1 INTRODUCTION
With the recently published 6th report of the Intergovernmental Panel on Climate Change, IPCC (IPCC 2022; https://www.ipcc.ch/report/ar6/wg2/), we have again been warned that ongoing global warming will expose plants and animals to conditions exceeding the limits of their historical and eventual evolutionary experience. Species inhabiting polar and alpine regions that are already being influenced by a warming climate will face challenges induced by these changes, including population decline (Descamps et al., 2017), habitat loss, and even extinction. These challenges surpass the limits of adaptation, even for less vulnerable species and systems (e.g. Román-Palacios & Wiens, 2020). Studying the potential of plant species to adapt to differing and rapidly changing conditions is now more relevant than ever. Habitats at the coldest margins of the Earth, such as Arctic and alpine ecosystems, are considered most affected by rapidly proceeding global warming (Ernakovich et al., 2014); this is especially true for arctic-alpine species at the limit of their distribution range (Lescia et al., 2004). Even though temperatures are rising on a global scale, winter at high latitudes and elevations is over-proportionally affected by climate change, as average winter temperatures are increasing more rapidly in Arctic and alpine habitats (ACIA, 2005; Kreyling, 2010). Winter warming has led to a decline in the amount and duration of snow cover, which is crucial to the survival of subnivean species in several regions. During the last 30 years, Arctic snow cover has already declined by 10%, and model projections expect an additional decrease of 10-20% until the end of this century (ACIA, 2005; Kreyling, 2010). In Europe, a 40-80% decline in days with snow coverage is predicted (Kreyling, 2010). The rate of snow cover reduction is among the most dramatic signs of climate change, and its impacts are severe. Snow cover is one of the most important factors affecting the survival of arctic and alpine plants. Its availability, depth, and duration determine environmental variables such as soil moisture, temperature, and freezing depth (Wipf & Rixen, 2010). Snow has an insulating effect because it contains a large amount of air, which protects species living underneath from wind and extreme winter temperatures (Pomeroy & Brun, 2001). This dynamic creates a relatively mild microclimate with temperatures only slightly below 0°C, whereas ambient temperatures may drop as low as -45°C, thereby ensuring the survival of many plant species (Bokhorst et al., 2009; Armstrong et al., 2015). Earlier snowmelt and decreased snow depth caused by a reduction in snowfall and mid-winter thawing events followed by cold periods are consequences of global warming that can threaten the survival of arctic and alpine plants. Plants may be exposed to considerably lower temperatures, cold winds, and temperature fluctuations, which can cause substantial frost damage and may lead to plant die-offs (Sonesson & Callaghan, 1991; Bokhorst et al., 2009). Winter warming has led to a lengthening of the vegetative growing season in the Northern Hemisphere, especially at higher latitudes (Parmesan, 2006). As plants often react to warming with increased productivity and less investment in protection, frost events after a period of warming can be fatal in many cases because they occur more frequently. Considering this dynamic, plant species inhabiting areas experiencing changes in snow cover may paradoxically require an increased tolerance to freezing temperatures, as these changes counteract the overall effect of global warming (Kreyling, 2010). As climatic conditions change drastically, plant populations may no longer be best adapted to ambient conditions (Franks et al., 2014). Reactions of plant species to changes in ecological conditions include strategies such as migration and adaptation through evolutionary changes or developing ample phenotypic plasticity (Aitken et al., 2008; Williams et al., 2008; Franks et al., 2014). Migration allows for the exploration of more suitable conditions. This is, however, a limited option, as migration attempts may not keep pace with rapidly progressing changes, and such explorations may require the movement of entire ecosystems (Loarie et al., 2009). The expected response of northern plant species to rising temperatures is relocation (ACIA, 2005). With improvements in livability for warm-loving plant species in arctic regions, poleward migrations are expected, leaving arctic species at risk of displacement; this risk is especially pressing for species whose northward migration is hindered by the Arctic Ocean. Similarly, a documented upward shift in the distribution of montane species has been caused by the inability of species to adapt to local conditions at their current elevation. In addition, successful colonization increases with longer growing seasons and higher temperatures (Walther et al., 2005; Parolo et al., 2008). These shifts in distribution patterns have led to increased species richness in both Arctic and high alpine regions, which may lead to increased competition for species originally inhabiting these areas (Loarie et al., 2009). If species currently adapted to the coldest regions and highest mountain summits, where no space for further migration is left, fail to adapt to unfavorable conditions in time, the eventual eradication of these species is a real possibility and a serious threat. Therefore, local adaptation may be a quicker and more efficient response to changing conditions. Several plant populations may need to respond to climate change through phenotypic plasticity, which refers to the ability of a genotype to express different phenotypes or adaptive evolution (Franks et al., 2014). The extent to which plant populations can adapt to locally changing conditions is highly relevant in predicting future responses and the chances of their survival in periods of rapidly progressing global warming. Nevertheless, these responses may be insufficient to keep pace with the current rate of climate change (Franks et al., 2014).
An important adaptation of plants inhabiting cold-characterized regions is their ability to protect against frost damage induced by minimum winter temperatures through a process called cold acclimation. Minimum winter temperatures remain a major factor in determining species distribution ranges (Armstrong et al., 2020). Tolerance to freezing temperatures is a trait most common in temperate as well as arctic and alpine plants, which are frequently exposed to sub-zero temperatures, whereas most tropical and subtropical plants suffer injuries from temperatures below 10 °C (Xin & Browse, 2000). Freeze-induced cellular dehydration caused by membrane destabilization is commonly accepted as the primary cause of freezing damage (Uemura et al., 1995). The formation of ice crystals in extracellular spaces and cell walls leads to decreased water potential outside the cells, thereby drawing water and electrolytes out by osmosis and eventually leading to cell death. Because freezing damage primarily occurs in cell membranes, changes in membrane behavior are critical for developing freezing tolerance (Xin & Browse, 2000). In nature, temperate plants can increase their ability to withstand freezing temperatures after being exposed to low but non-freezing temperatures for a certain period, which makes them tolerant against seasonal changes and protects them against freezing damage in winter and early spring (Ritonga & Chen, 2020). The process of cold acclimation is mostly initiated by decreasing temperatures in late fall and can take a few days to several weeks, depending on the plant species (Xin & Browse, 2000). For Arabidopsis thaliana , an acclimation period of a few days is sufficient to improve cold tolerance (Gilmour et al., 1988), whereas 2-4 weeks of cold acclimation is necessary for wheat or rye to develop an increased frost tolerance (Brule-Babel & Fowler, 1989). The molecular basis of cold acclimation has long been investigated but has not yet been fully resolved. The process involves regulating hundreds of genes and is accompanied by complex biochemical and physiological changes, as well as structural changes (Xin & Browse, 2000; Thomashow, 2010). Such changes include the accumulation of soluble sugars, which act as cryoprotectants for enzymes, prevent excessive dehydration, and promote membrane stability, as well as compositional and structural changes in the cell membrane (Reyez-Díaz et al., 2006; Uemura et al., 2006); cold acclimation is therefore clearly a multifactorial process in which many genes act in parallel to ensure maximum freezing tolerance (Renaut et al., 2005). There is natural variation in the freezing tolerances of different plants and their potential to acquire and increase tolerance following cold acclimation; this variation exists among species and even populations within species (Xin & Browse, 2000; Hannah et al., 2006); Some of this variation may be linked to the species’ or populations’ distribution (Armstrong et al., 2020). Varying environmental conditions in different geographic regions may expose plants to different selection pressures, which may lead to differences in phenotypic adaptation to cold in geographically distant populations (Zuther et al., 2012). Therefore, an increased selection for cold tolerance is to be expected in northern populations, whereas in southern regions, there may be less selection for cold tolerance (Armstrong et al., 2020). This cline has been reported for accessions of different Arabidopsis species (Hannah et al, 2006; Zuther et al., 2012), whereas other studies have not confirmed this phenomenon (Armstrong et al., 2015; Armstrong et al., 2020). Freezing tolerance may also be pronounced along longitudinal and altitudinal gradients (Zuther et al., 2012; Davey et al., 2018). Cold acclimation is not a constitutive trait but instead must be induced by specific environmental cues. Several researchers have hypothesized that the process may, therefore, be biologically costly, thereby leading to trade-offs between the degree of cold tolerance and other traits such as productivity. Constitutive cold tolerance may be costly in environments that rarely encounter freezing events, as extensive physiological and biochemical changes must be maintained (Zhen et al., 2011). For species with a distribution spanning steep temperature gradients, the same degree of cold tolerance would not be beneficial across the full distribution range (Meireles et al., 2017). This cost of cold tolerance has been reported for Olea europaea (Arias et al., 2017). For herbaceous plants such as Arabidopsis thaliana , there is no indication of a trade-off between the level of protection and productivity (Zhen et al., 2011; Wos & Willi, 2015; Wos & Willi, 2018). The ability to prepare for freezing events through cold acclimation is especially important for arctic and alpine plant species because they are exposed to a wider temperature range (Davey et al., 2018).
Although several studies have intensively investigated the freezing tolerance and acclimation potential of different Arabidopsisaccessions and ecotypes (e.g. Hannah et al., 2006), minimal information is known about this trait in diverse but evolutionarily related species assemblages, such as the tribe Cochlearieae from the Brassicaceae family (mustards). This tribe belongs to one of the few isolated lineages of the Brassicaceae family, comprising approximately 30 species (Koch, 2012). It represents a promising system to study cold adaptation, as it shows distinctive traits that have evolved over a short time span. These traits include adaptations to extreme bedrock types, heavy metal soils, salt habitats, and high alpine and Arctic regions (Wolf et al., 2021). The tribe includes two genera, Cochlearia and Ionopsidium , which were separated by a deep evolutionary split during the mid-Miocene, approximately 13.8 million years ago (Koch, 2012). WhereasIonopsidium species adapted to western Mediterranean bioclimatic conditions, progenitors of present-day Cochlearia expanded their distribution range to the northern hemisphere and rapidly diversified during the Pleistocene to ecological conditions in central and northern Europe and the circumarctic (Koch, 2012; Wolf et al., 2021). Even though these taxa are closely related, they have colonized substantially different ecological niches, such as salt marshes, sand dunes, coastal areas, cold and calcareous springs, and high alpine and arctic environments. Separating most Cochlearia species fromIonopsidium is the coldness of their habitats (Koch, 2012). MostCochlearia species are already endangered, as suitable habitats have become increasingly rare, and two species have become extinct in the wild (Cochlearia polonica , Cieślak et al., 2007; Cieślak et al., 2010; C. macrorrhiza , Koch et al., 2003; Koch & Bernhardt, 2004). Most Cochlearia species, having been forced into cold-adapted and geographically isolated niches, may face extinction if they are unable to quickly adapt to warming conditions. Evaluating the different Cochlearia and Ionopsidium taxa in their physiological responses to freezing temperatures offers the opportunity to gain insights into the evolution of cold adaptation and tolerance in the tribe Cochlearieae.
We previously demonstrated that both Cochlearia andIonopsidium show a strong but very similar cold metabolome response to cold treatment (Wolf et al., 2021). We hypothesized that adaptations to drought (Mediterranean habitats), salinity (coastal line habitats), and cold (arctic-alpine environments, cold spring water) may be achieved via the same primary response in herbaceous (non-woody) plants, thus posing the question of an evolutionary shared molecular-physiological trait among Ionopsidium andCochlearia .
The focus of this study was therefore to elaborate on this idea and to test explicitly for cold tolerance of the living system across evolutionary scales (genera and species), genomic complexity (diploids versus polyploids), and biogeographic and bioclimatic gradients (Mediterranean to the Arctic). We elaborate on the hypothesis thatCochlearia species are caught in its cold-adapted niche, and successful escape from this bioclimatic cage may require a complete shift in life history strategies, a shift from perennial/polycarpic to annual/monocarpic growth, which is the most severe difference betweenCochlearia and Ionopsidium .