIBUTIONS
F. Curtis Lubbe, Andrea Kučerová and Jitka Klimešová conceived
idea. F. Curtis Lubbe and Andrea Kučerová collected data. Martin
Bitomský analysed data. F. Curtis Lubbe led writing of the manu-
script. All authors contributed to writing of this manuscript.
ACKNOWLEDGEMENTS
The study was supported by Grant Agency of the Czech Republic
GAČR (No. 22- 10897S), by long- term research development pro-
ject of the Czech Academy of Sciences (No. RVO 67985939) and
by the Praemium Academiae award from the Czech Academy
of Sciences of the Czech Republic. M. Bitomský was supported
by the Programme for the Promotion of Prospective Human
Resources—Postdocs (PPLZ) of the Czech Academy of Sciences.
We are also grateful to P. Sklenář (Department of Botany, Charles
University, Prague) for providing the instruments for measure-
ments of freezing tolerance and J. Kvíderová (Department of
Algology, Institute of Botany) for the assistance with FluorPen
measurements.
CONFLICT OF INTEREST STATEMENT
We have no conflicts of interest.
DATA AVAIL AB I LI T Y STATE MEN T
Data available from Mendeley Data repository: h t t p s : / / d o i . o r g / 1 0 .
1 7 6 3 2 / w 2 3 d x z f 9 m 4 . 1 (Lubbe et al., 2024).
STATEMENT ON INCLUSION
The majority of authors represent the study region.
ORCID
F. Curtis Lubbe https://orcid.org/0000-0002-1272-6226
Jitka Klimešová https://orcid.org/0000-0003-0123-3263
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SUPPORTING INFORMATION
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
Appendix S1: Detailed freezing methods.
How to cite this article: Lubbe, F. C., Kučerová, A., Bitomský,
M., & Klimešová, J. (2024). New opportunities for grassland
species in warming temperate winters. Functional Ecology, 00,
1–13. https://doi.org /10.1111/1365-2435.14677