Article
Does the Age of Pinus sylvestris Mother Trees Influence
Reproductive Capacity and Offspring Seedling Survival?
Marta Pardos 1, * , Javier Vázquez-Piqué 2 , Luis Benito 1 , Guillermo Madrigal 1 , Reyes Alejano 2 ,
Manuel Fernández 2 and Rafael Calama 1
1
2
*
Citation: Pardos, M.; Vázquez-Piqué,
J.; Benito, L.; Madrigal, G.; Alejano,
Forest Dynamics and Management Department, CIFOR (INIA-CSIC), Crtra Coruña Km 7.5,
28040 Madrid, Spain; lusfbm@gmail.com (L.B.); madrigal@inia.csic.es (G.M.); rcalama@inia.csic.es (R.C.)
Agroforestry Science Department, Escuela Técnica Superior de Ingeniería, Universidad de Huelva,
Avda. Fuerzas Armadas s/n, 21007 Huelva, Spain; jpique@uhu.es (J.V.-P.); reyes.alejano@dcaf.uhu.es (R.A.);
nonoe@dcaf.uhu.es (M.F.)
Correspondence: pardos@inia.csic.es
Abstract: We assess how the age of Pinus sylvestris mother trees influences seed size, seed viability,
germination capacity and later offspring seedling survival under greenhouse conditions. Thirty trees
ranging from 30 to 219 years old were selected in the north facing slopes in the Sierra de Guadarrama,
where we could find the oldest Pinus sylvestris trees in central Spain. Forty cones per tree were
harvested to study cone and seed characteristics (size and weight), seed viability and germination
capacity related to the mother tree age. In addition, 25 germinated seeds per tree were grown in a
greenhouse to assess offspring seedling survival during a death trial, where watering was stopped.
Significant differences between trees in cone and seed morphological traits were observed. The age
of the mother tree had a significant effect on cone size, seed size, and seed weight, but there was
no effect on seed germination capacity and seed viability. Seedling survival was mainly affected
by the decrease in water availability. However, a significant effect of the tree age was found once
soil moisture had reached 0%. Our results show the ability of overmature Pinus sylvestris trees to
maintain a relatively high reproductive capacity that assures its persistence.
R.; Fernández, M.; Calama, R. Does
the Age of Pinus sylvestris Mother
Trees Influence Reproductive
Keywords: old-growth forests; seed size; seed viability; germination capacity; death trial; chlorophyll
fluorescence; Scots pine
Capacity and Offspring Seedling
Survival? Forests 2022, 13, 937.
https://doi.org/10.3390/f13060937
Academic Editor: Božena Šerá
Received: 30 May 2022
Accepted: 13 June 2022
Published: 15 June 2022
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Attribution (CC BY) license (https://
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4.0/).
1. Introduction
The large history of management and use of forests has shaped its landscape and
structure over time [1]. The consequence of such prolonged management is the difficulty of
finding old-growth forests but also even knowing their level of maturity [2]. In addition to
this, we must consider the longevity of some tree species, such as Pinus, whose lifespans
are over 4000 years (e.g., Pinus longaeva), which makes it difficult to elucidate whether
senescence has been reached [3].
We must not forget that the last stages of senescence are also part of forest dynamics.
Forest dynamics analyzes the changes over time in the structure, composition, functioning
and biodiversity of a stand. It includes a cycle with four differentiated stages, which are
“Stand Initiation”, “Stem Exclusion”, “Understorey Reinitiation” and “Old Growth” [4].
Research on forest management usually focuses on the two intermediate stages of forest
dynamics (i.e., “Stem Exclusion” and “Understorey Reinitiation”). However, under the new
paradigms of multifunctionality and close-to-nature management, and considering global
change scenarios, it is interesting to study old-growth forests, as well as the decay and
mortality-related processes that can condition their functioning and reproductive capacity.
In this sense, National Parks are privileged scenarios to study these processes. After the
declaration of a National Park, all intensive forest exploitation ceases; thus, forests evolve
close to their natural dynamics [5].
Forests 2022, 13, 937. https://doi.org/10.3390/f13060937
https://www.mdpi.com/journal/forests
Forests 2022, 13, 937
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The role of old-growth forests in forest dynamics is a well-known process in some
aspects. For example, old-growth forests are characterized by harboring high biodiversity [6], providing multiple ecosystem services [7], while increasing stand resilience after
extreme climate events. In addition, old-growth forests store carbon in the long term [8].
Although traditionally it has been thought that carbon storage is interrupted as trees get
older, different studies have demonstrated that old-growth forests continue storing carbon
for centuries and in great amounts. In fact, in temperate and boreal forests in the Northern
Hemisphere, old-growth forests provide as much as 10% of the ecosystem’s global net
productivity [9]. However, there are other old-growth forest aspects that are not so clear,
for example, their ability to maintain their reproductive capacity and to produce abundant
and viable seeds once they reach senescence [10].
Natural regeneration is a main phase of forest dynamics and the only way to guarantee
forest persistence in the long term [11]. The successful regeneration of a forest conforms
a temporal succession of phases that should be effectively realized, including: seed production, seed dispersal, seed predation, germination, emergence, seedling survival and
seedling initial growth [12]. Regeneration could be seen as a multistage process based in
this approach, with underlying consecutive subprocesses that can be identified as successive survival thresholds for potential seedlings [13,14]. Thus, seed availability and viability
are key traits necessary for assuring the persistence of old-growth forests. In this sense,
there is evidence that, when referring to seed production, tree senescence does not decrease
fecundity [15,16]. However, there is not a systematic study on the effect of senescence on
progeny viability. For instance, there could be an age-related mother tree effect that reduces
some critical stages in the regeneration process, such as seed germination and first-year
seedling viability. In this way, senescence will be related to effective reproduction, which is
determinant in the biological efficacy of plants and, thus, in the future forest structure and
diversity [5]. Results from the few studies available for Pinus species about the relationship
of seed viability and germination capacity with mother tree age are unclear, being strongly
dependent on the species and the senescence stage. For example, no significant mother
tree age-related differences in seed weight or seed germinability were found for Pinus
logaeva [17]. Meanwhile, Tíscar-Oliver [18] found that the oldest Pinus nigra trees showed a
higher number of empty seeds, with lighter seeds that showed lower germination vigor.
Nonetheless, old trees were able to maintain a high germination rate. Moreover, also for
Pinus nigra, Alejano et al. [10] found significant inter-individual variability in different coneand seed-related traits, but no mother tree age effect was identified.
Seedling performance once seeds have germinated is also an interesting trait of study.
In fact, seed mass, emergence rate and seedling growth rate have been suggested to play
important roles in Pinus sylvestris early fitness in dry Mediterranean regions [19]. The
expected negative influence of climate change on Sub-Mediterranean mountain Pinus
sylvestris forests [20] makes it necessary to investigate the effect of drought on Pinus
sylvestris seedling survival and how this effect can be mediated by the senescence stage of
trees. Although Pinus sylvestris is considered a xerophytic species, the limited plasticity of
its xylem and leaf properties may be behind its high vulnerability to the decline in water
availability [21].
This study is aimed to assess how the mother tree age in Pinus sylvestris influences seed
and cone biometric attributes, seed viability and germination capacity, as well as seedling
performance (in terms of physiology and survival) in a death trial under nursery conditions.
For this purpose, we focused on Pinus sylvestris old-growth forests in Sierra de Guadarrama
National Park (Central Mountain Range of Spain). Specifically, we hypothesized that: (1)
overmature trees maintain their germination capacity and seed viability with age; (2) the
inter-individual variability for the studied traits can be greater than the effect of the mother
tree age; (3) seed size plays a main role in the reproductive success, thus conditioning the
results; (4) seedling performance in terms of survival will be more affected by the water
availability level than by the mother tree age.
Forests 2022, 13, 937
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2. Materials and Methods
The study area is located on the north facing slopes of Valsaín Forest (40◦ 49′ N, 4◦ 1′ W),
which is home to the oldest Pinus sylvestris in the Sierra de Guadarrama (Central Mountain
Range of Spain), at a mean elevation of 1835 m. The climate is Sub-Mediterranean, the
mean annual temperature is 8.5 ◦ C, the mean monthly temperatures range between 1.2 ◦ C
(January) and 18.3 ◦ C (July) and the average annual precipitation is 1275 mm. The major
soil types are moderately deep dystric cambisols and ferric luvisols that have developed
over acidic bedrock. The prevailing soil texture type is sandy loam. Ninety percent of the
forested area is occupied by pure, even-aged Pinus sylvestris forests, with some scattered
alpine shrubs in the upper parts.
A sample of 30 trees was chosen over an area of 10 ha, representing all age classes
between 30 and 219 years old (See Table 1 for the description of the tree’s dasometric characteristics.). Individual tree age was determined following standard dendrochronological
procedures [22].
Table 1. Dasometric Characteristics of the Selected Trees.
Tree No.
Tree Age
(yrs)
Diameter
(cm)
Height (m)
Tree No.
Tree Age
(yrs)
Diameter
(cm)
Height (m)
801
802
803
804
805
806
807
808
809
810
811
812
813
814
816
154
48
137
50
181
57
215
46
216
30
186
62
180
64
62
62.0
34.1
29.0
29.2
64.5
29.2
68.0
40.1
64.3
23.7
58.6
13.1
66.0
28.2
35.6
12.9
12.1
14.4
13.7
11.1
13.7
14.7
14.3
14.6
15
15.1
7.9
13.8
12.5
11.3
817
818
819
820
821
822
823
824
825
826
827
828
829
830
832
204
108
205
89
199
117
204
185
194
71
219
203
195
112
121
82.0
37.0
62.0
37.0
76.2
40.0
76.0
38.5
72.5
31.0
57
25
56.5
29.5
41.5
14.2
13.1
15.5
12.1
15
104
14.3
13.6
13.8
13.7
10
10.4
15.6
16.1
13.7
In December 2019, when cones were mature, but before the start of seed dispersal, an
average of 40 cones were collected from each selected tree. Cones were cold stored (3 ◦ C)
for a week and then placed in trays at room temperature. The number of cones per tree,
together with individual cone fresh weight (in g), cone diameter (in cm) and cone length
(in cm), were measured. Cone volume (in cm3 ) was estimated from cone diameter and
length, assimilating the volume to an ellipsoid. After cone morphological characterization,
cones were oven-dried at 30 ◦ C for 40 h to accelerate their opening. Once cones had opened,
the total seed mass per cone (in g) was recorded. Additionally, seed mass was weighed in
five random samples per cone, containing 100 seeds in each sample. Each of the 100 seeds
in the five samples was individually weighed (in mg), and length (in mm) and diameter (in
mm) were measured. The rest of the seeds were stored in sealed glass containers under
cold conditions (3 ◦ C) until their use in the experiments described below.
One hundred seeds per tree were randomly selected to be used in the germination tests
done at the University of Huelva. First, seeds were disinfected by immersion in a 30 mL L−1
solution of H2 O2 for 10 min. Then, seeds were mixed with wetted perlite and sprayed with
a fungicide (a solution of Daconil (Clortalonil) 50%, 1 g L−1 and Captan 50%, 1.5 g L−1 ). The
mixture was placed inside plastic bags under cold conditions (3 ◦ C) and kept in the dark.
After one month, 100 seeds per tree were distributed in four 8-cm-diameter Petri dishes
(25 seeds per dish). Petri dishes had five sheets of absorbent paper at the bottom that had
been previously wetted with distilled water and sprayed with fungicide. Petri dishes were
kept at room temperature (18 to 25 ◦ C) with a 14 h light/10h dark photoperiod. Germination
Forests 2022, 13, 937
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was recorded twice a week for 4 weeks. Different parameters were assessed [23] (FAO 1985):
(1) germination energy (GE, which is the percentage of seeds germinated in the first week),
(2) germination percentage at the end of the test (GP) and (3) germination values (GVCZ
and GVDP) according to Czabator [24] and Djavanshir and Pourbeik [25]. Additionally, an
extra sample of 20 seeds per tree was used to perform a tetrazolium test to analyze seed
viability [23]. Seeds were soaked in distilled water for 24 h at room temperature and then
cut in half with a scalpel. Each half was immersed in a tetrazolium solution (5 g L−1 of
2,3,5 triphenyl-2H-tetrazoliumchloride) and kept in the dark for 80 min at 20 ◦ C. Viable
seeds (VS, i.e., seeds whose embryo and nutritive tissue stained red) from each tree were
counted, and the percentage of viable seeds (%VS) was calculated.
In September 2020, an additional sample of 25 seeds per tree was pre-germinated
inside Petri dishes in the plant laboratory at INIA, using the same protocol as described
above. Ten days after seeding, germinated seeds were transplanted to FP-300 containers
filled with a mixture of peat moss and vermiculite (80:20, v:v), adding 32 g NUTRICOTE
fertilizer per container. The 25 pre-germinated seeds from each tree were distributed in five
blocks of five seeds each. Seedlings were grown in the greenhouse and were irrigated to
field capacity three days per week. Once the first juvenile needles appeared (37 to 40 days
after transplanting), seedlings were left to dry until death. Seedling survival and volumetric
water content of the substrate (measured using a portable time-domain reflectometer (TDR)
equipped with two 16-cm rod probes (TDR100, Spectrum Technologies, Inc., Aurora, IL
60504, USA) were monitored once per week during 160 days. Volumetric water content at
the beginning of the drying process was 15%.
To study the influence of the mother tree age on morphological traits of cones (weight,
length, diameter and volume) and seeds (weight, length and diameter), germination
capacity and viability (variables GE, GP, GVCZ, GVDP and VS%), we first checked the significance of the between-tree variability by building up a random effect model considering
the tree as a random effect. The equation for the model was:
yij = t j + eij
where yij referred to the assessed variable from tree j, tj is the tree random effect with
tj ~N (0, σ2 t) and eij is the residual error with eij ~N (0, σ2 e).
First, we predicted for each measured trait the covariance parameter at the tree level
and checked the covariance parameter equal-to-zero hypothesis with a χ2 test. We then
calculated the percentage of variance explained by the tree effect. In a second step, for
those traits with a significant tree effect, we introduced the age of the mother tree as a covariate in a linear and quadratic form. Then, we assessed the significance of the coefficients
(p < 0.1) with an F test. We introduced the quadratic term to check for non-linear relationships of the mother tree age with the studied traits (morphological traits of cones and seeds,
germination capacity and seed viability). The equation for the final model was:
yij = β 0 + β 1 age + β 2 age2 + t j + eij
where β 0 , β 1 and β 2 are unknown but estimable parameters and tij and eij are as previously defined.
The influence of the mother tree age on seedling survival during the death trial was
assessed considering a survival rate for a given tree j at a given date t (Sjt ) as the response
variable, following a binomial distribution. As a first step, we considered an independent
analysis for each date, using logistic regression with tree age (in a linear and quadratic form)
included as explanatory covariates. As a second step, we carried out a repeated measures
analysis by means of a generalised linear mixed model, where date was considered a
random effect. The final expression of the generalised linear mixed model, assuming a logit
link function, was:
exp θ jt
S jt =
1 + exp θ jt
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where θ jt = β 0 + β 1 age + β 2 age2 + Dt , Dt is the measurement date random effect with
Dt ~N (0, σ2D)
For those traits where a significant effect of age was detected, the fitted model was
plotted against the observed data in order to describe the pattern of the relationship.
3. Results
3.1. Morphological Traits of Cones and Seeds
Results for the morphological traits of cones showed significant differences between
trees (p = 0.0001, Table 2). The tree effect accounted for 39 to 47% of the total variability.
These four cone traits were highly correlated (0.51 < r < 0.93, p < 0.0001). Mean values for
the studied traits were (SD, range): length = 38.2 cm (4.3, 31.6 to 48.3), diameter = 20.6 cm
(2.04, 17.1 to 25.2), weight = 5.8 g (1.29, 3.8 to 9.0) and volume = 71.2 cc (20.0, 39.1 to 113.0).
The age of the mother tree had a significant effect on the diameter, weight and volume of
cones (Table 2, Figure 1). For these traits, we observed a significant decrease with age, up
to a tree age over 150–175 years, when a slight increase in the values was detected.
Table 2. Between-Tree Variability and Effect of Tree Age on Morphological Parameters of Cones.
When the effect of age is significant (p < 0.1), the value is shown in bold.
Variables
Length
Diameter
Weight
Volume
Covariance Parameter Estimates
Tree (σ2 t )
Residual (σ2 e )
Total
Variance Explained
by Tree Effect (%)
p (σ2 t = 0)
18.02
3.96
1.61
385.71
20.14
6.28
2.01
498.71
38.15
10.24
3.62
884.42
47
39
44
44
0.0001
0.0001
0.0001
0.0001
(a)
Tree Age Effect
(b)
Figure 1. Cont.
Age (p > F)
Age2 (p > F)
0.16
0.07
0.07
0.08
0.15
0.19
0.09
0.12
Forests 2022, 13, 937
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(c)
(d)
Figure 1. Mean values (±SE) (points) and fitted line (red line) for the morphological parameters of
cones along the range of tree age analyzed: (a) length (in cm); (b) diameter (in cm); (c) weight (in g)
and (d) volume (in cc). Fitted line is only shown for those traits showing a significant relationship
with age.
Results for the morphological traits of seeds showed significant differences between
trees (p = 0.0001, Table 3). The tree effect accounted for 22 to 45% of the total variability.
These three seed traits were correlated (0.38 < r < 0.47, p < 0.0001). In addition, we recorded a
negative correlation of seed weight (r = −−0
0.07, p = 0.0003) and length (r = −−0
0.12, p < 0.0001)
with the age of the mother tree. Mean values for the studied traits were (SD, range):
length = 4.8 mm (0.4, 4.2 to 5.8), diameter = 2.6 mm (0.2, 2.1 to 3.0) and weight = 9.2 mg
(1.8, 6.6 to 14.2). The age of the mother tree had a significant effect on seed weight (Table 3,
Figure 2). As in the previous case, for this trait, we also observed a significant decrease with
age, up to a tree age over
– 150–175 years, when a slight increase in the values was detected.
Table 3. Between-Tree Variability and Effect of Tree Age on Morphological Parameters of Seeds.
When the effect of age is significant (p < 0.1), the value is shown in bold.
Variables
Length
Diameter
Weight
Covariance Parameter Estimates
Tree
(σ2
0.15
0.04
3.29
t)
Residual
(σ2
0.18
0.07
11.67
e)
Total
Variance Explained by
Tree Effect (%)
p (σ2 t = 0)
0.33
0.11
14.95
45
36
22
<0.0001
<0.0001
0.0001
Tree Age Effect
Age (p > F)
Age2 (p > F)
0.24
0.15
0.09
0.29
0.15
0.12
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(a)
(b)
(c)
Figure 2. Mean values (±SE) (points) and fitted line (red line) for the morphological parameters of
seeds along the range of tree age analyzed: (a) length (in mm); (b) diameter (in mm) and (c) weight
(in mg). Fitted line is only shown for those traits showing a significant relationship with age.
3.2. Germination Capacity and Seed Viability
In the case of seed germination-related parameters (GE, GP, GVCZ and GVDP) significant differences were detected between trees (p < 0.001, Table 4), the tree effect accounting
for 51 to 66% of the total variability. However, the age of the mother tree was not significant
(Table 4, Figure 3). These four parameters were highly correlated (0.51 < r < 0.96, p < 0.002,
n = 30), the correlation between GVCZ and GVDP being especially prominent (r = 0.96,
p < 0.001, n = 30). We observed the following mean values (SD, range): GE = 61.6% (17.3,
24 to 95); GP = 75.4% (13.6, 33 to 96); GVCZ = 34.0 (12.7, 10 to 56) and GVDP = 46.0 (16.7, 14
to 77). Additionally, the percentage of viable seed (VS) ranged from 60 to 100%.
Forests 2022, 13, 937
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Table 4. Between-Tree Variability and Effect of Tree Age on Germination Parameters and Seed
Viability. GE: Germination Energy. GP: Germination Percentage. GVCZ: Germination Values
according to Czabator. GVDP: Germination Value according to Djavanshir and Pourbeik. VS%:
Percentage of Viable Seed.
Variables
GE
GP
GVCZ
GVDP
VS
Covariance Parameter Estimates
Tree (σ2 t )
Residual (σ2 e )
Total
Variance Explained by
Tree Effect (%)
p (σ2 t = 0)
234.3
157.9
134.1
237.5
225.5
85.8
85.7
122.2
0.0698
459.8
242.7
219.8
359.7
51
65
61
66
<0.0001
<0.0001
<0.0001
<0.0001
Tree Age Effect
(a)
(b)
(c)
(d)
Age (p > F)
Age2 (p > F)
0.45
0.86
0.94
0.49
0.74
0.31
0.92
0.89
0.63
0.72
Figure 3. Mean values (±SE) (points) for the germination parameters of seeds along the range of tree
age analyzed: (a) GE; (b) GP; (c) GVCZ and (d) GVDP. No fitted line is only shown as traits do not
show a significant relationship with age.
Forests 2022, 13, 937
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3.3. Seedling Survival in the Death Trial
Results from the repeated measurements analysis of variance showed a significant
effect of drought on survival over time (p < 0.0001). Irrespective of the age of the mother
tree, seedlings showed a survival rate of over 90% during 113 days after watering was
interrupted, even if, by then, soil moisture had already reached 0% (Figure 4). From this
day, survival drastically decreased to 27.1% in a week, although some seedlings remained –
still alive for another 47 days until the end of the experiment (day 160).
Figure 4. Survival (lines) and soil moisture (bars) in the death trial for seedlings from young (red line)
and overmature (black line) mother trees. For the sake of clarity, trees are divided into two groups:
young (<150 years old) and overmature (>150 years old).
When analyzing each measuring date separately, significant differences (p < 0.10)
in seedling survival related to the age of the mother tree were recorded in two periods:
(i) from day 74 to day 110, when soil moisture ranged from 2.9 to 0.02%, and survival rate
was maintained over 90% and (ii) from day 120 to day 144, when soil moisture had already
reached 0%, and survival rate decreased from 27.1 to 4.1%. In the first period, higher
survival was related to increasing tree age, while in the second period, higher survival
was observed for lower tree age. In addition, the generalised linear mixed model revealed
a global significant effect of the age of the mother tree on the rate of seedling survival,
indicating that the rate of survival increases with mother tree age, up to an age range of
150–200 years old, decreasing from this age (Table 5, Figure 5).
Table 5. Parameter Estimates and Level of Significance of the Generalized Linear Mixed Model for
the Survival Rate in the Death Trial.
Fixed Effect
Intercept
Age
Age2
Random
effects
Date
Parameter
Estimate
Standard
Error
t Value
P > |t|
1.1180
0.003500
−0.00001
0.7036
0.001546
5.924 × 10−6
1.59
2.26
−1.92
0.1219
0.0238
0.0557
Covariance
estimate
Standard
error
Z
P>Z
15.9918
4.1338
3.87
<0.0001
0.001
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Figure 5. Marginal response for the survival rate along the range of tree age analyzed in the death trial.
4. Discussion
Mean values for the morphological parameters of cones and seeds, as well as for
seed germination-related parameters for the sampled Pinus sylvestris trees from the corresponding age range, were within the average range described for the species in the study
area [12,26]. The between-tree differences in cone (between 20 and 60 cones per tree) and
seed production (from 162 seeds in a 216-year-old tree to 1871 seeds in a 180-year-old tree)
can be expected to be mainly related to the masting habit of the species, irrespective of
tree age. For instance, Pinus sylvestris produces about two abundant seed yields every
seven years [27] that are greatly mediated by the spring precipitation of the two years
and highest
previous to cone maturation [28]. Additionally, the fact that both the lowest
−6
−0 001
−1
seed production were obtained in overmature trees (2l6 and 180 years old, respectively)
supports the idea that, when referring to seed production, tree senescence does not decrease
fecundity with aging [15,16].
The significant differences between trees in the morphological parameters of cones
and seeds bring out the high between-tree variability, which can be greatly influenced by
species genetics, physiology or the environmental conditions during the collection year,
even by cone location in the tree crown, rather than by the age of the mother tree [29–31].
In fact, in our study, the between-tree variability accounts for more than 50% of the total
variability. Intraspecific variation in seed mass is common in conifers, whose seed mass
could vary 3–5 fold among and within trees [32]. Nevertheless, we found a significant
negative effect of the age of the mother tree on different morphological parameters of
cones (diameter, weight and volume) and seeds (weight). These negative correlations
highlighted lower values for these morphological parameters in trees reaching senescence.
The observed nonlinear relationship indicates that this trend tends to be reverted in the
oldest trees (>200 years old). Similarly, Kaliniewicz et al. [33] also observed a drop in the
weight of Pinus sylvestris seeds at increasing ages up to 180 years old. Although, in our
study, differences were small in absolute terms, they were significant, indicating that the
sampled trees of advanced ages had passed beyond the stage of maturity and entered into
the senescence stage [34].
In general, seed germination is determined by both environmental conditions and seed
viability [35]. In our study, germination-related parameters and seed viability also showed
a high between-tree variability that could point out a mother tree effect. However, these
parameters were not related to the mother tree age; thus, there was not likely a decline in
seed germination or seed viability in the oldest trees studied, as has been recorded in other
species [10,36]. In addition, although seed weight decreased with tree age, no differential
Forests 2022, 13, 937
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germination rates were observed related to tree age; thus, the hypothesis that postulated
that larger seeds generally germinate more rapidly was not checked [37].
Seedling survival in the death trial was mainly affected by the decrease in water availability rather than by the age of the mother tree. Once soil moisture reached 0%, seedlings
still maintained survival rates of over 90% for two weeks. After this period, survival
drastically decreased to ca. 27% in all seedlings, irrespective of the age of the mother tree.
Nevertheless, we found an effect of the age of the mother tree on the survival rate, although
the marginal effect of tree age (once the date as a random effect had been discounted) was
small, accounting for differences in seedling survival rates of 4.8% (Figure 5). Interestingly,
once soil moisture had reached 0%, we observed during 3.5 weeks higher survival rates in
seedlings developed from younger trees (from day 120 to day 144). The greater seed weight
found in younger trees (Figure 2c) could partially explain such differences in survival rates
since larger seed size can confer seedlings with an initial advantage in terms of survival
and growth [38,39]. Seed mass strongly influences seedling establishment, with heavier
seeds usually exhibiting a higher capacity to survive under hazardous environments due
to larger embryo and energy reserves that are efficiently used [32]. In fact, seed mass is
a relevant life history trait, affecting crucial processes of species recruitment, including
drought [40]. Particularly, seed size effects are expected to be especially relevant in highly
competitive environments [41]. The positive association between seed mass and survival
under dry conditions has been explained by the fact that larger seeds have more reserves to
produce seedlings with larger growths and/or deeper roots (e.g., [42,43]). Thus, seedlings
developing from larger seeds containing more nutrients and carbon-based reserves could
show an improved ability to uptake the scarce resources and support respiration longer
under carbon starvation [44]. For instance, Ramírez-Valiente and Robledo-Arnuncio [45]
found a positive association between seed mass and seedling survival rate under dry conditions in Pinus sylvestris, which was explained by a lower needle-to-root ratio in seedlings
from heavier seeds. However, differences in seedling survival related to the mother tree
age were no longer significant when drought was prolonged beyond 144 days. At this
point, seedling survival solely depended on the ability of belowground organs to absorb
resources available in the soil [33]. Generally, Pinus sylvestris seedlings can maintain stable
shoot water content under different water conditions. However, the water content collapses
under severe drought stress, and seedlings die [46]. During severe drought stress, several
physiological adaptation mechanisms allow Pinus sylvestris seedlings to obtain the water
from the medium and maintain growth processes, though they are obviously affected by
water stress [47]. Therefore, until seedlings collapsed, the functioning of PSII remained
stable, thus indicating an undisturbed function of PSII even under drought stress. Accordingly, one of the main mechanisms used by Pinus sylvestris seedlings to adjust its hydraulic
system to climatic conditions has been suggested to be directly linked to stomatal control
instead of to the sensitivity of leaf physiology to drought [21].
5. Conclusions
For the studied Pinus sylvestris trees, ranging between 30 and 219 years old, the age of
the mother tree had significantly influenced different cone (diameter, weight and volume)
and seed (weight) morphological attributes, but not germination capacity or seed viability.
Although the significant effect of mother tree age on the morphological traits of cones and
seeds is small in absolute terms, it evidences that the sampled trees of advanced ages had
passed beyond the stage of maturity and entered into the senescence stage. In any case,
the production of good quality viable seeds, seed germination ability and the production
of potentially vigorous seedlings were not affected by the age of the mother tree. Thus,
the persistence of the studied overmature stands is guaranteed. It remains to study the
proportion of empty seeds that seems to increase with aging. Additionally, in the death
trial, seedlings from younger trees showed higher survival rates once soil moisture had
reached 0%. Similar to the morphological traits of cones and seeds, the marginal effect of
tree age was small. Seedling response, in terms of survival, to the suppression of water
Forests 2022, 13, 937
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supply revealed the ability of Pinus sylvestris seedlings to maintain the functioning of PSII
undisturbed even under severe drought stress until the moment when seedlings collapsed
and died.
Author Contributions: Conceptualization, M.P., J.V.-P., R.A. and R.C.; methodology, M.P., J.V.-P. and
R.C.; formal analysis, M.P., J.V.-P. and R.C.; investigation, M.P., J.V.-P., L.B., G.M., R.A., M.F. and
R.C.; resources, M.P., J.V.-P. and R.C.; writing—original draft preparation, M.P; writing—review and
editing, M.P., J.V.-P., R.A., M.F. and R.C.; visualization, M.P., J.V.-P. and R.C.; supervision, M.P., J.V.-P.
and R.C.; project administration, M.P., J.V.-P., R.A. and R.C.; funding acquisition, M.P., J.V.-P., R.A.
and R.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Ministerio de Ciencia e Innovación, grant number AGL201783828-C2.
Data Availability Statement: The dataset generated during and/or analyzed during the current
study are available from the corresponding author on reasonable request.
Acknowledgments: We thank the Forest Service at Sierra de Guadarrama National Park for providing
the sampling permits required for the area and for their interest in the project and Giorgia Fiorino for
helping in the seed viability and seed germination tests.
Conflicts of Interest: The authors declare no conflict of interest.
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