adaptation of the long-lived monocarpic perennial ...€¦ · 27 capacity for adaptation. here, we...

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1 Adaptation of the Long-Lived Monocarpic Perennial, Saxifraga longifolia to High 1 Altitude 2 Sergi Munné-Bosch 1* , Alba Cotado 1 , Melanie Morales 1 , Eva Fleta-Soriano 1 , Jesús 3 Villellas 2,3 , Maria B. Garcia 2 4 1 Department of Plant Biology, Faculty of Biology, University of Barcelona, Barcelona, 5 Spain 6 2 Pyrenean Institute of Ecology, CSIC, Zaragoza, Spain 7 3 Current address: Department of Zoology, Trinity College Dublin, Dublin, Ireland 8 *Correspondence: 9 Sergi Munné-Bosch, [email protected], tel.: +34-934021463; fax: +34934112842 10 11 Short title: Adaptation to Altitude in a Monocarpic Perennial 12 13 One sentence summary: Adaptation of an endemic long-lived monocarpic perennial to 14 high altitude is influenced by multiple mechanisms operating at various levels 15 16 Funding information: This work was supported by the Spanish Government (project 17 number BFU2015-64001-P to SMB and CGL 2010-21642 to MBG) and the Catalan 18 Government (Institució Catalana de Recerca i Estudis Avançats Academia award given 19 to S.M.B.). 20 The author responsible for distribution of materials integral to the findings presented in 21 this article in accordance with the policy described in the Instructions for Authors 22 (www.plantphysiol.org) is: Sergi Munné-Bosch ([email protected]) 23 24 Plant Physiology Preview. Published on July 20, 2016, as DOI:10.1104/pp.16.00877 Copyright 2016 by the American Society of Plant Biologists www.plantphysiol.org on May 1, 2020 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

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Page 1: Adaptation of the Long-Lived Monocarpic Perennial ...€¦ · 27 capacity for adaptation. Here, we aimed at unveiling mechanisms of adaptation to high 28 altitude in an endemic long-lived

1

Adaptation of the Long-Lived Monocarpic Perennial, Saxifraga longifolia to High 1

Altitude 2

Sergi Munné-Bosch1*, Alba Cotado1, Melanie Morales1, Eva Fleta-Soriano1, Jesús 3

Villellas2,3, Maria B. Garcia2 4

1Department of Plant Biology, Faculty of Biology, University of Barcelona, Barcelona, 5

Spain 6

2Pyrenean Institute of Ecology, CSIC, Zaragoza, Spain 7

3Current address: Department of Zoology, Trinity College Dublin, Dublin, Ireland 8

*Correspondence: 9

Sergi Munné-Bosch, [email protected], tel.: +34-934021463; fax: +34934112842 10

11

Short title: Adaptation to Altitude in a Monocarpic Perennial 12

13

One sentence summary: Adaptation of an endemic long-lived monocarpic perennial to 14

high altitude is influenced by multiple mechanisms operating at various levels 15

16

Funding information: This work was supported by the Spanish Government (project 17

number BFU2015-64001-P to SMB and CGL 2010-21642 to MBG) and the Catalan 18

Government (Institució Catalana de Recerca i Estudis Avançats Academia award given 19

to S.M.B.). 20

The author responsible for distribution of materials integral to the findings presented in 21

this article in accordance with the policy described in the Instructions for Authors 22

(www.plantphysiol.org) is: Sergi Munné-Bosch ([email protected]) 23

24

Plant Physiology Preview. Published on July 20, 2016, as DOI:10.1104/pp.16.00877

Copyright 2016 by the American Society of Plant Biologists

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ABSTRACT 25

Global change is exerting a major effect on plant communities altering their potential 26

capacity for adaptation. Here, we aimed at unveiling mechanisms of adaptation to high 27

altitude in an endemic long-lived monocarpic, Saxifraga longifolia, by combining 28

demographic and physiological approaches. Plants from three altitudes (570, 1100 and 29

2100 m a.s.l.) were investigated in terms of leaf water and pigment contents, and 30

activation of stress defense mechanisms. The influence of plant size on physiological 31

performance and mortality was also investigated. Levels of photoprotective molecules 32

(α-tocopherol, carotenoids and anthocyanins) increased in response to high altitude 33

(1100 relative to 570 m a.s.l.), which was paralleled by reduced soil and leaf water 34

contents and increased ABA levels. The more demanding effect of high altitude on 35

photoprotection was however partly abolished at very high altitudes (2100 m a.s.l.) due 36

to improved soil water contents, with the exception of α-tocopherol accumulation. α-37

Tocopherol levels increased progressively at increasing altitudes, which paralleled with 38

reductions in lipid peroxidation, thus suggesting plants from the highest altitude 39

effectively withstood high light stress. Furthermore, mortality of juveniles was highest 40

at the intermediate population, suggesting that drought stress was the main 41

environmental driver of mortality of juveniles in this rocky plant species. Population 42

structure and vital rates in the high population evidenced lower recruitment and 43

mortality in juveniles, activation of clonal growth, and absence of plant size-dependent 44

mortality. We conclude that, despite S. longifolia has evolved complex mechanisms of 45

adaptation to altitude at the cellular, whole-plant and population levels, drought events 46

may drive increased mortality in the framework of global change. 47

Keywords: antioxidants; carotenoids; chemical defenses; high altitude; photoprotection; 48

plant size effects; vitamin E; mortality rate 49

50

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INTRODUCTION 51

European mountains shelter a huge biodiversity, and are home to many endemic plants 52

and animals, i.e. species that occur nowhere else. Global change, and particularly 53

climatic change, is expected to exert a major effect on mountain plant communities, 54

altering their potential capacity for adaptation (Peñuelas and Boada, 2003; Franklin et 55

al., 2016). Under such scenario of environmental changes, populations of organisms 56

must either escape or get quickly adapted, otherwise they go extinct. For instance, 57

certain butterfly species have been migrating north, or to higher altitudes, to escape 58

rising temperatures (Breed et al., 2013). Plants, of course, cannot migrate as fast as 59

animals, and important shifts have already been found among plant communities 60

inhabiting mountain summits (Gottfried et al., 2012). When global air temperatures 61

increase, the number of cooler habitats will shrink, producing a crowding effect and 62

increased competition among some species in the remaining cooler areas; at the same 63

time, however, other habitat types will increase in abundance (Scherrer and Körner, 64

2011). Alpine habitats could prove more attractive to plant species than lowlands 65

because of their topography providing favorable microhabitats. However, certain rare 66

species may lose out in the long-term competition for space, especially those favoring 67

cooler climates (Körner, 2013). 68

Leaves of high-mountain plants are highly resistant to photoinhibitory damage. 69

Tocochromanols (particularly tocopherols and plastochromanol-8) are found in 70

thylakoids and play an antioxidant function in protecting lipids from the propagation of 71

lipid peroxidation in chloroplasts. Together with carotenoids, they also prevent 72

photosystem II damage as a result of singlet oxygen attack (Munné-Bosch and Alegre, 73

2002; Falk and Munné-Bosch, 2010; Zbierzak et al., 2010; Kruk et al., 2014). A higher 74

tocochromanol content, particularly of α-tocopherol, and an increased capacity for non-75

radiative dissipation of excitation energy by activation of the xanthophyll cycle have 76

been found in high-mountain plants, thus supporting such a role (Streb et al., 1997, 77

1998, 2003a, 2003b; García-Plazaola et al., 2015). Furthermore, although the number of 78

studies is still very limited for plants in their natural habitat, high-mountain plants tend 79

to accumulate large amounts of ABA (Bano et al., 2009), a phytohormone that is known 80

to mediate the acclimation/adaptation of plants to temperature extremes by modulating 81

the up- and downregulation of numerous genes (Gilmour et al., 1991). The activation of 82

other chemical defenses, such as the accumulation of salicylates and jasmonates, which 83

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serve against biotrophs and necrotrophs, can also be affected by extreme temperatures 84

(Kosova et al., 2012; Dong et al., 2014; Miura and Tada, 2014), but it has not been 85

investigated thus far in high-mountain plants. 86

Some degree of plasticity in physiological traits is ubiquitous among plants, so 87

that environmental growth conditions are generally considered essential factors 88

governing the physiological performance of plants and their organs (Larcher, 1994). A 89

number of recent studies with trees, shrubs and herbs, including vascular epiphytes 90

(Mencuccini and Grace 1996; Zotz, 1997; Schmidt et al., 2001; Schmidt and Zotz, 91

2001; Munné-Bosch and Lalueza, 2007; Morales et al., 2014) point out, however, to 92

another source of intraspecific variation that many studies in the past have inadvertently 93

missed, i.e. substantial variation in physiological traits related to plant size rather than 94

changing environmental conditions (Zotz et al., 2001). In trees, increased plant size 95

leads to increased hydraulic resistance causing reductions in relative leaf growth rates 96

(Mencuccini and Grace, 1996). Furthermore, photo-oxidative stress has been shown to 97

increase during periods of low precipitation combined with high light in the 98

Mediterranean shrub, Cistus clusii as a function of plant size, therefore suggesting an 99

increased vulnerability to photo-oxidative stress in the largest individuals (Munné-100

Bosch and Lalueza, 2007). To our knowledge, no studies are however available to 101

unveil the possible influence of plant size on photoprotection and activation of chemical 102

defenses in high-mountain plants. 103

Saxifraga longifolia Lapeyruse (Saxifragaceae) is an endemic species of the 104

western Mediterranean mountains, ranging from the Pyrenees (plus a couple of 105

populations in the Cantabric Mountains) through eastern Spain to reach its southern 106

limit in the high Atlas of Morocco (Webb and Gornall, 1989). This long-lived 107

monocarpic plant develops a basal rosette growing in limestone rocky places, mainly on 108

cliffs, offering a unique sight in years of intensive blooming. Reproduction occurs when 109

plants are at least 6 years old in greenhouse conditions (Webb and Gornall, 1989) and it 110

is thought to be much later in natural conditions. This orophyte plant shows striking 111

variation in plant size, with a diameter of the rosette up to 30 cm in the largest 112

individuals. It has been shown that flower and seed production increase as a function of 113

plant size with female success being maximum in intermediate sized plants (García, 114

2003). 115

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In the present study, with the aim of getting new insights into the mechanisms of 116

adaptation to high altitudes and the influence of plant size on this adaptation capacity, 117

we examined the physiological response of S. longifolia growing at three contrasted 118

populations spanning its whole altitudinal range. We described population structure, 119

calculated mortality rates, and analyzed physiological performance, including water 120

contents and activation of photoprotection mechanisms and chemical defenses. We 121

aimed at understanding the effect of varying altitude on the expression of defense 122

mechanisms that govern adaptive processes in high-mountain plants. 123

RESULTS 124

Physiological Performance and Mortality in Populations at Various Altitudes 125

S. longifolia is a monocarpic species; therefore, plants die as a consequence of 126

reproduction. We wondered, however, whether mortality of juveniles is also influenced 127

by stressful conditions across an altitudinal range by monitoring three plant populations 128

growing at 570, 1100 and 2100 m a.s.l. The population occurring at the highest altitude 129

(Las Blancas) had the largest plants, the less frequency of small ones (Supplemental 130

Fig. S1A), and the lowest mortality rate in juveniles (average across 4 years: 4.8 %, 131

Table 1). Mortality of juveniles was higher in the lowest population (Pantano de la 132

Peña, 6.9 %) and highest in the intermediate one (San Juan de la Peña, 11.5 %, Table 1), 133

both of them showing a similar population size distribution (Supplemental Fig. S1A). 134

Therefore, altitude, which, as expected, resulted in lower temperatures (Supplemental 135

Fig. S2), does not seem to be associated to mortality rates in juveniles. Rather, mortality 136

rates in juveniles seemed to be more associated with reduced soil water contents. 137

Among the three populations studied, the volumetric soil water content was the highest 138

in the high population and lowest in the intermediate one (Supplemental Fig. S2). Plants 139

growing in San Juan de la Peña were exposed to more stressful conditions during the 140

day of measurements compared to the other two populations, as indicated not only by 141

reduced soil water contents, but also higher solar radiation and air temperatures, which 142

may in turn contribute to drought stress. Mortality due to flowering was much lower 143

than among juvenile plants (Table 1), and flowering rates were rather stochastic across 144

years and populations, with the highest population showing the higher flowering rates 145

during 2013/14, but also the smaller ones during 2014/15 (Table 1). 146

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Physiological stress indicators, including leaf water, pigment and lipid 147

peroxidation levels (Fig. 1), antioxidant protection (Fig. 2) and stress-related 148

phytohormones (Fig. 3), revealed that the physiological performance in juvenile plants 149

differed in the three populations, some parameters changing as a function of altitude and 150

others following the mortality rate pattern more linked to drought stress (the 151

intermediate population showed the lowest leaf water contents among the three 152

populations, Fig. 1). Intriguingly, the intermediate population was the one showing the 153

highest pigment levels, including those of chlorophylls (Fig. 1), carotenoids and 154

anthocyanins (Fig. 2). However, when antioxidants were expressed on a chlorophyll 155

basis, the intermediate population showed the highest carotenoid/chlorophyll ratio but 156

the lowest α-tocopherol/chlorophyll ratios (Supplemental Fig. S3), which was observed 157

together with the lowest chlorophyll a/b ratios (Fig. 1). Furthermore, the intermediate 158

population was the one showing the highest levels of all stress-related phytohormones, 159

including ABA, salicylic acid and jasmonic acid, while the lowest levels of the jasmonic 160

acid precursor, 12-oxo-phytodienoic acid (Fig. 3), thus confirming that the intermediate 161

population was the one experiencing the highest physiological stress in juvenile plants, 162

which is in agreement with the highest mortality rates observed (Table 1). 163

Levels of photoprotective molecules (α-tocopherol, carotenoids and 164

anthocyanins) increased significantly in response to high altitude (1100 relative to 570 165

m a.s.l., Fig. 2), which was paralleled by reduced leaf water contents (Fig. 1) and 166

increased ABA levels (Fig. 3). The more demanding effect of high altitude on 167

photoprotection was however abolished (except for α-tocopherol increases) at very high 168

altitudes (2100 m a.s.l.), these plants showing improved water contents (Fig. 1), and a 169

reduced need for photoprotection (driven by anthocyanins and carotenoids, Fig. 2) and 170

activation of chemical defenses (including the three aforementioned classes of stress-171

related phytohormones, Fig. 3). α-Tocopherol levels increased (Fig. 2), and lipid 172

hydroperoxide levels (an indicator of lipid peroxidation) decreased (Fig. 1) as a function 173

of altitude. Furthermore, Las Blancas was the population showing the lowest leaf mass 174

per area ratio (Fig. 1), carotenoid/chlorophyll ratio (Supplemental Fig. S3) and levels of 175

ABA and jasmonic acid (Fig. 3). 176

Size Influences Physiological Performance and Plant Death 177

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Logistic models showed that, for juveniles, the effect of individual plant size on 178

mortality was significantly important for the low and intermediate populations, where 179

smaller plants had a higher probability to die than larger ones (estimated β1 parameter: -180

0.12 and -0.05 respectively; p<0.001 in both cases; Fig. 4). Mortality rate in the highest 181

population was independent on plant size, with dead plants less concentrated in small 182

plants and more uniformly distributed across total size distribution (Fig. 4, 183

Supplemental Fig. S4). 184

Correlation analyses revealed that the relative leaf water content varied as a 185

function of plant size in the high population (r=0.528, P<0.001), but not in the 186

intermediate and low populations. The larger the plant, the higher the leaf water content 187

in Las Blancas (Table 2, Fig. 5), a correlation that was not significant at lower altitudes. 188

In should be noted, however, that the lowest leaf water contents were observed in San 189

Juan de la Peña (with values around 50% lower than in the other two populations), 190

although the large variability observed prevented the correlation to be significant (Fig. 191

5). Other, more moderate, correlations were observed between plant size and leaf mass 192

area ratio for the intermediate and high populations, and between plant size and the 193

chlorophyll a/b ratio for the low population (r=0.34-0.35, P<0.05, Table 2). 194

Plant Maturity and Death 195

As a monocarpic species, S. longifolia dies right after blooming. During flowering, 196

leaves serve as an important source of photoassimilates, but then the plant enters into a 197

programmed, senescing process leading to death. We were interested in evaluating 198

possible differences in plant physiological performance between juvenile and mature 199

plants, and particularly between mature plants growing at different altitudes. With this 200

purpose, we measured stress indicators in both juvenile and mature plants (at a 201

flowering stage) in the low and intermediate populations (no sufficient individuals could 202

be sampled for analyses in the high population due to extremely low reproductive 203

events during 2015). Plant maturity increased the leaf mass area ratio, and the levels of 204

antioxidants (carotenoids and α-tocopherol), ABA and jasmonic acid, while decreased 205

those of 12-oxo-phytodienoic acid in the two populations studied (Table 3), thus 206

indicating that plant maturity led to enhanced physiological stress. Furthermore, mature 207

plants of the intermediate population showed lower leaf water contents, higher α-208

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tocopherol levels and a lower extent of lipid peroxidation, as estimated by lipid 209

hydroperoxides, compared to mature plants from the low population (Table 3). 210

Clonal Growth, Reproduction and Death 211

Rosettes might split into two or many more smaller rosettes in a given year, in a kind of 212

clonal growth process but without the presence of rhizomes because the single 213

axonomorphic root keeps inside the crevice (see Supplemental Fig. S1B). The 214

frequency of multiple-rosette individuals ranged 7-11% across the 4-years of study in 215

Las Blancas, whereas it was as low as 2% in San Juan de la Peña, and absent in Pantano 216

de la Peña. Although clonal growth might happen at any plant size, it is more frequent 217

among large plants (average diameter of non-clonal and clonal plants: 48.2 mm and 218

83.4 mm, respectively; n=264 and 18 plants recorded in 2015, respectively). Survival 219

did not differ between individuals with or without clonal reproduction (Fisher's exact 220

test, P=0.141, n=1075), and none of the physiological parameters measured differed 221

between non-clonal and clonal plants, except for the relative leaf water content, which 222

was significantly lower in clonal plants (Table 4). 223

DISCUSSION 224

Our study of three populations of the long lived monocarpic Mediterranean S. longifolia 225

located at different altitudes in the Pyrenees, has revealed that despite the complexity of 226

mechanisms of adaptation to high altitude, which operate at the cellular, whole-plant 227

and population levels, this species may be vulnerable to drought stress events (periods 228

of low precipitation combined with high solar radiation and high temperatures) during 229

the summer in the framework of climate change. 230

Physiological Adaptation to Altitude: Photo- and Antioxidant Protection 231

Exposure to high solar radiation is known to induce photo-oxidative stress, particularly 232

when it is accompanied by other stress conditions, such as extreme temperatures, as it 233

occurs at high altitude (Streb et al., 1997, 2003a, 2003b; Pintó-Marijuan and Munné-234

Bosch, 2014). Furthermore, global change may increase the frequency of drought 235

events, which occur irregularly and may therefore affect plant populations from a given 236

species in a rather different way just depending on its specific location, leading to 237

increased photo-oxidative stress. In chloroplasts, production of reactive oxygen species 238

under excess light conditions is mainly mediated by the triplet excitation state of 239

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chlorophyll, which can lead to singlet oxygen formation, as well as by the 240

photoreduction of oxygen through photosynthetic electron transport in the Mehler 241

reaction, leading to the production of superoxide anions (Asada 2006). Plants have 242

developed a variety of protective systems, which allow them to control ROS levels, so 243

that oxidative damage can be prevented. Among them, carotenoids, acting as scavengers 244

of triplet chlorophyll and singlet oxygen, and mediating the harmless dissipation of 245

excess excitation energy as heat (Demmig-Adams and Adams 1993; Demmig-Adams et 246

al., 2013, 2014), anthocyanins, acting as a filter for high-level energy from the blue and 247

UV light region of the spectrum (Landi et al., 2015), and tocochromanols, both 248

quenching and scavenging singlet oxygen, and inhibiting the propagation of lipid 249

peroxidation (Havaux et al., 2005; Munné-Bosch, 2005; Triantaphylidès and Havaux, 250

2009; Falk and Munné-Bosch, 2010), play a key role. Results shown here for an 251

orophyte endemism of the western Mediterranean illustrates that despite the complex 252

mechanisms evolved by plants at the cellular level to survive high-mountain conditions, 253

drought stress is one of the main triggers of mortality in S. longifolia. The intermediate 254

population was the one showing the highest mortality rates, which paralleled with the 255

lowest soil and leaf water contents and the activation of defense responses (e.g. ABA 256

accumulation). Increased water availability in the high population (compared to the 257

other ones) most likely led to a reduced need for photoprotection, despite increased high 258

light exposure due to the altitudinal gradient. Interestingly, however, α-tocopherol 259

levels increased as a function of altitude, their biosynthesis being mostly governed by 260

high light exposure (Havaux et al., 2005). Such pattern was paralleled with reductions 261

in lipid hydroperoxides, thus indicating the protective role of vitamin E in preventing 262

the propagation of lipid peroxidation in the chloroplasts, which is in agreement with 263

previous studies on other high-mountain plants (Streb et al., 1997; 2003a, 2003b). 264

Unfortunately, chlorophyll fluorescence measurements could not be performed in intact 265

leaves from this species in the field due to the high reflectance of the epidermis, an 266

aspect that warrants further investigation. 267

Activation of chemical defenses, such as the biosynthesis of salicylates and 268

jasmonates, are known to be influenced by both biotic and abiotic stress factors (Davies, 269

2010). In the present study, both groups of compounds increased with altitude 270

(comparing the intermediate and low populations), but its accumulation was abolished 271

at the highest altitude, most likely due, at least in part, to improved soil and leaf water 272

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contents (compared to the other two populations). Other factors may however also 273

influence the accumulation of salicylates and jasmonates. In the high population, 274

salicylic acid accumulation was similar to that of the low population, and that of 275

jasmonic acid was even smaller, thus indicating a reduced need for chemical defense 276

against necrotrophs at 2100 m a.s.l. (Davies, 2010). It is also likely that reduced 277

jasmonic acid levels result from a trade-off between activation of different defense 278

pathways in plants (photoprotection versus potential chemical defense to necrotrophs 279

through jasmonates), so that enhanced vitamin E accumulation at the highest altitude 280

may negatively influence the biosynthesis of jasmonates, which is in agreement with 281

previous studies (Demmig-Adams et al., 2013, 2014; Morales et al., 2015; Simancas 282

and Munné-Bosch, 2015). Enhanced jasmonic acid accumulation in the intermediate 283

population may reflect activation of acclimation responses, but also increased cell death, 284

as shown in other studies (Shumbe et al., 2016). Enhanced jasmonic acid levels may be 285

triggered by increased biotic stress, but also by abiotic factors, such as drought stress 286

(Brossa et al., 2011; de Ollas et al., 2013). Thus, it is very likely that enhanced ABA, 287

salicylic acid and jasmonic acid levels, all respond to an increased drought stress that 288

activates acclimation responses, but that ultimately lead to increased cell death and 289

mortality in the intermediate population. Results suggest that enhanced physiological 290

stress and mortality in the intermediate population was caused by increased drought 291

stress during 2015. If more drought events occur in the other two populations, which are 292

indeed likely to increase in the frame of global change (IPCC, 2014), it is expected they 293

will also result in an increased mortality. More frequent snowfalls leads however to an 294

increased water availability in the highest population. It may therefore be anticipated 295

that, as precipitation patterns suggest (Supplemental Fig. S2), the populations found at 296

the two lowest altitudes will be the ones showing the highest sensitivity to drought 297

stress-induced mortality. 298

Adaptation at the Population Level: Plant Size, Clonal Growth and Population 299

Size Structure 300

The population at highest altitude showed some demographic differences compared to 301

the other two sampled populations: lower frequency of small individuals, size-302

independent mortality rate, the largest plants of all recorded across populations, and a 303

particular trait that was almost absent in the other two: clonal growth (Supplemental 304

Fig. S1B). The lower frequency of small-sized plants in the high population is the 305

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consequence of lower recruitment (very few new individuals enter each year in the 306

monitoring plots; M.B. García, unpublished). Interestingly, small rosettes at high 307

altitude survive better than at lower altitudes (Supplemental Fig. S4), and mortality was 308

more uniformly distributed with size in the high population compared to the other two 309

populations. Higher survival in the highest population may be associated with improved 310

soil and leaf water contents, despite being exposed to higher light intensity. 311

Furthermore, plants from this species seem to escape from increased size-dependent 312

mortality, as it has been shown in other plant species, mostly woody perennials (shrubs 313

and trees), in which increased plant size make larger individuals more vulnerable to 314

environmental constraints (Mencuccini et al., 2005, 2007; Baudisch et al., 2013; 315

Salguero et al., 2013; Munné-Bosch, 2014, 2015). This was not observed here in either 316

studied population. Small sizes throughout their lifespan, as it happens in perennial 317

herbs (García et al., 2011; Morales et al., 2013; Morales and Munné-Bosch, 2015), may 318

protect S. longifolia plants from the potential negative effects of aging. It appears, 319

therefore, that whole-plant senescence in this species may be attributed to reproduction 320

and extrinsic factors, such as drought stress, but not to ageing, as only very small 321

individuals (<30 mm diameter) die more frequently in the two lowest populations. 322

Interestingly, those populations are the more exposed ones to summer drought. It may 323

be speculated that an increase in temperatures and drought events in the framework of 324

global change may be a serious threat for this species. Furthermore, population 325

recruitment is lowest at the highest population, and reduced recruitment would translate 326

into a negative population dynamics, a real limitation for adaptation in the framework of 327

global change. 328

The fact that plants get larger in the high population could be related to the 329

existence of clonal growth, another interesting mechanism observed mainly at the 330

highest altitude. Newly formed rosettes never become fully independent because they 331

all share the same root system, but they can behave independent in the sense that not all 332

daughter rosettes die or reproduce at the same time (see Supplemental Fig. S1B). 333

Considering that this is a monocarpic plant, forming new rosettes might help the plant to 334

reduce mortality as a consequence of reproduction, and extend the fecundity period like 335

a polycarpic organism, spreading fitness over time. Flowering plants with one single 336

rosette inevitably die the same year of reproduction, whereas 31% of multiple-rosette 337

individuals survived. Therefore, having more than one rosette allows the plant to decide 338

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which ones to "sacrifice", which translates into survival of the individual if not all 339

rosettes synchronously flower in a given year. Clonal growth, thus, constitutes an 340

additional process operating at the individual level in terms of enhancing survival, 341

which could also help to explain the lower mortality rate of this population compared to 342

the other ones. The particular habitat or environment has been shown to be an important 343

selective factor in predicting the evolutionary stable reproductive strategy in natural 344

populations of monocarpic plants (Hesse et al., 2008), and S. longifolia constitutes a 345

clear example of reproductive strategy variability along an altitudinal gradient. 346

It is concluded that, despite the endemic Mediterranean plant, S. longifolia has 347

evolved complex mechanisms of adaptation to altitude (including e.g. enhanced α-348

tocopherol levels or changes in reproductive strategy like activation of clonal growth), 349

this species is rather sensitive to drought stress, and consequently drought events may 350

drive increased mortality in populations from this species in the framework of global 351

change. Further research is however needed to better understand the mechanisms 352

underlying the influence of altitude and drought on size-dependent mortality, how they 353

interact, and how this will in turn be affected by global warming in this and other 354

endemic plants in the near future. 355

MATERIALS AND METHODS 356

Plant Populations, Treatments and Sampling 357

The study was carried out in three natural populations of Saxifraga longifolia Lapeyruse 358

located in central Pyrenees, the area of highest abundance within its distribution range. 359

The three populations were located across an altitudinal range spanning 50 km in 360

straight line. The first population was located in rocky walls of limestone near Pantano 361

de la Peña (570 m a.s.l., coordinates: 42°22'58.4"N 0°44'02.1"W), the second one 362

occurred on a very sloppy conglomerated area near San Juan de la Peña (1100 m a.s.l., 363

42°30'30.6"N 0°40'21.3"W), and the third one in the uppermost needles of calcareous 364

mountain named Las Blancas (2100 m a.s.l., 42°44'49.3"N 0°33'26.4"W). This long-365

lived monocarpic perennial plant develops a basal rosette growing in the crevices of 366

limestone rocky places, mainly on cliffs, offering a unique sight in years of intensive 367

blooming. 368

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Samplings in at least 70 individuals randomly selected within each population 369

among plants larger than 30 mm of diameter were performed for biochemical analyses 370

in 2015, including both juvenile and reproductive plants, except in Las Blancas, where 371

too few individuals flowered during 2015. Sixty-five, 64 and 83 juveniles were sampled 372

in Pantano de la Peña, San Juan de la Peña and Las Blancas, respectively, and 373

additionally 8 and 10 mature individuals were sampled in the two former populations. 374

Samplings were performed on fully expanded leaves at midday (12 a.m. solar time) on 375

22 June, 3 July and 18 August in Pantano de la Peña, San Juan de la Peña and Las 376

Blancas, respectively, just after flowering in mature plants, so that all plants were at the 377

same phenological stage. Rosette leaves were used to estimate leaf water contents, 378

pigment concentrations (including chlorophylls, carotenoids and anthocyanins), levels 379

of vitamin E, the extent of lipid peroxidation, as well as the endogenous concentrations 380

of stress-related phytohormones, including ABA, salicylates and jasmonates. Samples 381

for biochemical analyses were collected, immediately frozen in liquid nitrogen in situ, 382

and stored at -80°C upon arrival to the laboratory. 383

Mortality 384

Between two and three hundred plants per location were marked and annually 385

monitored from 2011 through 2015 to estimate survival rates with the aid of grid plots. 386

In summer each year, all numbered plants were checked, and if alive their diameter 387

were recorded. Annual flowering and mortality rates were compared after pooling all 388

the events recorded along pairs of consecutive years (2011/2012, 2012/2013, 2013/2014 389

and 2014/2015). Furthermore, in order to explore possible reasons and consequences of 390

clonal reproduction in the highest population, we tested if juvenile and mature plants 391

with multiple rosettes had a different survival probability than singled-rosette ones. 392

Leaf Water Contents, Pigment Levels, and Lipid Peroxidation 393

To estimate leaf water contents, samples were collected, kept humid in small bags in 394

darkness during transport to the laboratory, and then weighed to estimate fresh matter 395

(FW). They were immersed in distilled water at 4°C for 24h to estimate the turgid 396

matter (TW), and then oven-dried at 80°C to constant weight to estimate the dry matter 397

(DW). Relative water content (RWC) was then calculated as 100 x (FW-DW)/(TW-398

DW). 399

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For pigment analysis, measurements were performed spectrophotometrically on 400

methanolic extracts to estimate chlorophyll and carotenoid levels, as described by 401

Lichtenthaler (1987), which were then acidified with 30% HCl to estimate total 402

anthocyanin levels as described by Gitelson et al. (2001). The extent of lipid 403

peroxidation was estimated by measuring the levels of lipid hydroperoxides in leaves. 404

Lipid hydroperoxide levels were estimated spectrophotometrically following a modified 405

ferrous oxidation-xylenol orange (FOX) assay, as described (DeLong et al., 2002). 406

Tocochromanols 407

For analyses of tocochromanol (tocopherols and plastochromanol-8) contents, leaf 408

samples were ground in liquid nitrogen and extracted with cold methanol containing 409

0.01% butylated hydroxyltoluene using ultrasonication. After centrifuging at 12000 rpm 410

for 10 min and 4°C, the supernatant was collected and the pellet re-extracted with the 411

same solvent until it was colorless; then, supernatants were pooled, filtered and injected 412

into the HPLC. Tocopherols and tocotrienols were separated isocratically on a normal-413

phase HPLC system using a fluorescent detector as described (Cela et al., 2011). 414

Compounds were identified by co-elution with authentic standards and quantified by 415

using a calibration curve. From all tocochromanols investigated (α-, β-, γ- and δ-416

tocopherols and tocotrienols, and plastochromanol-8), α-tocopherol was the only 417

compound present at quantifiable amounts in leaves. 418

Stress-related Phytohormones 419

For analyses of ABA, salicylic acid and jasmonates, leaf samples were ground in liquid 420

nitrogen and extracted with cold methanol using ultrasonication. After centrifuging at 421

12000 rpm for 10 min and 4°C, the supernatant was collected and the pellet re-extracted 422

with the same solvent until it was colorless; then, supernatants were pooled, filtered and 423

injected into the UHPLC-MS/MS. Phytohormones were separated using an elution 424

gradient on a reverse-phase UHPLC system and quantified using tandem mass 425

spectrometry in multiple reaction monitoring mode as described (Müller and Munné-426

Bosch, 2011). Recovery rates were calculated for each hormone on every sample by 427

using deuterated compounds. 428

Statistical Analysis 429

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To determine the effect of altitude, mean values were tested by one-way factorial 430

analysis of variance (ANOVA) and additionally Bonferroni posthoc tests. Mean values 431

were compared between clonal and non-clonal plants by means of Student's t-test. In all 432

cases, differences were considered significant at a probability level of P<0.05. 433

Spearman rank's correlation analyses were performed between plant size (estimated as 434

rosette diameter) and all biochemical parameters, and Bonferroni correction applied to 435

determine significant differences. Statistical tests were carried out using the SPSS 20.0 436

statistical package. The effect of individual size on mortality probability was explored 437

by fitting logistic regression models (logit link function, binomial distribution, in R 438

version 2.15.2, Core Team) for all the annual transitions recorded over that period, for 439

each population separately. 440

441

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ACKNOWLEDGEMENTS 442

We are very grateful to Maren Müller and Serveis Científico-tècnics for technical help 443

with biochemical analyses. We are also indebted to M. Paz Errea and Ricardo García 444

González for providing environmental data, and the fieldwork assistance of Juanlu, Iker, 445

P. Sánchez and P. Bravo. 446

AUTHOR CONTRIBUTIONS 447

S.M.-B., A.C., M.M. and M.B.G. conceived the research plan. A.C., M.M., E.F.S., J.V. 448

and M.B.G. performed the experiments. S.M.-B. wrote the article with the help of 449

M.B.G. 450

451

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Table 1. Mortality rates (numbers and percentage) during the last 4 years in the long-452

lived monocarpic plant, S. longifolia. Monitoring of individual plants was carried out 453

between June and August of the given periods (indicated in parentheses). Mortality rates 454

refer to juveniles (Juv) and flowering (R). 455

456

457

458

459

Ju v R Juv R Ju v R N (2 0 1 1 -2 0 1 2 ) Dead 6 2 14 12 5 0 % mo rtality 3.1 1.0 6.9 5.9 1.8 0.0

N (2 0 1 2 -2 0 1 3 ) Dead 33 0 22 0 13 3 % mo rtality 14.3 0.0 10.7 0.0 4.6 1.1

N (2 0 1 3 -2 0 1 4 ) Dead 11 1 30 0 13 42 % mo rtality 5.2 0.5 14.9 0.0 4.7 15.2

N (2 0 1 4 -2 0 1 5 ) Dead 10 12 27 10 18 2 % mo rtality 4.9 5.9 13.6 5.1 8.3 0.9

A v e r a g e (2 0 1 1 -2 0 1 5 ) 6.9 11.5 4.8

Pa n t a n o d e la Peña San Juan de la Peña L a s Bl a n ca s

281

285

276

217

194

230

21 1

204

204

205

201

198

1.8 2.8 4.3

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Table 2. Spearman rank's correlation analyses between plant size (estimated as rosette 460

diameter) and all measured parameters in the long-lived monocarpic plant, S. longifolia. 461

All data from juvenile plants, including the three populations, was pooled for analyses, 462

but also analyzed separately. rho and P values are indicated in bold when correlations 463

were significant (Bonferroni adjusted, P<0.0033). RWC, relative water content; LMA, 464

leaf mass per area ratio; Chl, chlorophyll. LOOH, lipid hydroperoxides; Ant, 465

anthocyanins; Car, carotenoids; α-Toc, α-tocopherol; ABA, abscisic acid; SA, salicylic 466

acid; OPDA, oxo-phytodienoic acid; JA, jasmonic acid. 467

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) α-Toc -0.147 (0.017) -0.331 (0.004) -0.224 (0.039) 0.091 (0.207)

α-Toc/Chl -0.046 (0.253) 0.013 (0.459) -0.057 (0.327) -0.089 (0.213) 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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Table 3. Influence of maturity for all measured parameters in the long-lived monocarpic 469

plant, S. longifolia. Data correspond to the mean ± SE. n=64 juvenile and n=8 mature 470

plants in Pantano de la Peña, and n=64 juvenile and n=10 mature plants in San Juan de 471

la Peña. An asterisk indicates differences between juvenile and mature plants (Student's 472

t-test, P<0.05). Different letters indicate differences between populations, either in 473

juvenile or mature plants, the latter indicated in capital letters(Student's t-test, P<0.05). 474

No sufficient plants in a mature stage were found in Las Blancas, so data are not 475

available for the population at the highest altitude. RWC, relative water content; LMA, 476

leaf mass per area ratio; Chl, chlorophyll. LOOH, lipid hydroperoxides; Ant, 477

anthocyanins; Car, carotenoids; α-Toc, α-tocopherol; ABA, abscisic acid; SA, salicylic 478

acid; OPDA, oxo-phytodienoic acid; JA, jasmonic acid. 479

480

481

Pantano de la Peña San Juan de la Peña Juvenile Mature Juvenile Mature Diameter (mm) 72.1 ± 3.2a 96.3 ± 20.0A 71.8 ± 3.9a 116.0 ± 14.6*A RWC (%) 92.4 ± 0.8a 93.0 ± 2.5A 78.9 ± 1.5b 65.0 ± 5.6*B LMA (g/m2) 1561 ± 47a 725 ± 79*A 1611 ± 51a 780 ± 66*A Chl a+b (μmol/g DW) 1.33 ± 0.08a 1.39 ± 0.25A 1.70 ± 0.07b 1.61 ± 0.22A Chl a/b 2.26 ± 0.02a 1.88 ± 0.04*A 2.01 ± 0.02b 1.79 ± 0.07*A LOOH (μmol/g DW) 6.94 ± 0.49a 11.86 ± 2.84A 5.84 ± 0.71a 5.33 ± 1.23B Ant (μmol/g DW) 0.61 ± 0.03a 0.60 ± 0.11A 0.71 ± 0.04b 0.78 ± 0.12A Ant /Chl 0.49 ± 0.03a 0.45 ± 0.04A 0.42 ± 0.01b 0.52 ± 0.10A Car (μmol/g DW) 0.29 ± 0.02a 0.43 ± 0.07*A 0.44 ± 0.02b 0.56 ± 0.06*ACar/Chl 0.23 ± 0.01a 0.32 ± 0.02*A 0.26 ± 0.01b 0.37 ± 0.03*Aα-Toc (μmol/g DW) 0.28 ± 0.01a 0.39 ± 0.05*A 0.32 ± 0.01b 0.56 ± 0.03*B α-Toc /Chl 0.26 ± 0.02a 0.34 ± 0.06A 0.20 ± 0.01b 0.40 ± 0.05*A ABA (ng/g DW) 424.5 ± 21.3a 937.3 ± 153.5*A 598.3 ± 38.7b 848.8 ± 63.9*A SA (ng/g DW) 375.9 ± 12.0a 674.4 ± 89.9*A 472.6 ± 24.6b 568.4 ± 58.3AOPDA (ng/g DW) 2788 ± 225a 440 ± 103*A 1289 ± 158b 256 ± 38*A JA (ng/g DW) 187.5 ± 7.9a 379.3 ± 74.6*A 216.2 ± 14.4b 550.1 ± 273.7A

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Table 4. Influence of clonal growth for all measured parameters in the highest 482

population (Las Blancas) of the long-lived monocarpic plant, S. longifolia. Data 483

correspond to the mean ± SE of n=74 non-clonal and n=9 clonal plants. An asterisk 484

indicates differences between juvenile and mature plants (Student's t-test, P<0.05). 485

RWC, relative water content; LMA, leaf mass per area ratio; Chl, chlorophyll. LOOH, 486

lipid hydroperoxides; Ant, anthocyanins; Car, carotenoids; α-Toc, α-tocopherol; ABA, 487

abscisic acid; SA, salicylic acid; OPDA, oxo-phytodienoic acid; JA, jasmonic acid. 488

489

Non-Clonal Clonal

Diameter (mm) 67.63 ± 2.72 66.71 ± 7.75

RWC (%) 84.01 ± 1.04 75.74 ± 1.81*

LMA(g/m2) 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

OPDA(ng/g DW) 2428.69 ± 216.78 1609.05 ± 357.17

JA(ng/g DW) 119.57 ± 5.02 100.95 ± 15.27

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FIGURE LEGENDS 490

Figure 1. Plant size (estimated as rosette diameter), relative leaf water content (RWC), 491

leaf mass per area ratio (LMA), chlorophyll (Chl) a+b, Chl a/b ratio and lipid 492

hydroperoxide levels (an estimation of lipid peroxidation) in plants of the long-lived 493

monocarpic plant, S. longifolia growing at three altitudes (570, 1100 and 2100 m a.s.l. 494

in Pantano de la Peña, San Juan de la Peña and Las Blancas, respectively). Data shows 495

the mean ± SE of n=65, 64 and 83 juvenile individuals for the three populations at 496

increasing altitude, respectively. Results of one-way ANOVA are shown in the inlets. 497

Different letters indicate significant differences between populations (P<0.05) using 498

Bonferroni posthoc tests. NS, not significant. 499

Figure 2. Antioxidant protection, including levels of anthocyanins, carotenoids and α-500

tocopherol, in plants of the long-lived monocarpic plant, S. longifolia growing at three 501

altitudes (570, 1100 and 2100 m a.s.l. in Pantano de la Peña, San Juan de la Peña and 502

Las Blancas, respectively). Data show the mean ± SE of n=65, 64 and 83 juvenile 503

individuals for the three populations. Results of one-way ANOVA are shown in the 504

inlets. Different letters indicate significant differences between populations (P<0.05) 505

using Bonferroni posthoc tests. 506

Figure 3. Endogenous concentrations of stress-related phytohormones, including 507

abscisic acid (ABA), salicylic acid (SA), oxo-phytodienoic acid (OPDA) and jasmonic 508

acid (JA) in plants of the long-lived monocarpic plant, S. longifolia growing at three 509

altitudes (570, 1100 and 2100 m a.s.l. in Pantano de la Peña, San Juan de la Peña and 510

Las Blancas, respectively). Data shows the mean ± SE of n=65, 64 and 83 juvenile 511

individuals for the three populations at increasing altitude, respectively. Results of one-512

way ANOVA are shown in the inlets. Different letters indicate significant differences 513

between populations (P<0.05) using Bonferroni posthoc tests. 514

Figure 4. Logistic mortality regression models for the three populations studied. The 515

“x” axis corresponds to the diameter (measured in mm) of plants in year “t”, and the “y” 516

axis to the recorded fate in year “t+1” (0=alive, 1=dead). Dots show individual yearly 517

events (dead or alive) from 2011 till 2015. A total of 824, 786 and 1012 events are 518

plotted in the low, intermediate and high population respectively. All dots should fit the 519

“0” or “1” values, but were not forced to lie on a line for illustrative purposes. 520

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Figure 5. Spearman rank's correlation analyses between plant size of juvenile plants 521

(estimated as rosette diameter) and the relative water content (RWC) in three 522

populations of the long-lived monocarpic plant, S. longifolia. rho (r) and P values are 523

indicated in the inlets (correlation was significant in the population at the highest 524

altitude only, Las Blancas, P<0.0033, Bonferroni adjusted). 525

526

527

528

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SUPPLEMENTAL DATA 529

Supplemental Figure S1. Saxifraga longifolia population structure in 2011, according to rosette diameter 530 (seedlings excluded). 531

Supplemental Figure S2. Monthly average temperatures, soil water contents and precipitation recorded 532 in the three studied populations. 533

Supplemental Figure S3. Levels of anthocyanins, carotenoids and α-tocopherol, expressed per 534 chlorophyll (Chl) unit, in plants of the long-lived monocarpic plant, S. longifolia growing at three 535 altitudes (570, 1100 and 2100 m.a.s.l. in Pantano de la Peña, San Juan de la Peña and Las Blancas, 536 respectively). 537

Supplemental Figure S4. Mortality rate for plants of different sizes. 538

539

540

Supplemental Figure S1. (A) Saxifraga longifolia population structure in 2011, 541

according to rosette diameter (seedlings excluded). (B) The left sided plant is an 542

example of clonal growth in a plant from Las Blancas (multiple rosette individual), 543

whereas the right sided plant shows a typical, single rosette individual. 544

Supplemental Figure S2. (A) Monthly average temperatures recorded in the three 545

studied populations. In the lowest and highest ones (Pantano de la Peña and Las Blancas 546

respectively), temperature was recorded by tinny thermometers (Maxim’s ibutton 547

devices) placed inside populations, and the average monthly values recorded over 3 548

years (2012-2015) is shown. For the intermediate population (San Juan de la Peña), we 549

averaged monthly temperatures from the closest meteorological station, located 5 km 550

away. (B) Soil water contents, daily solar radiation and maximum diurnal air 551

temperatures during the days of measurements (22 June, 3 July and 18 August from the 552

highest to the lowest population, respectively). (C) Monthly average precipitation in the 553

three sites of study (precipitation during months of measurements for each population is 554

indicated in red). 555

Supplemental Figure S3. Levels of anthocyanins, carotenoids and α-tocopherol, 556

expressed per chlorophyll (Chl) unit, in plants of the long-lived monocarpic plant, S. 557

longifolia growing at three altitudes (570, 1100 and 2100 m.a.s.l. in Pantano de la Peña, 558

San Juan de la Peña and Las Blancas, respectively). Data show the mean ± SE of n=65, 559

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64 and 83 juvenile individuals for the three populations at increasing altitude, 560

respectively. Results of one-way ANOVA are shown in the inlets. Different letters 561

indicate significant differences between populations (P<0.05) using Duncan posthoc 562

tests. NS, not significant. 563

Supplemental Figure S4. Mortality rate for plants of different sizes. Small: x<30 mm, 564

Medium: 60>x>30, Large: x≥60 mm. Populations: PP: Pantano de la Peña, SJP: San 565

Juan de la Peña, LB: Las Blancas. 566

567

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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.

Dia

me

ter

(mm

)

0

50

60

70

80

NS

RW

C (

%)

70

80

90

LM

A (

gD

W·m

-2)

1300

1400

1500

1600

1700

a

b

c

a

b

b

Ch

l a

+b

(

mo

l·g

DW

-1)

0,0

1,0

1,2

1,4

1,6

1,8

a

a

b

Ch

l a

/b

0,0

1,6

1,8

2,0

2,2

2,4

LO

OH

(

mo

l e

q H

2O

2·g

DW

-1)

0

4

5

6

7

Pantano de

la Peña

San Juan de

la Peña

Las Blancas

Location

a

b

c

a

ab

b

P<0.001

P<0.001 P<0.001

P=0.006 P=0.048

Pantano de

la Peña

San Juan de

la Peña

Las Blancas

Location

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Figure 2. Antioxidant protection, including levels of anthocyanins, carotenoids and a-

tocopherol, 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.

An

tho

cya

nin

s (

mo

l·g

DW

-1)

0,0

0,4

0,5

0,6

0,7

0,8

Pantano de

la Peña

San Juan de

la Peña

Las Blancas

Location

Ca

rote

no

ids (

mo

l·g

DW

-1)

0,0

0,2

0,3

0,4

a-T

oco

ph

ero

l (

mo

l·g

DW

-1)

0,00

0,20

0,25

0,30

0,35

P<0.001

a

b

a

P=0.002

a

b

b

P<0.001

a a

b

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Page 33: Adaptation of the Long-Lived Monocarpic Perennial ...€¦ · 27 capacity for adaptation. Here, we aimed at unveiling mechanisms of adaptation to high 28 altitude in an endemic long-lived

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.

AB

A (

ng

·gD

W-1

)

0

300

400

500

600

700

SA

(n

g·g

DW

-1)

0

300

400

500

JA

(n

g·g

DW

-1)

0

100

150

200

Pantano de

la Peña

San Juan de

la Peña

Las Blancas

Location

P<0.001

a

b

c

P<0.001

a a

b

P<0.001

a

b

c

OP

DA

(n

g·g

DW

1)

0

1000

1500

2000

2500

3000

a

P<0.001b

b

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0

50

10

0

15

0

20

0

25

0

0

1

Pantano de la Peña

Size

Fa

te

0

50

10

0

15

0

20

0

25

0

0

1

San Juan de la Peña

Size

0

50

10

0

15

0

20

0

25

0

0

1

Las Blancas

Size

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.

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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).

Diameter (mm)

20 40 60 80 100 120 140 160

RW

C (

%)

0

75

80

85

90

95

100

Pantano de la Peña

Diameter (mm)

0 50 100 150 200 250

RW

C (

%)

0

40

50

60

70

80

90

100

San Juan de la Peña

Diameter (mm)

20 40 60 80 100 120 140 160

RW

C (

%)

0

60

70

80

90

100

Las Blancas

r = 0.293

p = 0.009

r = 0.256

p = 0.021 r = 0.528

p < 0.001

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