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).