3 RESULTS
3.1 Bioclimatic niche characteristics of analyzed Cochlearia andIonopsidium accessions show severe differences
The principal coordinate analysis revealed a cumulative proportion of
variance of 91% for the first three principal components (PC1,65.9%;
PC2,19.2%; PC3,5.9%). As shown in Fig. 2a, four Cochleariaecotype groups were defined, with Ionopsidium forming a fifth and
separate group of accessions. Mostly along PC1, alpine accessions
(C. excelsa and C. tatrae ) were grouped into cluster 1.
Cluster 2 was comprised primarily by inland taxa such as C.
polonica and C. pyrenaica , but also included arctic C.
groenlandica . A third cluster combined polycarpic coastal taxa
(C. anglica and C. aestuaria ). Monocarpic accessions ofC. danica were considered as a separate cluster 4, which shows
closest affinities with Mediterranean Ionopsidium representing
cluster 5 also defined exclusively by monocarpic taxa. These results are
consistent with the findings of Wolf et al. (2021), who identified the
same groups based on nine of the 19 bioclimatic variables. These results
show that Cochlearia and Ionopsidium accessions are
exposed to varying environmental conditions, which is expected
considering their geographically distant distribution.Ionopsidium accessions can be aggregated into a single group,
suggesting that they inhabit regions with similar precipitation rates
and temperatures. Cochlearia accessions, however, were separated
into different groups, suggesting a wider distribution and habitat sites
that vary in their bioclimatic character along PC1. The orientation and
length of loading vectors of variables indicate the method through which
variables contribute to principal components, and therefore to separate
different clusters (Fig. 2b). Notably, precipitation- and
temperature-dominated variables separate clusters on the left (mostly
polycarpic Cochlearia accessions) from clusters on the right
(monocarpic coastal, Mediterranean Ionopsidium ) in a very similar
way. The differentiation of clusters along axis 1 suggests that the
habitats of alpine and inland Cochlearia species may be strongly
influenced by the amount and regularity of precipitation under cold
conditions, whereas for coastal and Mediterranean species, temperature,
especially warm temperatures, may play a more prominent role in
ecological separation. This raises the question of whether
continentality, which is defined by a strong seasonality of temperature
and lower precipitation (Bruch et al., 2011), strongly influences the
inland distribution of Cochlearia species. Based on these
results, it would be expected for different ecological groups to vary in
their physiological adaptation to cold; this is especially relevant for
groups that were most clearly separated, such as alpineCochlearia and Ionopsidium accessions, as differences in
these groups´ bioclimatic niches are supposedly the strongest.Cochlearia danica and coastal Cochlearia species may
exhibit a cold response that is more similar to Ionopsidiumspecies, as these groups were positioned closely together in the PCoA
analysis.
3.2 Cold acclimation similarly enhanced the freezing tolerances ofIonopsidium and Cochlearia accessions
Cold acclimation for five days at 4°C enhanced the freezing tolerance of
all Cochlearia and Ionopsidium accessions.LT50 and LT100 values were
significantly lower for acclimated samples than for non-acclimated
samples (LT50 : t = -6.5886, df = 61.131,p -value = 5.824e-09; LT100 t = -5.8435, df
= 79.099, p -value = 5.437e-08). AcclimatedLT50 ranged from -2.82°C to -12.82°C with a mean
value of -7.06°C, whereas non-acclimated LT50values were generally higher, ranging from -2.34°C to -9.24°C with a
mean of -4.17°C (Table 1). This was also true forLT100 values, with acclimated values ranging from
-4.24°C to -18.09°C (mean: -11.56°C) and non-acclimated values ranging
from -2.12°C to -11.78°C (mean: -7.37°C) (Table 2). There was
substantial variation in the lethal values for all the measured
accessions (Table 2). Cold acclimation potential is indicated by the
difference between LT values of acclimated and non-acclimated
samples, as it shows how the freezing tolerance of individuals increases
through exposing plants to low but non-freezing temperatures for a
certain period (in this study, 4°C for five days). This difference was
expressed in terms of the LT50 and ΔLT100 values (Tables 1 and 2). The larger this
difference, the greater the freezing tolerance of plants through cold
acclimation. The LT50 andLT100 values were exclusively positive, which
supports the observation that acclimated lethal values were considerably
lower than non-acclimated values. There was substantial variation in the
cold acclimation potential of different accessions, withLT50 ranging from 0.48°C to 6.59°C (mean:2.91°C)
and LT100 ranging from 0.06°C to 10.22°C
(mean:4.18°C). Generally, ΔLT100 values were
significantly higher than Δ LT50 values
(p -value 0.004), which could be expected because freezing damage
is not a linear function. Examples of freezing tolerance measurement
experiments are provided in Fig. 3, and individual measurements are
shown in Suppl. Mat. Table 2. The mean values for our internal controlArabidopsis thaliana Col0 were -8.1°C (SD:0.7) and -4.1 (SD:0.3)
(LT50 , acclimated and non-acclimated,
respectively), which is also within the range of previously reported
values for this ecotype (-9.7°C and -5.5°C; Hannah et al., 2006).
3.3 Freezing tolerance variation within and between species: not
taxonomic group specific
Substantial variation in LT50 andLT100 values were observed within species (Tables
1 and 2). The highest range of LT50 values was
exhibited by acclimated C. tatrae samples, spanning a difference
(min-max) of 6.92°C, and non-acclimated I. abulense samples, at
4.6°C. For LT100 values, C. tatrae showed
the highest range of values among species for both acclimated and
non-acclimated samples, at 8.21°C and 9.3°C, respectively. If standard
deviations are used to compare variability in the data, considering
varying sample sizes, there was also substantial variation within (and
among) species (Fig. 4). The various species showed, on average, a much
lower standard deviation in non-acclimated LT50values compared to all other values. For acclimated samples, C.
tatrae had the highest standard deviation for bothLT50 and LT100 values.
This species also showed the highest standard deviation forLT50 values of the non-acclimated samples.
Although LT50 values of non-acclimated samples
presented the lowest standard deviation values, there was also minimal
difference among species, with only I. abulense showing the
highest value. Notably, a small sample size did not necessarily result
in a higher standard deviation, as was expected. Cochlearia
pyrenaica (n = 9) and C. tatrae (n = 6) showed higher standard
deviation values than some species with lower sample sizes, such asC. excelsa (n = 2). This suggests that, besides varying sample
sizes, other differences, such as genetic variation within species,
likely influence the variation in the measured data. Because only one
accession was measured for I. glastifolium and I.
megalospermum , no standard deviation could be calculated for these
species. Given that these two species are genetically very similar and
are sometimes referred to as subspecies (Vogt, 1987; Koch, 2012) and as
they are also distributed in the same regions, they may be compared here
as if they were a single species. A similar cold response with very
similar lethal values was observed for these two taxa (Fig. 4).
Comparisons among species revealed that C. danica (coasts of the
Atlantic Ocean and Northern Sea) and I. abulense (Spain mainland)
showed the lowest LT50 values for acclimated
samples (-10.72°C and -9.96°C, respectively) (Table 1). Both species are
adapted to hot, dry, and in the case of C. Danica , coastal,
high-salt conditions (Fig. 1). This result supports the assertion thatC. danica exhibits a similar cold response compared toIonopsidium owing to the similar environmental conditions of
their habitats (Fig. 3). This was contrary to the expectation that these
species would display the lowest cold tolerance. We expected that
species such as arctic C. groenlandica and high alpine C.
tatrae and C. excelsa exposed to the lowest ambient temperatures
would display the highest frost tolerance and the lowest lethal values.
Interestingly, a different species pair showed the lowest acclimatedLT100 values: I. abulense with -15.48°C
and C. excelsa with -15.26°C, which shows that even thoughC. excelsa did not show the lowest LT50 value, it may still be able to withstand more extreme temperatures
than other species.
Coastal C. danica , which also occurs in Portugal and Spain, may
exhibit a cold response similar to that of Ionopsidium species,
as these species may be exposed to similar environmental conditions.
However, this assumption was not supported by the measured data (Fig.
4). Because lethal values vary considerably within theIonopsidium group, comprehensive comparisons of this group other
species proved difficult. Notably, I. abulense showed much lower
lethal values (both LT50 andLT100 ) than the remaining species of this group.
As stated above, C. danica did exhibit lethal values similar to
those of I. abulense . Coastal C. anglica and C.
aestuaria exhibited high lethal values that were most similar to those
of other Ionopsidium species. However, no obvious distinction
between alpine/arctic Cochlearia species and coastal species,
such as Ionopsidium , could be identified. ANOVA showed that,
generally, there was a significant difference in lethal values between
species (LT50 acclim: ***,LT50 non-acclim: **, LT100acclim: **, LT100 non-acclim: **, df = 12).
However, multiple t-tests revealed mostly insignificant differences. As
Table 3 shows, only nine of the 55 comparisons were significant.
The two evolutionary lineages leading to the sister generaCochlearia and Ionopsidium diverged from each other
approximately ten million years ago. However, comparisons of lethal
values between the two genera revealed insignificant differences, as
shown in Fig. 5 (LT50 acclimated, p =
0.822; LT100 acclimated, p = 0.883;LT50 non-acclimated, p = 0.0599;LT100 non-acclimated, p = 0.249). This
further substantiates the finding that Cochlearia andIonopsidium species respond similarly to freezing temperatures.
This is contrary to the expectation that, as western MediterraneanIonopsidium forms a separate bioclimatically defined group (Fig.
2), the genus would respond differently to freezing temperatures.
Similarly, there were no differences when comparing polycarpic versus
monocarpic and diploid versus polyploid accessions (Table 4).
3.3 Freezing tolerance shows weak geographically defined trends
Low temperatures, especially winter minimum temperatures, are important
in determining the geographic boundaries of plant species distributions.
Therefore, it is expected that a species’ tolerance to freezing
temperatures is often correlated with its geographic distribution
(Armstrong et al., 2020). There is indeed an environmental gradient
strongly associated with temperature, which may create a gradient in
natural selection with strong selection pressures for an increased cold
tolerance towards the north (Wos & Willi, 2015; Armstrong et al.,
2020). A significant correlation between lethal values and longitude has
been demonstrated for A. thaliana accessions, suggesting that
there may also be a continentality factor influencing the response to
cold, as conditions become colder and drier with increasing distance
from the coast (Bruch et al., 2011; Zuther et al., 2012). Therefore,
geographically distant taxa are expected to differ in their responses to
cold (Davey et al., 2018). As shown in the previous chapter,Cochlearia and its sister clade Ionopsidium exhibit
similar responses to freezing temperatures, despite being distributed in
different geographic regions with varying bioclimatic conditions. To
elaborate further on this spatial distribution,LT50 and LT100 values for
the different Cochlearia and Ionopsidium accessions have
been plotted on a map (Fig. 6). The range of lethal values (acclimated
and non-acclimated) is indicated by a color gradient. We determined that
accessions at high latitudes would display higher cold tolerances and
therefore show lower lethal values than accessions at low latitudes,
thereby forming a gradient of cold tolerance. As inland species may be
exposed to a colder winter climate than coastal species owing to
continentality, a longitudinal gradient may also occur. No such gradient
was initially identified for non-acclimated samples, as these plants did
not undergo the process of cold acclimation that would naturally occur.
Acclimated LT50 values showed a weak trend
towards a gradient that spanned from southwest to northeast with lower
lethal values, indicating an increase in freezing tolerance; this was
partly mirrored by LT100 values for acclimated
samples. However, the northwestern arctic C. groenlandica was an
outlier in both cases, showing the highest freezing tolerance. In
addition, I.abulense accessions located in the southwest showed a
comparatively high freezing tolerance, thereby suggesting that the
supposed gradient is extremely weak. This also suggests that a
combination of latitude and longitude might influence freezing tolerance
instead of a single factor. Correlation analysis supports these
observations. Both the correlation between lethal values and latitude
(LT50 acclim: R = -0.3; p = 0.057,LT100 acclim: R = -0.2; p = 0.22,LT50 non-acclim: R = 0.023; p = 0.88,LT100 non-acclim: R = -0.12; p = 0.44) and
the correlation between lethal values and longitude
(LT100 acclim: R = 0.056; p = 0.73,LT50 non-acclimated: R = 0.3; p = 0.059,LT100 acclim: R = -0.0094; p = 0.95,LT100 non-acclim: R = 0.21; p = 0.19) were
not significant. However, in reviewing the correlation analysis between
lethal values and these two factors, the lethal values of the acclimated
samples seemed to be slightly more correlated with latitude.