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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
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Short title: Adaptation to Altitude in a Monocarpic Perennial 12
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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
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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
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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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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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