plants Article Comparative Germination Ecology of Two Endemic Rhaponticum Species (Asteraceae) in Different Climatic Zones of the Ligurian and Maritime Alps (Piedmont, Italy) Valentina Carasso 1,* , Marco Mucciarelli 2, Francesco Dovana 2 and Jonas V Müller 3 1 Centro Regionale Biodiversità Vegetale, Ente di gestione delle Aree Protette delle Alpi Marittime, Via S. Anna, 34, 12013 Chiusa di Pesio, Italy 2 Department of Life Sciences and Systems Biology, Viale P.A. Mattioli, 25, Università di Torino, 10125 Torino, Italy; [email protected] (M.M.); [email protected] (F.D.) 3 Royal Botanic Gardens Kew, Millennium Seed Bank, Conservation Science, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK; [email protected] * Correspondence: [email protected]; Tel.: +39-171-734021 Received: 17 April 2020; Accepted: 30 May 2020; Published: 2 June 2020 Abstract: Comparative studies of seed germination of closely related taxa can help increase our understanding of the ecological limitations of cold-adapted plants and forecast how they might respond to global warming. No studies exist on the relationship between thermoclimatic belts that classify mountain life zones according to bioclimatic criteria and the germination strategy of alpine plants. The aim of this study was to assess this relationship using two closely related species growing in different thermotypes and to test whether their germination responses were related to the climate at natural sites. Fresh Rhaponticum bicknellii and R. scariosum seeds were cold stratified for 0, 30, 60 and 90 days and tested for germination at 10, 15 and 20 ◦C. At the same time, seed burial experiments were run in the field and in the plant nursery. A GLM analysis showed that the length of cold stratification affected significantly only the germination of R. bicknellii seeds, while increasing temperatures prompted germination in both species. We found that R. bicknellii adopts a drought-avoiding germination strategy, while R. scariosum germination is favoured by warm temperatures. Our findings support the general view that alpine plants do not share common germination requirements and that any conclusions should be interpreted from a biogeographical and bioclimatic perspective. Therefore, seed germination and seedling establishment of endemic alpine species can also be predicted by looking at the bioclimate of the species’ range. Keywords: cold stratification; comparative germination ecology; maritime and ligurian alps; Rhaponticum; seed dormancy and germination; soil seed bank; thermoclimatic belts 1. Introduction Seed dormancy plays a pivotal role in timing germination and seedling emergence and establishment [1,2], the most important phases in the life cycle of every plant species. Like other life stages, seed dormancy and germination are sensitive to variations in the environmental conditions [3–5]. Depending on variation in flowering time, seed mass, parental investment and maternal effects caused by genetic, epigenetic and/or environmental factors, the mechanisms that regulate seed dormancy release can vary dramatically, even within the same species [6,7]. As pointed out by [8], germination depends to a great extent on natural selection pressure, thereby it is associated with the ecological niche and the range of the plant species. Numerous studies Plants 2020, 9, 708; doi:10.3390/plants9060708 www.mdpi.com/journal/plants Plants 2020, 9, 708 2 of 18 have documented that variations in dormancy and germination traits among and within species are associated with the habitat (reviewed in [9]). For example, for some species, germination is impaired by the absence of a particular environmental cue (such as light, fire, low or high temperatures), and the permanent absence of these signals may preclude the establishment of the species in a particular location [8]. It is well known that many alpine plants increase their final germination after a cold period (cold stratification), which is necessary to break the physiological component of dormancy (physiological dormancy) [9]. In fact, dormancy is the result of inhibiting physiological mechanisms that act within the embryo until their complete removal. In nature, embryo inhibition is gradually suppressed after exposure to winter temperatures, and the dormancy status is removed at a rate that depends on the intensity of inhibition [10–13]. Most alpine species germinate immediately after the snowmelt, while their germination in autumn is normally precluded by the physiological dormancy and the combination of low temperatures and short day lengths [5,14,15]. However, the role and efficacy of the cold stratification for seed germination of alpine species is not always obvious and related to the mean duration of the snow cover in a given habitat. Based on detailed, albeit contrasting studies, it was shown that alpine species are less dormant and require higher germination temperatures than subalpine species [9]. This dependence on relatively high temperatures was interpreted as a reproductive strategy that is used to postpone germination and seedling emergence until late spring or early summer when climatic conditions are more favourable and the seedling is less likely to be exposed to freezing temperatures [16,17]. This finding is documented for many alpine and tundra species that show surprisingly high germination temperature optima. However, rapid germination at relatively high temperatures was also documented for some species in the Mediterranean mountains [18]. Thus, the extent to which germination strategies adopted by different taxa and environmental conditions (associated with their habitats) are correlated is still not fully understood. Yet another germination strategy, characterised by seeds germinating at cold temperatures, was proposed for certain mountain species growing at lower elevations [19]. In fact, seed sensing of winter duration could be crucial for a plant in warmer climates as it allows seed germination to happen immediately after snowmelt when the soil moisture is high and when it is more likely that the seedling can establish itself successfully [19]. Such a strategy could be advantageous for those plants that experience severe drought conditions at the onset of summer, such as plants in many dry parts of the western Alps. In brief, many studies suggest that alpine and subalpine species lack a common germination strategy [20] and that their germination requirements can vary greatly over space and time [6,21,22]. It is difficult to associate precise germination traits to specific alpine environments/habitats as this depends also on the number of microhabitats [14,20]. In this regard, small scale variations in temperature and precipitation are just as important as environmental signals. In this paper, we investigated the germination responses of Rhaponticum scariosum (Asteraceae), which is endemic to the south-western Alps, and of Rhaponticum bicknellii, which is narrow endemic to the Ligurian and Maritime Alps (Figure1)[23,24]. These two species are closely related (Supplementary file S1) but belong to two different thermotypes [25–30]. The range of R. scariosum falls within the orotemperate belt between 1700 and 2500 m a.s.l. [23,31,32] with a relatively low annual temperature threshold (Tp = 381–800 ◦C, whereby Tp is the yearly positive temperature, calculated as the sum of the monthly average temperatures above 0 ◦C, [24,33]). The range of R. bicknellii corresponds to the supratemperate (Submediterranean) bioclimatic belt between 800 and 1700 m a.s.l., with a relatively higher temperature threshold (Tp = 801–1400 ◦C). To the best of our knowledge, no previous studies exist which analyse the relationship between thermoclimatic belts (thermotypes) and the germination strategy of rare alpine species. Therefore, the rarity, vulnerability and threat status of the two Rhaponticum species makes them ideal candidates to investigate this relationship and, possibly, to forecast the impact of global warming on seed germination. Plants 2020, 9, 708 3 of 18 Plants 2020, 9, x FOR PEER REVIEW 3 of 19 Figure 1. The location of the two study areas in the south south-western-western Alps. OurThese previous two species work are on Rhaponticumclosely relatedendemics (Supplementary showed that file the S1) final but seed belong germination to two different of these speciesthermotypes increases [25– after30]. The a cold range stratification of R. scariosum (V. Carasso, falls within unpublished the orotemperate data). This belt finding between is in 1700 line withand the2500 general m a.s.l. pattern [23,31,32] for with species a relatively with physiological low annual dormancy. temperature threshold (Tp = 381–800 °C, whereby Tp isOur the overallyearly positive hypothesis temperature, was that the calculated two Rhaponticum as the sumspecies of the would monthly show average different temperat adaptations,ures inabove terms 0°C, of temperature[24,33]). The requirementsrange of R. bicknellii for dormancy corresponds break, to according the supratemperate to the recorded (Submediterranean) air temperatures atbioclimatic their growing belt between site. In 800 particular, and 1700 we m expecteda.s.l., with that a relatively (i) both R. higher scariosum temperatureand R. bicknelliithresholdwould (Tp = show801–1400 physiological °C). dormancy and, specifically, that the orotemperate R. scariosum would exhibit (ii) a higherTo dependencethe best of our of germinationknowledge, no percentages previous studies and rates exist on which a period analyse of cold