4 DISCUSSION
Cold acclimation is an inducible process, and researchers have
demonstrated that induction of the cold acclimation pathway occurs
within the first 15 min of exposure to low, non-freezing temperatures
such as 4°C for the monocarpic and annual Brassicaceae speciesArabidopsis thaliana . The highest frost tolerance was reached in
a few days (2-5) in previous studies as multiple mechanisms worked in
parallel, sometimes interacting to confer maximum tolerance to frost
(Gilmour et al., 1988; Xin & Browse, 2000; Hincha et al., 2014). For
all Cochlearia and Ionopsidium individuals assessed in
this study, an acclimation period of five days at a temperature of 4°C
was sufficient for the studied species to develop a profound cold
acclimation and a largely enhanced freezing tolerance. This tolerance is
built upon similar physiological principles and primary metabolites
(Wolf et al., 2021), especially carbohydrates and amino acids. These
significant increases in carbohydrate levels, a widely known reaction to
cold stress in plants, have been demonstrated in Cochlearia andIonopsidium through metabolome analyses (Wolf et al., 2021).
Carbohydrates play a crucial role as cryoprotectants and signalling
molecules in plant cold responses (Janská et al., 2010; Davey et al.,
2008). Similarly, among analyzed amino acids, proline is recognized for
its role in plant responses to various abiotic stresses, including low
temperatures (Ashraf & Foolad, 2007). Increased levels of glutamic acid
and aspartic acid are associated with a typical stress response, which
is consistent with the cold metabolomes of A. thaliana (Kaplan et
al., 2004).
In contrast to A. thaliana sampled across Europe and analyses of
various “ecotypes” with a demonstrably low within-accession variation
(e.g. Hannah et al., 2006; Zuther et al., 2012), there was a
substantially larger variation in the freezing tolerance within the
accessions of Cochlearia and Ionopsidium analysed herein.
In contrast to A. thaliana ecotypes with comparably minimal
within-accession genetic variation (often inbred lines obtained from
stock centers), the material used in this study most often reflects its
natural genetic diversity because (i) the seeds originated from the
wild, (ii) most species are outbreeding, and (iii) polyploidy may
contribute to increased genetic variation. Furthermore, we may also
assume a larger phenotypic plasticity, at least for polycarpicCochlearia species, compared to annual taxa. However, we did not
find any significant difference (p > 0.01 neither
for ploidal-level variation (LT50 accl.,LT50 non-acclim., LT100accl., LT100 nonaccl.; p =
0.152/0.489/0.019/0.138) or in comparing monocarpic and polycarpic life
forms (LT50 accl., LT50non-acclim., LT100 accl.,LT100 nonaccl.; p =
0.315/0.361/0.623/0.437) in the Cochlearia /Ionopsidiumalliance; this suggests that multiple factors contributed to its higher
plasticity compared to A. thaliana . Additional electrolytic
leakage data are available for Arabidopsis lyrata , a polycarpic
species spanning a distribution range from lowland sites in Central
Europe to the Arctic region across the Northern Hemisphere (Schmickl et
al., 2010; Hohmann et al., 2014; Koch, 2018; Hohmann & Koch, 2017). For
this polycarpic, diploid, and largely outbreeding taxon, significant
differences in the survival of sub-zero temperatures from different
geographic regions have been demonstrated, with the majority of plants
not surviving temperatures below -10°C (Davey et al., 2018). In North
America, A. lyrata (Wos & Willi, 2015) demonstrated that
resistance to frost and heat varies significantly with latitude.
However, in this study, aside from resistance as quantified by leaf
damage (electrolytic leakage), tolerance to frost and cold measured as
the phenotypic plasticity of an entire plant grown under varying
temperature regimes did not increase in the northern region; therefore,
the cost of frost tolerance may be an important component of the limits
of species distributions (Wos & Willi, 2015).
Cochlearia and Ionopsidium can be clustered into separate
groups according to the bioclimatic character of their habitats, proving
that different species experience varying bioclimatic environmental
conditions. In the distribution range of Ionopsidium , hot and dry
conditions prevail, and arctic-alpine Cochlearia species may be
exposed to extremely low winter temperatures paired with a strong
temperature seasonality and winter dryness. Inland as well as coastalCochlearia species experience conditions between these extremes.
We assumed that varying selection pressures in these geographically
distant habitats would lead to differences in the species´ responses to
cold. Even though considerable variation in lethal values was detected
within species assessed in this thesis, there was minimal significant
variation in freezing tolerance among species. Even though the species
supposedly experience different environmental conditions, they exhibited
a similar responses to freezing temperatures in this study. We expected
that northern species such as C. groenlandica would display a
much higher freezing tolerance compared to southern Ionopsidiumspecies, proving the existence of a latitudinal gradient of selection
for or against cold tolerance. However, the data measured in this
experimental setting did not support this hypothesis. NorthernCochlearia species did not show considerably higher lethal values
than southern Ionopsidium species (see Fig. 6). Temperature
seasonality and winter dryness intensify with increasing distance from
the coast, which may increase the need for cold tolerance in inland
species. Continentality can therefore create a longitudinal gradient of
frost tolerance. However, the analysis indicated that a combination of
latitude and longitude may influence the freezing tolerance of different
accessions. There was only a demonstrably weak trend in decreasing
lethal values following a southwest to northeast gradient. Considering
this result, key adjustments of the Cochlearia species may
instead be adaptations to winter dryness, as the response to low
temperatures seemingly did not vary significantly between mostCochlearia species. Accordingly, principal coordinate analysis
(Fig. 2) showed that the distributions of northern, alpine, and inland
Cochlearia species were strongly influenced by precipitation variables.
Arctic and alpine Cochlearia species may be covered by an
insulating layer of snow during winter, which suggests another reason
for the lack of differences in freezing tolerance between these and
other species. Snow protects plants growing underneath from freezing
damage as it creates a relatively mild microclimate. Plants growing
underneath a layer of snow are sheltered from ambient temperatures that
can drop as low as -45°C, whereas temperatures below the snow cover may
only be as low as -5°C (Bokhorst et al., 2009; Armstrong et al., 2015).
This suggests that northern and alpine species may not be exposed to
lower temperatures than other Cochlearia species, thereby
decreasing the need for increased frost tolerance compared to other
species. Even though Cochearia species are spatially widespread,
they mostly inhabit cold-characterized habitat sites (Wolf et al.,
2021). The lack of a correlation between latitude or longitude and the
species´ response to cold supports the distribution of Cochleariaspecies among often azonally distributed habitats, such as cold
calcareous springs, wet meadows, or wet bedrock, where local conditions
are formative rather than climate zones. Even though the ambient climate
of the greater region may be warmer, plants growing in or along
cold-characterized habitats share a need for cold adaptation, similar to
arctic and alpine species. Central European Cochlearia species,
except for C. danica , are highly endangered and are mostly
threatened by habitat loss. In the alpine system, however, C.
excelsa occurs only as a southeastern alpine-endemic species at two
high mountain peaks (Seckauer Zinken, Eisenhut; Austria); during the
last 25 years, scattered populations have declined rapidly and
elevational occurrence has lost roughly 250 m at its lower distribution
limits from 1900 m to 2150 m a.s.l. (Koch, unpublished data); this is in
accordance with general observations of alpine flora shifts affected by
global warming (e.g. Auld et al., 2022; and reference provided therein).
The similar cold responses exhibited by different Cochleariaspecies may therefore be a result of several factors acting in concert.
It still unknown why Ionopsidium species respond similarly toCochlearia species in mitigating freezing temperatures despite
being exposed to hot and dry conditions. Although cold acclimation is
likely a useful tool in protecting against freezing damage in mostCochlearia species, Ionopsidium species should not be
expected to require cold acclimation, considering their Mediterranean
distribution. Researchers have hypothesized that cold acclimation comes
with a biological cost to the plant in environments that rarely
encounter freezing events, such as the Mediterranean (Zhen et al., 2011;
Meireles et al., 2017). As the process of cold acclimation includes
extensive physiological and biochemical changes, a trade-off should be
expected between the degree of cold tolerance and other metabolically
challenging processes, such as growth or reproductive rates. The cost of
cold tolerance may explain latitudinal selection gradients. If cold
tolerance does not come at a cost to the plant, it should be generally
high, even in species that are seldom exposed to freezing temperatures
(Armstrong et al., 2020). This cold tolerance cost has not been observed
in several studies evaluating the freezing tolerances of different plant
species (Zhen et al., 2011; Wos & Willi, 2018; Armstrong et al., 2020).
This could explain why Ionopsidium species exhibit similar cold
responses to those of Cochlearia species. Constitutive cold
tolerance in Ionopsidium could be maintained, even though these
species rarely encounter freezing events. Wolf et al. (2021) revealed
that although different Cochlearia and Ionopsidium species
can be clustered into ecological groups, they do not show significantly
different metabolomic responses to cold stress. The same ecological
groups were identified herein and support the observations of Wolf et
al., in that all species seem to exhibit a similar tolerance to cold. We
speculate that the observed magnitude of the cold response may predate
the origin of Cochlearia and Ionopsidium and may have
resulted from an ancient preadaptation in a remote tribe (Cochlearieae)
in the Brassicaceae family (Walden et al., 2020). Wolf et al. (2021)
argued the continuous connection of Cochlearia to
cold-characterised habitats since its diversification during the
Pleistocene glaciation and deglaciation cycles, in which it migrated to
the northern regions. This early-evolved cold tolerance may have not
been lost secondarily in Cochlearia explaining the low lethal
values in southern and coastal species such as C. danica .
However, cold tolerance appears to be accompanied by sensitivity to
increased temperature, which was not analyzed in this study. However,
some species, such as C. pyrenaica and C. polonica , are
critically endangered according to the IUCN Red List of Endangered
Species, as their habitats have become increasingly rare. With global
warming, endemic species such as C. polonica may face extinction,
they may not have enough time to adapt to rapidly warming conditions.
Cochlearia danica and the genus Ionopsidium may exemplify
the escape route needed to migrate from increased temperature and
drought, as all species are monocarpic and may survive uncomfortable
seasons as seeds in the soil seed bank. Cochearia danica , for
example, is highly adapted to coastal sand dune habitats and can manage
high levels of salinity, drought, and disturbance (Koch, 2012). This
species exhibited the highest freezing tolerance, which supports the
assertion that adaptations to salt or drought stress may also confer
tolerance to cold. Other studies have evaluated the ability ofCochlearia seedlings to cope with salt stress (Levi Yant,
Nottingham, unpublished), showing that salt stress can be managed by
cold-adapted species that do not naturally occur in regions with high
salinity. Unfortunately, all present-day Ionopsidium are
monocarpic; therefore, we cannot test the hypothesis of convergent
evolution (as referred to in C. danica ) changing the life cycle,
such as with the onset of the Mediterranean salinity crisis (MSC) appr.
6 -5.3 mya during the Late Miocene (Mascle & Mascle, 2019).