Plant Physiology Preview. Published on July 20, 2016, as DOI:10.1104/pp.16.00877
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Adaptation of the Long-Lived Monocarpic Perennial, Saxifraga longifolia to High
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Altitude
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Sergi Munné-Bosch1*, Alba Cotado1, Melanie Morales1, Eva Fleta-Soriano1, Jesús
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Villellas2,3, Maria B. Garcia2
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Department of Plant Biology, Faculty of Biology, University of Barcelona, Barcelona,
Spain
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Pyrenean Institute of Ecology, CSIC, Zaragoza, Spain
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Current address: Department of Zoology, Trinity College Dublin, Dublin, Ireland
*Correspondence:
Sergi Munné-Bosch, [email protected], tel.: +34-934021463; fax: +34934112842
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Short title: Adaptation to Altitude in a Monocarpic Perennial
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One sentence summary: Adaptation of an endemic long-lived monocarpic perennial to
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high altitude is influenced by multiple mechanisms operating at various levels
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Funding information: This work was supported by the Spanish Government (project
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number BFU2015-64001-P to SMB and CGL 2010-21642 to MBG) and the Catalan
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Government (Institució Catalana de Recerca i Estudis Avançats Academia award given
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to S.M.B.).
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The author responsible for distribution of materials integral to the findings presented in
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this article in accordance with the policy described in the Instructions for Authors
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(www.plantphysiol.org) is: Sergi Munné-Bosch ([email protected])
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ABSTRACT
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Global change is exerting a major effect on plant communities altering their potential
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capacity for adaptation. Here, we aimed at unveiling mechanisms of adaptation to high
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altitude in an endemic long-lived monocarpic, Saxifraga longifolia, by combining
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demographic and physiological approaches. Plants from three altitudes (570, 1100 and
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2100 m a.s.l.) were investigated in terms of leaf water and pigment contents, and
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activation of stress defense mechanisms. The influence of plant size on physiological
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performance and mortality was also investigated. Levels of photoprotective molecules
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(α-tocopherol, carotenoids and anthocyanins) increased in response to high altitude
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(1100 relative to 570 m a.s.l.), which was paralleled by reduced soil and leaf water
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contents and increased ABA levels. The more demanding effect of high altitude on
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photoprotection was however partly abolished at very high altitudes (2100 m a.s.l.) due
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to improved soil water contents, with the exception of α-tocopherol accumulation. α-
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Tocopherol levels increased progressively at increasing altitudes, which paralleled with
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reductions in lipid peroxidation, thus suggesting plants from the highest altitude
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effectively withstood high light stress. Furthermore, mortality of juveniles was highest
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at the intermediate population, suggesting that drought stress was the main
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environmental driver of mortality of juveniles in this rocky plant species. Population
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structure and vital rates in the high population evidenced lower recruitment and
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mortality in juveniles, activation of clonal growth, and absence of plant size-dependent
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mortality. We conclude that, despite S. longifolia has evolved complex mechanisms of
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adaptation to altitude at the cellular, whole-plant and population levels, drought events
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may drive increased mortality in the framework of global change.
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Keywords: antioxidants; carotenoids; chemical defenses; high altitude; photoprotection;
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plant size effects; vitamin E; mortality rate
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INTRODUCTION
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European mountains shelter a huge biodiversity, and are home to many endemic plants
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and animals, i.e. species that occur nowhere else. Global change, and particularly
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climatic change, is expected to exert a major effect on mountain plant communities,
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altering their potential capacity for adaptation (Peñuelas and Boada, 2003; Franklin et
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al., 2016). Under such scenario of environmental changes, populations of organisms
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must either escape or get quickly adapted, otherwise they go extinct. For instance,
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certain butterfly species have been migrating north, or to higher altitudes, to escape
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rising temperatures (Breed et al., 2013). Plants, of course, cannot migrate as fast as
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animals, and important shifts have already been found among plant communities
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inhabiting mountain summits (Gottfried et al., 2012). When global air temperatures
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increase, the number of cooler habitats will shrink, producing a crowding effect and
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increased competition among some species in the remaining cooler areas; at the same
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time, however, other habitat types will increase in abundance (Scherrer and Körner,
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2011). Alpine habitats could prove more attractive to plant species than lowlands
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because of their topography providing favorable microhabitats. However, certain rare
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species may lose out in the long-term competition for space, especially those favoring
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cooler climates (Körner, 2013).
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Leaves of high-mountain plants are highly resistant to photoinhibitory damage.
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Tocochromanols (particularly tocopherols and plastochromanol-8) are found in
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thylakoids and play an antioxidant function in protecting lipids from the propagation of
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lipid peroxidation in chloroplasts. Together with carotenoids, they also prevent
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photosystem II damage as a result of singlet oxygen attack (Munné-Bosch and Alegre,
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2002; Falk and Munné-Bosch, 2010; Zbierzak et al., 2010; Kruk et al., 2014). A higher
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tocochromanol content, particularly of α-tocopherol, and an increased capacity for non-
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radiative dissipation of excitation energy by activation of the xanthophyll cycle have
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been found in high-mountain plants, thus supporting such a role (Streb et al., 1997,
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1998, 2003a, 2003b; García-Plazaola et al., 2015). Furthermore, although the number of
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studies is still very limited for plants in their natural habitat, high-mountain plants tend
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to accumulate large amounts of ABA (Bano et al., 2009), a phytohormone that is known
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to mediate the acclimation/adaptation of plants to temperature extremes by modulating
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the up- and downregulation of numerous genes (Gilmour et al., 1991). The activation of
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other chemical defenses, such as the accumulation of salicylates and jasmonates, which
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serve against biotrophs and necrotrophs, can also be affected by extreme temperatures
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(Kosova et al., 2012; Dong et al., 2014; Miura and Tada, 2014), but it has not been
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investigated thus far in high-mountain plants.
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Some degree of plasticity in physiological traits is ubiquitous among plants, so
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that environmental growth conditions are generally considered essential factors
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governing the physiological performance of plants and their organs (Larcher, 1994). A
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number of recent studies with trees, shrubs and herbs, including vascular epiphytes
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(Mencuccini and Grace 1996; Zotz, 1997; Schmidt et al., 2001; Schmidt and Zotz,
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2001; Munné-Bosch and Lalueza, 2007; Morales et al., 2014) point out, however, to
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another source of intraspecific variation that many studies in the past have inadvertently
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missed, i.e. substantial variation in physiological traits related to plant size rather than
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changing environmental conditions (Zotz et al., 2001). In trees, increased plant size
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leads to increased hydraulic resistance causing reductions in relative leaf growth rates
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(Mencuccini and Grace, 1996). Furthermore, photo-oxidative stress has been shown to
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increase during periods of low precipitation combined with high light in the
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Mediterranean shrub, Cistus clusii as a function of plant size, therefore suggesting an
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increased vulnerability to photo-oxidative stress in the largest individuals (Munné-
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Bosch and Lalueza, 2007). To our knowledge, no studies are however available to
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unveil the possible influence of plant size on photoprotection and activation of chemical
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defenses in high-mountain plants.
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Saxifraga longifolia Lapeyruse (Saxifragaceae) is an endemic species of the
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western Mediterranean mountains, ranging from the Pyrenees (plus a couple of
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populations in the Cantabric Mountains) through eastern Spain to reach its southern
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limit in the high Atlas of Morocco (Webb and Gornall, 1989). This long-lived
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monocarpic plant develops a basal rosette growing in limestone rocky places, mainly on
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cliffs, offering a unique sight in years of intensive blooming. Reproduction occurs when
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plants are at least 6 years old in greenhouse conditions (Webb and Gornall, 1989) and it
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is thought to be much later in natural conditions. This orophyte plant shows striking
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variation in plant size, with a diameter of the rosette up to 30 cm in the largest
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individuals. It has been shown that flower and seed production increase as a function of
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plant size with female success being maximum in intermediate sized plants (García,
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2003).
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In the present study, with the aim of getting new insights into the mechanisms of
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adaptation to high altitudes and the influence of plant size on this adaptation capacity,
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we examined the physiological response of S. longifolia growing at three contrasted
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populations spanning its whole altitudinal range. We described population structure,
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calculated mortality rates, and analyzed physiological performance, including water
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contents and activation of photoprotection mechanisms and chemical defenses. We
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aimed at understanding the effect of varying altitude on the expression of defense
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mechanisms that govern adaptive processes in high-mountain plants.
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RESULTS
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Physiological Performance and Mortality in Populations at Various Altitudes
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S. longifolia is a monocarpic species; therefore, plants die as a consequence of
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reproduction. We wondered, however, whether mortality of juveniles is also influenced
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by stressful conditions across an altitudinal range by monitoring three plant populations
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growing at 570, 1100 and 2100 m a.s.l. The population occurring at the highest altitude
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(Las Blancas) had the largest plants, the less frequency of small ones (Supplemental
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Fig. S1A), and the lowest mortality rate in juveniles (average across 4 years: 4.8 %,
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Table 1). Mortality of juveniles was higher in the lowest population (Pantano de la
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Peña, 6.9 %) and highest in the intermediate one (San Juan de la Peña, 11.5 %, Table 1),
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both of them showing a similar population size distribution (Supplemental Fig. S1A).
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Therefore, altitude, which, as expected, resulted in lower temperatures (Supplemental
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Fig. S2), does not seem to be associated to mortality rates in juveniles. Rather, mortality
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rates in juveniles seemed to be more associated with reduced soil water contents.
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Among the three populations studied, the volumetric soil water content was the highest
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in the high population and lowest in the intermediate one (Supplemental Fig. S2). Plants
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growing in San Juan de la Peña were exposed to more stressful conditions during the
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day of measurements compared to the other two populations, as indicated not only by
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reduced soil water contents, but also higher solar radiation and air temperatures, which
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may in turn contribute to drought stress. Mortality due to flowering was much lower
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than among juvenile plants (Table 1), and flowering rates were rather stochastic across
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years and populations, with the highest population showing the higher flowering rates
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during 2013/14, but also the smaller ones during 2014/15 (Table 1).
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Physiological stress indicators, including leaf water, pigment and lipid
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peroxidation levels (Fig. 1), antioxidant protection (Fig. 2) and stress-related
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phytohormones (Fig. 3), revealed that the physiological performance in juvenile plants
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differed in the three populations, some parameters changing as a function of altitude and
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others following the mortality rate pattern more linked to drought stress (the
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intermediate population showed the lowest leaf water contents among the three
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populations, Fig. 1). Intriguingly, the intermediate population was the one showing the
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highest pigment levels, including those of chlorophylls (Fig. 1), carotenoids and
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anthocyanins (Fig. 2). However, when antioxidants were expressed on a chlorophyll
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basis, the intermediate population showed the highest carotenoid/chlorophyll ratio but
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the lowest α-tocopherol/chlorophyll ratios (Supplemental Fig. S3), which was observed
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together with the lowest chlorophyll a/b ratios (Fig. 1). Furthermore, the intermediate
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population was the one showing the highest levels of all stress-related phytohormones,
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including ABA, salicylic acid and jasmonic acid, while the lowest levels of the jasmonic
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acid precursor, 12-oxo-phytodienoic acid (Fig. 3), thus confirming that the intermediate
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population was the one experiencing the highest physiological stress in juvenile plants,
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which is in agreement with the highest mortality rates observed (Table 1).
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Levels
of
photoprotective
molecules
(α-tocopherol,
carotenoids
and
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anthocyanins) increased significantly in response to high altitude (1100 relative to 570
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m a.s.l., Fig. 2), which was paralleled by reduced leaf water contents (Fig. 1) and
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increased ABA levels (Fig. 3). The more demanding effect of high altitude on
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photoprotection was however abolished (except for α-tocopherol increases) at very high
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altitudes (2100 m a.s.l.), these plants showing improved water contents (Fig. 1), and a
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reduced need for photoprotection (driven by anthocyanins and carotenoids, Fig. 2) and
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activation of chemical defenses (including the three aforementioned classes of stress-
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related phytohormones, Fig. 3). α-Tocopherol levels increased (Fig. 2), and lipid
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hydroperoxide levels (an indicator of lipid peroxidation) decreased (Fig. 1) as a function
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of altitude. Furthermore, Las Blancas was the population showing the lowest leaf mass
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per area ratio (Fig. 1), carotenoid/chlorophyll ratio (Supplemental Fig. S3) and levels of
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ABA and jasmonic acid (Fig. 3).
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Size Influences Physiological Performance and Plant Death
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Logistic models showed that, for juveniles, the effect of individual plant size on
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mortality was significantly important for the low and intermediate populations, where
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smaller plants had a higher probability to die than larger ones (estimated β1 parameter: -
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0.12 and -0.05 respectively; p<0.001 in both cases; Fig. 4). Mortality rate in the highest
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population was independent on plant size, with dead plants less concentrated in small
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plants and more uniformly distributed across total size distribution (Fig. 4,
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Supplemental Fig. S4).
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Correlation analyses revealed that the relative leaf water content varied as a
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function of plant size in the high population (r=0.528, P<0.001), but not in the
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intermediate and low populations. The larger the plant, the higher the leaf water content
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in Las Blancas (Table 2, Fig. 5), a correlation that was not significant at lower altitudes.
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In should be noted, however, that the lowest leaf water contents were observed in San
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Juan de la Peña (with values around 50% lower than in the other two populations),
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although the large variability observed prevented the correlation to be significant (Fig.
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5). Other, more moderate, correlations were observed between plant size and leaf mass
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area ratio for the intermediate and high populations, and between plant size and the
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chlorophyll a/b ratio for the low population (r=0.34-0.35, P<0.05, Table 2).
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Plant Maturity and Death
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As a monocarpic species, S. longifolia dies right after blooming. During flowering,
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leaves serve as an important source of photoassimilates, but then the plant enters into a
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programmed, senescing process leading to death. We were interested in evaluating
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possible differences in plant physiological performance between juvenile and mature
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plants, and particularly between mature plants growing at different altitudes. With this
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purpose, we measured stress indicators in both juvenile and mature plants (at a
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flowering stage) in the low and intermediate populations (no sufficient individuals could
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be sampled for analyses in the high population due to extremely low reproductive
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events during 2015). Plant maturity increased the leaf mass area ratio, and the levels of
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antioxidants (carotenoids and α-tocopherol), ABA and jasmonic acid, while decreased
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those of 12-oxo-phytodienoic acid in the two populations studied (Table 3), thus
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indicating that plant maturity led to enhanced physiological stress. Furthermore, mature
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plants of the intermediate population showed lower leaf water contents, higher α-
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tocopherol levels and a lower extent of lipid peroxidation, as estimated by lipid
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hydroperoxides, compared to mature plants from the low population (Table 3).
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Clonal Growth, Reproduction and Death
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Rosettes might split into two or many more smaller rosettes in a given year, in a kind of
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clonal growth process but without the presence of rhizomes because the single
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axonomorphic root keeps inside the crevice (see Supplemental Fig. S1B). The
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frequency of multiple-rosette individuals ranged 7-11% across the 4-years of study in
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Las Blancas, whereas it was as low as 2% in San Juan de la Peña, and absent in Pantano
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de la Peña. Although clonal growth might happen at any plant size, it is more frequent
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among large plants (average diameter of non-clonal and clonal plants: 48.2 mm and
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83.4 mm, respectively; n=264 and 18 plants recorded in 2015, respectively). Survival
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did not differ between individuals with or without clonal reproduction (Fisher's exact
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test, P=0.141, n=1075), and none of the physiological parameters measured differed
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between non-clonal and clonal plants, except for the relative leaf water content, which
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was significantly lower in clonal plants (Table 4).
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DISCUSSION
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Our study of three populations of the long lived monocarpic Mediterranean S. longifolia
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located at different altitudes in the Pyrenees, has revealed that despite the complexity of
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mechanisms of adaptation to high altitude, which operate at the cellular, whole-plant
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and population levels, this species may be vulnerable to drought stress events (periods
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of low precipitation combined with high solar radiation and high temperatures) during
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the summer in the framework of climate change.
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Physiological Adaptation to Altitude: Photo- and Antioxidant Protection
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Exposure to high solar radiation is known to induce photo-oxidative stress, particularly
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when it is accompanied by other stress conditions, such as extreme temperatures, as it
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occurs at high altitude (Streb et al., 1997, 2003a, 2003b; Pintó-Marijuan and Munné-
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Bosch, 2014). Furthermore, global change may increase the frequency of drought
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events, which occur irregularly and may therefore affect plant populations from a given
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species in a rather different way just depending on its specific location, leading to
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increased photo-oxidative stress. In chloroplasts, production of reactive oxygen species
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under excess light conditions is mainly mediated by the triplet excitation state of
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chlorophyll, which can lead to singlet oxygen formation, as well as by the
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photoreduction of oxygen through photosynthetic electron transport in the Mehler
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reaction, leading to the production of superoxide anions (Asada 2006). Plants have
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developed a variety of protective systems, which allow them to control ROS levels, so
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that oxidative damage can be prevented. Among them, carotenoids, acting as scavengers
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of triplet chlorophyll and singlet oxygen, and mediating the harmless dissipation of
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excess excitation energy as heat (Demmig-Adams and Adams 1993; Demmig-Adams et
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al., 2013, 2014), anthocyanins, acting as a filter for high-level energy from the blue and
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UV light region of the spectrum (Landi et al., 2015), and tocochromanols, both
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quenching and scavenging singlet oxygen, and inhibiting the propagation of lipid
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peroxidation (Havaux et al., 2005; Munné-Bosch, 2005; Triantaphylidès and Havaux,
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2009; Falk and Munné-Bosch, 2010), play a key role. Results shown here for an
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orophyte endemism of the western Mediterranean illustrates that despite the complex
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mechanisms evolved by plants at the cellular level to survive high-mountain conditions,
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drought stress is one of the main triggers of mortality in S. longifolia. The intermediate
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population was the one showing the highest mortality rates, which paralleled with the
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lowest soil and leaf water contents and the activation of defense responses (e.g. ABA
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accumulation). Increased water availability in the high population (compared to the
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other ones) most likely led to a reduced need for photoprotection, despite increased high
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light exposure due to the altitudinal gradient. Interestingly, however, α-tocopherol
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levels increased as a function of altitude, their biosynthesis being mostly governed by
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high light exposure (Havaux et al., 2005). Such pattern was paralleled with reductions
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in lipid hydroperoxides, thus indicating the protective role of vitamin E in preventing
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the propagation of lipid peroxidation in the chloroplasts, which is in agreement with
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previous studies on other high-mountain plants (Streb et al., 1997; 2003a, 2003b).
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Unfortunately, chlorophyll fluorescence measurements could not be performed in intact
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leaves from this species in the field due to the high reflectance of the epidermis, an
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aspect that warrants further investigation.
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Activation of chemical defenses, such as the biosynthesis of salicylates and
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jasmonates, are known to be influenced by both biotic and abiotic stress factors (Davies,
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2010). In the present study, both groups of compounds increased with altitude
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(comparing the intermediate and low populations), but its accumulation was abolished
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at the highest altitude, most likely due, at least in part, to improved soil and leaf water
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contents (compared to the other two populations). Other factors may however also
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influence the accumulation of salicylates and jasmonates. In the high population,
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salicylic acid accumulation was similar to that of the low population, and that of
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jasmonic acid was even smaller, thus indicating a reduced need for chemical defense
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against necrotrophs at 2100 m a.s.l. (Davies, 2010). It is also likely that reduced
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jasmonic acid levels result from a trade-off between activation of different defense
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pathways in plants (photoprotection versus potential chemical defense to necrotrophs
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through jasmonates), so that enhanced vitamin E accumulation at the highest altitude
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may negatively influence the biosynthesis of jasmonates, which is in agreement with
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previous studies (Demmig-Adams et al., 2013, 2014; Morales et al., 2015; Simancas
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and Munné-Bosch, 2015). Enhanced jasmonic acid accumulation in the intermediate
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population may reflect activation of acclimation responses, but also increased cell death,
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as shown in other studies (Shumbe et al., 2016). Enhanced jasmonic acid levels may be
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triggered by increased biotic stress, but also by abiotic factors, such as drought stress
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(Brossa et al., 2011; de Ollas et al., 2013). Thus, it is very likely that enhanced ABA,
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salicylic acid and jasmonic acid levels, all respond to an increased drought stress that
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activates acclimation responses, but that ultimately lead to increased cell death and
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mortality in the intermediate population. Results suggest that enhanced physiological
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stress and mortality in the intermediate population was caused by increased drought
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stress during 2015. If more drought events occur in the other two populations, which are
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indeed likely to increase in the frame of global change (IPCC, 2014), it is expected they
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will also result in an increased mortality. More frequent snowfalls leads however to an
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increased water availability in the highest population. It may therefore be anticipated
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that, as precipitation patterns suggest (Supplemental Fig. S2), the populations found at
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the two lowest altitudes will be the ones showing the highest sensitivity to drought
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stress-induced mortality.
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Adaptation at the Population Level: Plant Size, Clonal Growth and Population
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Size Structure
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The population at highest altitude showed some demographic differences compared to
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the other two sampled populations: lower frequency of small individuals, size-
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independent mortality rate, the largest plants of all recorded across populations, and a
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particular trait that was almost absent in the other two: clonal growth (Supplemental
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Fig. S1B). The lower frequency of small-sized plants in the high population is the
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consequence of lower recruitment (very few new individuals enter each year in the
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monitoring plots; M.B. García, unpublished). Interestingly, small rosettes at high
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altitude survive better than at lower altitudes (Supplemental Fig. S4), and mortality was
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more uniformly distributed with size in the high population compared to the other two
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populations. Higher survival in the highest population may be associated with improved
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soil and leaf water contents, despite being exposed to higher light intensity.
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Furthermore, plants from this species seem to escape from increased size-dependent
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mortality, as it has been shown in other plant species, mostly woody perennials (shrubs
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and trees), in which increased plant size make larger individuals more vulnerable to
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environmental constraints (Mencuccini et al., 2005, 2007; Baudisch et al., 2013;
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Salguero et al., 2013; Munné-Bosch, 2014, 2015). This was not observed here in either
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studied population. Small sizes throughout their lifespan, as it happens in perennial
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herbs (García et al., 2011; Morales et al., 2013; Morales and Munné-Bosch, 2015), may
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protect S. longifolia plants from the potential negative effects of aging. It appears,
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therefore, that whole-plant senescence in this species may be attributed to reproduction
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and extrinsic factors, such as drought stress, but not to ageing, as only very small
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individuals (<30 mm diameter) die more frequently in the two lowest populations.
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Interestingly, those populations are the more exposed ones to summer drought. It may
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be speculated that an increase in temperatures and drought events in the framework of
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global change may be a serious threat for this species. Furthermore, population
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recruitment is lowest at the highest population, and reduced recruitment would translate
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into a negative population dynamics, a real limitation for adaptation in the framework of
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global change.
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The fact that plants get larger in the high population could be related to the
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existence of clonal growth, another interesting mechanism observed mainly at the
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highest altitude. Newly formed rosettes never become fully independent because they
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all share the same root system, but they can behave independent in the sense that not all
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daughter rosettes die or reproduce at the same time (see Supplemental Fig. S1B).
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Considering that this is a monocarpic plant, forming new rosettes might help the plant to
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reduce mortality as a consequence of reproduction, and extend the fecundity period like
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a polycarpic organism, spreading fitness over time. Flowering plants with one single
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rosette inevitably die the same year of reproduction, whereas 31% of multiple-rosette
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individuals survived. Therefore, having more than one rosette allows the plant to decide
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which ones to "sacrifice", which translates into survival of the individual if not all
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rosettes synchronously flower in a given year. Clonal growth, thus, constitutes an
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additional process operating at the individual level in terms of enhancing survival,
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which could also help to explain the lower mortality rate of this population compared to
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the other ones. The particular habitat or environment has been shown to be an important
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selective factor in predicting the evolutionary stable reproductive strategy in natural
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populations of monocarpic plants (Hesse et al., 2008), and S. longifolia constitutes a
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clear example of reproductive strategy variability along an altitudinal gradient.
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It is concluded that, despite the endemic Mediterranean plant, S. longifolia has
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evolved complex mechanisms of adaptation to altitude (including e.g. enhanced α-
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tocopherol levels or changes in reproductive strategy like activation of clonal growth),
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this species is rather sensitive to drought stress, and consequently drought events may
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drive increased mortality in populations from this species in the framework of global
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change. Further research is however needed to better understand the mechanisms
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underlying the influence of altitude and drought on size-dependent mortality, how they
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interact, and how this will in turn be affected by global warming in this and other
355
endemic plants in the near future.
356
MATERIALS AND METHODS
357
Plant Populations, Treatments and Sampling
358
The study was carried out in three natural populations of Saxifraga longifolia Lapeyruse
359
located in central Pyrenees, the area of highest abundance within its distribution range.
360
The three populations were located across an altitudinal range spanning 50 km in
361
straight line. The first population was located in rocky walls of limestone near Pantano
362
de la Peña (570 m a.s.l., coordinates: 42°22'58.4"N 0°44'02.1"W), the second one
363
occurred on a very sloppy conglomerated area near San Juan de la Peña (1100 m a.s.l.,
364
42°30'30.6"N 0°40'21.3"W), and the third one in the uppermost needles of calcareous
365
mountain named Las Blancas (2100 m a.s.l., 42°44'49.3"N 0°33'26.4"W). This long-
366
lived monocarpic perennial plant develops a basal rosette growing in the crevices of
367
limestone rocky places, mainly on cliffs, offering a unique sight in years of intensive
368
blooming.
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369
Samplings in at least 70 individuals randomly selected within each population
370
among plants larger than 30 mm of diameter were performed for biochemical analyses
371
in 2015, including both juvenile and reproductive plants, except in Las Blancas, where
372
too few individuals flowered during 2015. Sixty-five, 64 and 83 juveniles were sampled
373
in Pantano de la Peña, San Juan de la Peña and Las Blancas, respectively, and
374
additionally 8 and 10 mature individuals were sampled in the two former populations.
375
Samplings were performed on fully expanded leaves at midday (12 a.m. solar time) on
376
22 June, 3 July and 18 August in Pantano de la Peña, San Juan de la Peña and Las
377
Blancas, respectively, just after flowering in mature plants, so that all plants were at the
378
same phenological stage. Rosette leaves were used to estimate leaf water contents,
379
pigment concentrations (including chlorophylls, carotenoids and anthocyanins), levels
380
of vitamin E, the extent of lipid peroxidation, as well as the endogenous concentrations
381
of stress-related phytohormones, including ABA, salicylates and jasmonates. Samples
382
for biochemical analyses were collected, immediately frozen in liquid nitrogen in situ,
383
and stored at -80°C upon arrival to the laboratory.
384
Mortality
385
Between two and three hundred plants per location were marked and annually
386
monitored from 2011 through 2015 to estimate survival rates with the aid of grid plots.
387
In summer each year, all numbered plants were checked, and if alive their diameter
388
were recorded. Annual flowering and mortality rates were compared after pooling all
389
the events recorded along pairs of consecutive years (2011/2012, 2012/2013, 2013/2014
390
and 2014/2015). Furthermore, in order to explore possible reasons and consequences of
391
clonal reproduction in the highest population, we tested if juvenile and mature plants
392
with multiple rosettes had a different survival probability than singled-rosette ones.
393
Leaf Water Contents, Pigment Levels, and Lipid Peroxidation
394
To estimate leaf water contents, samples were collected, kept humid in small bags in
395
darkness during transport to the laboratory, and then weighed to estimate fresh matter
396
(FW). They were immersed in distilled water at 4°C for 24h to estimate the turgid
397
matter (TW), and then oven-dried at 80°C to constant weight to estimate the dry matter
398
(DW). Relative water content (RWC) was then calculated as 100 x (FW-DW)/(TW-
399
DW).
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400
For pigment analysis, measurements were performed spectrophotometrically on
401
methanolic extracts to estimate chlorophyll and carotenoid levels, as described by
402
Lichtenthaler (1987), which were then acidified with 30% HCl to estimate total
403
anthocyanin levels as described by Gitelson et al. (2001). The extent of lipid
404
peroxidation was estimated by measuring the levels of lipid hydroperoxides in leaves.
405
Lipid hydroperoxide levels were estimated spectrophotometrically following a modified
406
ferrous oxidation-xylenol orange (FOX) assay, as described (DeLong et al., 2002).
407
Tocochromanols
408
For analyses of tocochromanol (tocopherols and plastochromanol-8) contents, leaf
409
samples were ground in liquid nitrogen and extracted with cold methanol containing
410
0.01% butylated hydroxyltoluene using ultrasonication. After centrifuging at 12000 rpm
411
for 10 min and 4°C, the supernatant was collected and the pellet re-extracted with the
412
same solvent until it was colorless; then, supernatants were pooled, filtered and injected
413
into the HPLC. Tocopherols and tocotrienols were separated isocratically on a normal-
414
phase HPLC system using a fluorescent detector as described (Cela et al., 2011).
415
Compounds were identified by co-elution with authentic standards and quantified by
416
using a calibration curve. From all tocochromanols investigated (α-, β-, γ- and δ-
417
tocopherols and tocotrienols, and plastochromanol-8), α-tocopherol was the only
418
compound present at quantifiable amounts in leaves.
419
Stress-related Phytohormones
420
For analyses of ABA, salicylic acid and jasmonates, leaf samples were ground in liquid
421
nitrogen and extracted with cold methanol using ultrasonication. After centrifuging at
422
12000 rpm for 10 min and 4°C, the supernatant was collected and the pellet re-extracted
423
with the same solvent until it was colorless; then, supernatants were pooled, filtered and
424
injected into the UHPLC-MS/MS. Phytohormones were separated using an elution
425
gradient on a reverse-phase UHPLC system and quantified using tandem mass
426
spectrometry in multiple reaction monitoring mode as described (Müller and Munné-
427
Bosch, 2011). Recovery rates were calculated for each hormone on every sample by
428
using deuterated compounds.
429
Statistical Analysis
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430
To determine the effect of altitude, mean values were tested by one-way factorial
431
analysis of variance (ANOVA) and additionally Bonferroni posthoc tests. Mean values
432
were compared between clonal and non-clonal plants by means of Student's t-test. In all
433
cases, differences were considered significant at a probability level of P<0.05.
434
Spearman rank's correlation analyses were performed between plant size (estimated as
435
rosette diameter) and all biochemical parameters, and Bonferroni correction applied to
436
determine significant differences. Statistical tests were carried out using the SPSS 20.0
437
statistical package. The effect of individual size on mortality probability was explored
438
by fitting logistic regression models (logit link function, binomial distribution, in R
439
version 2.15.2, Core Team) for all the annual transitions recorded over that period, for
440
each population separately.
441
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442
ACKNOWLEDGEMENTS
443
We are very grateful to Maren Müller and Serveis Científico-tècnics for technical help
444
with biochemical analyses. We are also indebted to M. Paz Errea and Ricardo García
445
González for providing environmental data, and the fieldwork assistance of Juanlu, Iker,
446
P. Sánchez and P. Bravo.
447
AUTHOR CONTRIBUTIONS
448
S.M.-B., A.C., M.M. and M.B.G. conceived the research plan. A.C., M.M., E.F.S., J.V.
449
and M.B.G. performed the experiments. S.M.-B. wrote the article with the help of
450
M.B.G.
451
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452
Table 1. Mortality rates (numbers and percentage) during the last 4 years in the long-
453
lived monocarpic plant, S. longifolia. Monitoring of individual plants was carried out
454
between June and August of the given periods (indicated in parentheses). Mortality rates
455
refer to juveniles (Juv) and flowering (R).
456
Pa ntano de la Peña
Ju v
R
194
6
2
3.1
1.0
N (2011-2 012)
Dead
% mo rtality
N (2012-2013)
Dead
% mo rtality
33
14.3
N (2013-2014)
Dead
% mo rtality
11
5.2
N (2014-2015)
Dead
% mo rtality
Average (2011-2015)
San Juan de la Peña
Juv
R
204
14
12
6.9
5.9
230
Las Blancas
Juv
R
281
5
0
1.8
0.0
285
205
0
0.0
22
10.7
0
0.0
13
4.6
1
0.5
30
14.9
0
0.0
13
4.7
10
4.9
12
5.9
27
13.6
10
5.1
18
8.3
2
0.9
6.9
1.8
11.5
2.8
4.8
4.3
211
276
201
204
42
15.2
217
198
457
458
459
17
3
1.1
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460
Table 2. Spearman rank's correlation analyses between plant size (estimated as rosette
461
diameter) and all measured parameters in the long-lived monocarpic plant, S. longifolia.
462
All data from juvenile plants, including the three populations, was pooled for analyses,
463
but also analyzed separately. rho and P values are indicated in bold when correlations
464
were significant (Bonferroni adjusted, P<0.0033). RWC, relative water content; LMA,
465
leaf mass per area ratio; Chl, chlorophyll. LOOH, lipid hydroperoxides; Ant,
466
anthocyanins; Car, carotenoids; α-Toc, α-tocopherol; ABA, abscisic acid; SA, salicylic
467
acid; OPDA, oxo-phytodienoic acid; JA, jasmonic acid.
468
All data
Pantano de la Peña San Juan de la Peña
Las Blancas
RWC
0.370 (<0.001)
0.293 (0.009)
0.256 (0.021)
0.528 (<0.001)
LMA
0.347 (<0.001)
0.331 (0.004)
0.347 (0.003)
0.344 (0.001)
Chl a+b
-0.028 (0.345)
-0.186 (0.071)
-0.170 (0.092)
0.243 (0.014)
Chl a/b
0.098 (0.080)
0.354 (0.002)
0.244 (0.027)
-0.084 (0.227)
LOOH
-0.166 (0.009)
0.016 (0.451)
0.120 (0.178)
0.291 (0.004)
Ant
-0.034 (0.314)
-0.206 (0.052)
-0.172 (0.089)
0.221 (0.023)
Ant/Chl
-0.025 (0.359)
-0.084 (0.255)
-0.032 (0.401)
-0.039 (0.364)
Car
-0.081 (0.123)
-0.313 (0.006)
-0.087 (0.249)
0.169 (0.064)
Car/Chl
-0.093 (0.090)
-0.261 (0.019)
0.067 (0.301)
-0.203 (0.034)
-0.147 (0.017)
-0.331 (0.004)
-0.224 (0.039)
0.091 (0.207)
α-Toc
0.013 (0.459)
-0.057 (0.327)
-0.089 (0.213)
α-Toc/Chl -0.046 (0.253)
ABA
-0.037 (0.298)
-0.044 (0.366)
-0.152 (0.130)
-0.073 (0.257)
SA
0.030 (0.334)
0.183 (0.074)
-0.220 (0.050)
0.086 (0.221)
OPDA
-0.005 (0.471)
0.070 (0.292)
-0.079 (0.281)
-0.032 (0.387)
JA
0.008 (0.453)
-0.010 (0.468)
-0.115 (0.196)
-0.018 (0.437)
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469
Table 3. Influence of maturity for all measured parameters in the long-lived monocarpic
470
plant, S. longifolia. Data correspond to the mean ± SE. n=64 juvenile and n=8 mature
471
plants in Pantano de la Peña, and n=64 juvenile and n=10 mature plants in San Juan de
472
la Peña. An asterisk indicates differences between juvenile and mature plants (Student's
473
t-test, P<0.05). Different letters indicate differences between populations, either in
474
juvenile or mature plants, the latter indicated in capital letters(Student's t-test, P<0.05).
475
No sufficient plants in a mature stage were found in Las Blancas, so data are not
476
available for the population at the highest altitude. RWC, relative water content; LMA,
477
leaf mass per area ratio; Chl, chlorophyll. LOOH, lipid hydroperoxides; Ant,
478
anthocyanins; Car, carotenoids; α-Toc, α-tocopherol; ABA, abscisic acid; SA, salicylic
479
acid; OPDA, oxo-phytodienoic acid; JA, jasmonic acid.
Diameter (mm)
RWC (%)
LMA (g/m2)
Chl a+b (μmol/g DW)
Chl a/b
LOOH (μmol/g DW)
Ant (μmol/g DW)
Ant /Chl
Car (μmol/g DW)
Car/Chl
α-Toc (μmol/g DW)
α-Toc /Chl
ABA (ng/g DW)
SA (ng/g DW)
OPDA (ng/g DW)
JA (ng/g DW)
Pantano de la Peña
Juvenile
Mature
72.1 ± 3.2a
96.3 ± 20.0A
92.4 ± 0.8a
93.0 ± 2.5A
1561 ± 47a
725 ± 79*A
1.33 ± 0.08a
1.39 ± 0.25A
2.26 ± 0.02a
1.88 ± 0.04*A
6.94 ± 0.49a
11.86 ± 2.84A
0.61 ± 0.03a
0.60 ± 0.11A
0.49 ± 0.03a
0.45 ± 0.04A
0.29 ± 0.02a
0.43 ± 0.07*A
0.23 ± 0.01a
0.32 ± 0.02*A
0.28 ± 0.01a
0.39 ± 0.05*A
0.26 ± 0.02a
0.34 ± 0.06A
424.5 ± 21.3a 937.3 ± 153.5*A
375.9 ± 12.0a 674.4 ± 89.9*A
2788 ± 225a
440 ± 103*A
187.5 ± 7.9a
379.3 ± 74.6*A
San Juan de la Peña
Juvenile
Mature
71.8 ± 3.9a
116.0 ± 14.6*A
78.9 ± 1.5b
65.0 ± 5.6*B
1611 ± 51a
780 ± 66*A
1.70 ± 0.07b
1.61 ± 0.22A
2.01 ± 0.02b
1.79 ± 0.07*A
5.84 ± 0.71a
5.33 ± 1.23B
0.71 ± 0.04b
0.78 ± 0.12A
0.42 ± 0.01b
0.52 ± 0.10A
0.44 ± 0.02b
0.56 ± 0.06*A
0.26 ± 0.01b
0.37 ± 0.03*A
0.32 ± 0.01b
0.56 ± 0.03*B
0.20 ± 0.01b
0.40 ± 0.05*A
598.3 ± 38.7b
848.8 ± 63.9*A
472.6 ± 24.6b
568.4 ± 58.3A
1289 ± 158b
256 ± 38*A
216.2 ± 14.4b
550.1 ± 273.7A
480
481
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
482
Table 4. Influence of clonal growth for all measured parameters in the highest
483
population (Las Blancas) of the long-lived monocarpic plant, S. longifolia. Data
484
correspond to the mean ± SE of n=74 non-clonal and n=9 clonal plants. An asterisk
485
indicates differences between juvenile and mature plants (Student's t-test, P<0.05).
486
RWC, relative water content; LMA, leaf mass per area ratio; Chl, chlorophyll. LOOH,
487
lipid hydroperoxides; Ant, anthocyanins; Car, carotenoids; α-Toc, α-tocopherol; ABA,
488
abscisic acid; SA, salicylic acid; OPDA, oxo-phytodienoic acid; JA, jasmonic acid.
489
Non-Clonal
Clonal
Diameter (mm)
67.63 ± 2.72
66.71 ± 7.75
RWC (%)
84.01 ± 1.04
75.74 ± 1.81*
1442.94 ± 35.48
1313.10 ± 75.29
Chl a + b(μmol/g DW)
1.52 ± 0.06
1.34 ± 0.11
Chl a / b
2.17 ± 0.02
2.24 ± 0.04
LOOH(μmol/g DW)
5.38 ± 0.37
3.94 ± 0.60
Ant(μmol/g DW)
0.53 ± 0.02
0.57 ± 0.10
Ant / Chl
0.36 ± 0.01
0.46 ± 0.13
Car (μmol/g DW)
0.29 ± 0.01
0.30 ± 0.01
Car / Chl
0.20 ± 0.01
0.22 ± 0.01
α-Toc (μmol/g DW)
0.34 ± 0.01
0.30 ± 0.02
α-Toc / Chl
0.25 ± 0.01
0.24 ± 0.02
ABA(ng/g DW)
336.22 ± 15.49
349.76 ± 28.53
SA(ng/g DW)
383.11 ± 10.38
415.69 ± 46.77
2428.69 ± 216.78
1609.05 ± 357.17
119.57 ± 5.02
100.95 ± 15.27
LMA(g/m2)
OPDA(ng/g DW)
JA(ng/g DW)
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490
FIGURE LEGENDS
491
Figure 1. Plant size (estimated as rosette diameter), relative leaf water content (RWC),
492
leaf mass per area ratio (LMA), chlorophyll (Chl) a+b, Chl a/b ratio and lipid
493
hydroperoxide levels (an estimation of lipid peroxidation) in plants of the long-lived
494
monocarpic plant, S. longifolia growing at three altitudes (570, 1100 and 2100 m a.s.l.
495
in Pantano de la Peña, San Juan de la Peña and Las Blancas, respectively). Data shows
496
the mean ± SE of n=65, 64 and 83 juvenile individuals for the three populations at
497
increasing altitude, respectively. Results of one-way ANOVA are shown in the inlets.
498
Different letters indicate significant differences between populations (P<0.05) using
499
Bonferroni posthoc tests. NS, not significant.
500
Figure 2. Antioxidant protection, including levels of anthocyanins, carotenoids and α-
501
tocopherol, in plants of the long-lived monocarpic plant, S. longifolia growing at three
502
altitudes (570, 1100 and 2100 m a.s.l. in Pantano de la Peña, San Juan de la Peña and
503
Las Blancas, respectively). Data show the mean ± SE of n=65, 64 and 83 juvenile
504
individuals for the three populations. Results of one-way ANOVA are shown in the
505
inlets. Different letters indicate significant differences between populations (P<0.05)
506
using Bonferroni posthoc tests.
507
Figure 3. Endogenous concentrations of stress-related phytohormones, including
508
abscisic acid (ABA), salicylic acid (SA), oxo-phytodienoic acid (OPDA) and jasmonic
509
acid (JA) in plants of the long-lived monocarpic plant, S. longifolia growing at three
510
altitudes (570, 1100 and 2100 m a.s.l. in Pantano de la Peña, San Juan de la Peña and
511
Las Blancas, respectively). Data shows the mean ± SE of n=65, 64 and 83 juvenile
512
individuals for the three populations at increasing altitude, respectively. Results of one-
513
way ANOVA are shown in the inlets. Different letters indicate significant differences
514
between populations (P<0.05) using Bonferroni posthoc tests.
515
Figure 4. Logistic mortality regression models for the three populations studied. The
516
“x” axis corresponds to the diameter (measured in mm) of plants in year “t”, and the “y”
517
axis to the recorded fate in year “t+1” (0=alive, 1=dead). Dots show individual yearly
518
events (dead or alive) from 2011 till 2015. A total of 824, 786 and 1012 events are
519
plotted in the low, intermediate and high population respectively. All dots should fit the
520
“0” or “1” values, but were not forced to lie on a line for illustrative purposes.
21
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521
Figure 5. Spearman rank's correlation analyses between plant size of juvenile plants
522
(estimated as rosette diameter) and the relative water content (RWC) in three
523
populations of the long-lived monocarpic plant, S. longifolia. rho (r) and P values are
524
indicated in the inlets (correlation was significant in the population at the highest
525
altitude only, Las Blancas, P<0.0033, Bonferroni adjusted).
526
527
528
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
529
SUPPLEMENTAL DATA
530
Supplemental Figure S1. Saxifraga longifolia population structure in 2011, according to rosette diameter
531
(seedlings excluded).
532
Supplemental Figure S2. Monthly average temperatures, soil water contents and precipitation recorded
533
in the three studied populations.
534
Supplemental Figure S3. Levels of anthocyanins, carotenoids and α-tocopherol, expressed per
535
chlorophyll (Chl) unit, in plants of the long-lived monocarpic plant, S. longifolia growing at three
536
altitudes (570, 1100 and 2100 m.a.s.l. in Pantano de la Peña, San Juan de la Peña and Las Blancas,
537
respectively).
538
Supplemental Figure S4. Mortality rate for plants of different sizes.
539
540
541
Supplemental Figure S1. (A) Saxifraga longifolia population structure in 2011,
542
according to rosette diameter (seedlings excluded). (B) The left sided plant is an
543
example of clonal growth in a plant from Las Blancas (multiple rosette individual),
544
whereas the right sided plant shows a typical, single rosette individual.
545
Supplemental Figure S2. (A) Monthly average temperatures recorded in the three
546
studied populations. In the lowest and highest ones (Pantano de la Peña and Las Blancas
547
respectively), temperature was recorded by tinny thermometers (Maxim’s ibutton
548
devices) placed inside populations, and the average monthly values recorded over 3
549
years (2012-2015) is shown. For the intermediate population (San Juan de la Peña), we
550
averaged monthly temperatures from the closest meteorological station, located 5 km
551
away. (B) Soil water contents, daily solar radiation and maximum diurnal air
552
temperatures during the days of measurements (22 June, 3 July and 18 August from the
553
highest to the lowest population, respectively). (C) Monthly average precipitation in the
554
three sites of study (precipitation during months of measurements for each population is
555
indicated in red).
556
Supplemental Figure S3. Levels of anthocyanins, carotenoids and α-tocopherol,
557
expressed per chlorophyll (Chl) unit, in plants of the long-lived monocarpic plant, S.
558
longifolia growing at three altitudes (570, 1100 and 2100 m.a.s.l. in Pantano de la Peña,
559
San Juan de la Peña and Las Blancas, respectively). Data show the mean ± SE of n=65,
23
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560
64 and 83 juvenile individuals for the three populations at increasing altitude,
561
respectively. Results of one-way ANOVA are shown in the inlets. Different letters
562
indicate significant differences between populations (P<0.05) using Duncan posthoc
563
tests. NS, not significant.
564
Supplemental Figure S4. Mortality rate for plants of different sizes. Small: x<30 mm,
565
Medium: 60>x>30, Large: x≥60 mm. Populations: PP: Pantano de la Peña, SJP: San
566
Juan de la Peña, LB: Las Blancas.
567
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568
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569
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DeLong JM, Prange RK, Hodges DM, Forney CF, Bishop MC, Quilliam M (2002)
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Figure 1. Plant size (estimated as rosette diameter),relative leaf water content (RWC),
leaf mass per area ratio (LMA), chlorophyll (Chl) a+b, Chl a/b ratio and lipid
hydroperoxide levels (an estimation of lipid peroxidation) in plants of the long-lived
monocarpic plant, S. longifolia growing at three altitudes (570, 1100 and 2100 m a.s.l.
in Pantano de la Peña, San Juan de la Peña and Las Blancas, respectively). Data
shows the mean ± SE of n=65, 64 and 83 juvenile individuals for the three populations
at increasing altitude, respectively. Results of one-way ANOVA are shown in the inlets.
Different letters indicate significant differences between populations (P<0.05) using
Bonferroni posthoc tests.NS, not significant.
80
P<0.001
b
-1
70
Diameter (mm)
1,8
Chl a+b (mol·gDW )
NS
a
1,6
a
1,4
60
1,2
50
1,0
0
0,0
P<0.001
P<0.001
c
c
2,4
b
2,2
a
b
2,0
a
80
1,8
Chl a/b
RWC (%)
90
1,6
70
0,0
b
P=0.048
ab
7
-2
LMA (gDW·m )
1600
a
a
1500
6
5
1400
1300
4
0
Pantano de
la Peña
San Juan de
la Peña
Location
Las Blancas
Pantano de
la Peña
San Juan de
la Peña
Las Blancas
Location
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
-1
b
b
LOOH (mol eq H2O2·gDW )
P=0.006
1700
Figure 2. Antioxidant protection, including levels of anthocyanins, carotenoids and atocopherol, in plants of the long-lived monocarpic plant, S. longifolia growing at three
altitudes (570, 1100 and 2100 m a.s.l. in Pantano de la Peña, San Juan de la Peña
and Las Blancas, respectively). Data show the mean ± SE of n=65, 64 and 83 juvenile
individuals for the three populations. Results of one-way ANOVA are shown in the
inlets. Different letters indicate significant differences between populations (P<0.05)
using Bonferroni posthoc tests.
-1
Anthocyanins (mol·gDW )
0,8
0,7
b
P<0.001
a
0,6
a
0,5
0,4
0,0
-1
Carotenoids (mol·gDW )
b
P<0.001
0,4
a
a
0,3
0,2
0,0
P=0.002
b
b
-1
a-Tocopherol (mol·gDW )
0,35
a
0,30
0,25
0,20
0,00
Pantano de
la Peña
San Juan de
la Peña
Las Blancas
Location
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Figure 3. Endogenous concentrations of stress-related phytohormones, including
abscisic acid (ABA), salicylic acid (SA), oxo-phytodienoic acid (OPDA) and jasmonic
acid (JA) in plants of the long-lived monocarpic plant, S. longifolia growing at three
altitudes (570, 1100 and 2100 m a.s.l. in Pantano de la Peña, San Juan de la Peña
and Las Blancas, respectively). Data shows the mean ± SE of n=65, 64 and 83
juvenile individuals for the three populations at increasing altitude, respectively. Results
of one-way ANOVA are shown in the inlets. Different letters indicate significant
differences between populations (P<0.05) using Bonferroni posthoc tests.
700
c
P<0.001
-1
ABA (ng·gDW )
600
500
b
400
a
300
0
P<0.001
b
-1
SA (ng·gDW )
500
400
a
a
b
P<0.001
300
0
3000
1
OPDA (ng·gDW )
b
2500
2000
a
1500
1000
0
c
b
-1
JA (ng·gDW )
200
P<0.001
150
a
100
0
Pantano de
la Peña
San Juan de
la Peña
Las Blancas
Location
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Figure 4. Logistic mortality regression models for the three populations studied. The
“x” axis corresponds to the diameter (measured in mm) of plants in year “t”, and the “y”
axis to the recorded fate in year “t+1” (0=alive, 1=dead). Dots show individual yearly
events (dead or alive) from 2011 till 2015. A total of 824, 786 and 1012 events are
plotted in the low, intermediate and high population respectively. All dots should fit the
“0” or “1” values, but were not forced to lie on a line for illustrative purposes.
Pantano de la Peña
San Juan de la Peña
Las Blancas
1
1
0
0
0
Size
Size
Size
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
250
200
150
100
50
0
250
200
150
100
50
0
250
200
150
100
50
0
Fate
1
Figure 5. Spearman rank's correlation analyses between plant size of juvenile plants
(estimated as rosette diameter) and the relative water content (RWC) in three
populations of the long-lived monocarpic plant, S. longifolia. rho (r) and P values are
indicated in the inlets (correlation was significant in the population at the highest
altitude only, Las Blancas, P<0.0033, Bonferroni adjusted).
San Juan de la Peña
Pantano de la Peña
Las Blancas
100
100
100
90
90
85
r = 0.256
p = 0.021
80
90
RWC (%)
r = 0.293
p = 0.009
RWC (%)
RWC (%)
95
70
60
r = 0.528
p < 0.001
80
70
80
50
75
40
0
0
20
40
60
80
100
Diameter (mm)
120
140
160
60
0
0
50
100
150
Diameter (mm)
200
250
20
40
60
80
100
Diameter (mm)
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120
140
160
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