1

Seasonal and inter-annual variability of bud development as related to

climate in two co-existing Mediterranean Quercus species

Arben Q. Alla1*, J. Julio Camarero

2, and Gabriel Montserrat-Martí

1

1Instituto Pirenaico de Ecología (CSIC). Avda. Montañana 1005, 50080 Zaragoza, Spain.

2ARAID-Instituto Pirenaico de Ecología (CSIC). Avda. Montañana 1005, 50080 Zaragoza,

Spain.

Running title:

Seasonal and inter-annual variability of bud development in oaks

*Corresponding author:

Arben Q. Alla

Instituto Pirenaico de Ecología, CSIC.

Avenida Montañana 1005, 50080 Zaragoza, Spain.

E-mail: benialla@gmail.com

Tel. 0034-976-716031, Fax: 0034-976-716019

2

Summary

In trees, bud development is driven by endogenous and exogenous factors such as

species and climate, respectively. However, there is scarce knowledge on how these factors

drive the changes in bud size across different time scales.

We describe the seasonal patterns of apical bud enlargement as related to primary and

secondary growth in two coexisting Mediterranean oaks with contrasting leaf habit (Quercus

ilex, evergreen; Quercus faginea, deciduous) over three years. Furthermore, we determine the

climatic factors driving changes in bud size of both oak species by correlating bud mass with

climatic variables at different time scales (from 5 to 30 days) over a 15-year period.

We found that the maximum enlargement rate of buds is reached between late July and

mid-August in both species. Moreover, the apical bud size increased as minimum air

temperatures did during the period of maximum bud enlargement rates.

The forecasted rising minimum air temperatures predicted by climatic models may

affect bud size and consequently alter crown architecture differentially in sympatric

Mediterranean oaks. However, the involvement of several drivers controlling the final size of

buds makes it difficult to predict the changes in bud size as related to ongoing climate

warming.

Key words: bud size, current-year stem, Quercus faginea, Quercus ilex subsp. ballota,

temperature, summer growth.

3

Introduction

The buds formed within an individual tree show different sizes (Gill, 1971). Bud size

determines primary growth and reproduction ability in plants with rhythmic growth (Nitta and

Ohsawa, 1998). This suggests that bud size is modulated by several drivers including the

individual tree size and vigour, the position of the bud in the shoot, and ontogenetic factors

(Remphrey and Powell, 1984; Sabatier and Barthélémy, 2001; Puntieri et al., 2002a, 2002b;

Ishihara and Kikuzawa, 2009; Alla et al., 2011). Among the endogenous factors, secondary

growth has been found to drive bud size since changes in current-year stem cross-sectional

area are linked to the stem hydraulic conductivity and the size of buds (Cochard et al., 2005;

Lauri et al., 2008; Alla et al., 2011). However, it remains unclear when the secondary growth

of trunks and current-year stems occur in relation to the pattern of bud enlargement, whether

these processes overlap in time, and if climate indirectly affects the development of buds

through the control of secondary growth. Climate and species are two of the main exogenous

and endogenous factors, respectively, affecting bud size in trees (Kozlowski et al., 1973;

Salminen and Jalkanen, 2004; Meier and Leuschner, 2008; Sanz-Pérez and Castro-Díez,

2010).

In species with preformed growth, shoot primordia are initiated inside the buds in a

growing season but remain un-extended during the autumn-winter resting period until the

following spring (Puntieri et al., 2002b). Many temperate Quercus species present preformed

growth of their shoots (Fontaine et al., 1999). Meristems and preformed primordia generally

are enclosed and protected by specialized elements, which determine the type of bud (Puntieri

et al., 2007). Quercus species have typical scaled buds (Keator, 1998) which show different

protective elements (hypsophylls, scales and cataphylls) and present a regular pattern of

development along the year (Nitta and Ohsawa, 1998, 1999). In spring, once shoot extension

is accomplished, the shoot presents a small hypsophyllary bud protected by hypsophylls and

4

located at the apex. A bud consists of many scales developed in the following weeks,

switching to a small scaled bud (Nitta and Ohsawa, 1999). Scaled buds enlarge until early or

mid-autumn, when they enter dormancy (Arora et al., 2003; Horvath et al., 2003). This

resting stage is broken in late winter or early spring when buds start swelling (Marks, 1975;

Montserrat-Martí et al., 2009). Indeed, the adaptive value of scaled buds seems to be the

protection of the shoot primordia against climatic stressors such as low temperatures during

the cold winter season (Nitta and Ohsawa, 1998).

Despite the relevance of buds as organs determining the aboveground growth and

reproduction patterns of trees, the driving factors controlling the seasonal pattern of bud

enlargement have received little attention except in the case of conifers (Kozlowski et al.,

1973; Lanner, 1985; Chen et al., 1996). Such drivers must be characterised to understand how

variations in bud size relate to bud composition and primary growth within a tree species. In a

previous study, Montserrat-Martí et al., (2009) analysed the seasonal patterns of bud

enlargement in two co-existing Mediterranean Quercus species with contrasting leaf habit,

namely Quercus ilex L. subsp. ballota (Desf.) Samp. (evergreen; hereafter abbreviated as Q.

ilex) and Q. faginea Lam. (winter deciduous). They showed that the buds of both species

reached the maximum growth rate in summer, although their data lacked the temporal

resolution to determine the precise period of maximum growth rate. In addition, they observed

that these two species present a reduced growth activity in response to drought during summer

when shoot and secondary growth are almost arrested and acorns enlarge at a very low rate.

Their study also showed that budburst and shoot extension occurred earlier in Q. faginea than

in Q. ilex, so the buds of Q. faginea grow during more time than those of Q. ilex. Montserrat-

Martí et al. (2009) also reported an alternating yearly pattern of bud and shoot size in Q. ilex.

Such pattern may be related to the different seasonal patterns of bud enlargement in the two

studied species.

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In this study we aimed: (i) to quantify the seasonal patterns of bud enlargement and

secondary growth of main (trunk) and current-year stems of Q. ilex and Q. faginea over three

years with contrasting climatic conditions, and (ii) to identify the climatic factors which most

influence bud size of both species over a 15-year period. Our hypotheses were: (i) buds will

attain their maximum enlargement rate when the climatic conditions are harsh enough for

cessation of other aboveground growth types (primary, secondary and reproductive), usually

occurring in mid-summer, and (ii) the main climatic drivers of bud enlargement will be those

more important for growth in summer, namely water availability. In this study, other growth

types happening in summer (lammas growth and acorn development) were discarded as they

tend to occur in a small percentage of branches (Alla et al., unpubl. res.). Furthermore, the

size of apical buds did not differ between shoots bearing and not bearing acorns (Alla et al.

2012).

Material and methods

Study area and species

The study site is located in Agüero, Aragón, north eastern Spain (42° 18’ N, 0° 47’ W, 750 m

a.s.l.). Climate is Mediterranean and continental being characterized by dry summers and cold

winters, with 544 mm and 13.8 ºC of total annual precipitation and mean annual temperature,

respectively. Soil in this area is a Calcisol (FAO, 1998), formed on Miocene clays with

bedrock of calcareous sandstone. Vegetation is a scrubland dominated by Q. ilex, Q. faginea,

Arbutus unedo L. and Pinus halepensis Mill., approximately with similar dominance, and

other less abundant woody species (see Montserrat-Martí et al., 2009 for further details on the

study site).

Quercus ilex is an evergreen oak tree with semi-ring to diffuse porous wood (Campelo

et al., 2007), which grows preferentially inland under continental conditions in the Iberian

6

Peninsula (Amaral Franco, 1990). Q. faginea is a deciduous oak tree with ring-porous wood

(Corcuera et al., 2004a) and with a wide distribution in sub-Mediterranean areas of the

Iberian Peninsula (Amaral Franco, 1990). Both oak species frequently coexist in the study

area forming multi-stemmed trees (Table 1).

Table 1. Morphological and structural features of the studied species and trees (means ± SE).

Species Diameter at

1.3 m (cm) Height (m)

No. stems

per tree

Shoot length (mm)

2006 2007 2008

Q. ilex 10.2 ± 1.2b 3.4 ± 0.4b 6.5 ± 1.0a 29.4 ± 2.0aC 43.2 ± 2.2aA 35.7 ± 2.0aB

Q. faginea 14.5± 1.8a 5.7 ± 0.9a 4.0 ± 1.2b 17.7 ± 1.8bB 24.7 ± 2.6bA 23.4 ± 1.9bA

Small letters correspond to significant differences between species and capital letters indicate

significant differences among years in the case of shoot length (P < 0.05, Mann-Whitney U tests).

Figure 1. Monthly climatic data (maximum, mean and minimum temperatures; total

precipitation) recorded during the study period (1997-2011).

Field sampling and variables measured

Two different approaches were undertaken: the seasonal development measurements were

made over three years (2006-2008) and the inter-annual measurements were taken over fifteen

Months

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Pre

cip

ita

tio

n (

mm

)

0

20

40

60

80

100

120

140

Te

mp

era

ture

(ºC

)

0

5

10

15

20

25

30

35

Precipitation

Temperature

7

years (1997-2011). Climate varied markedly among the studied years. During the monitoring

period of seasonal development higher summer temperature and lower precipitation in 2007

as compared with 2006 and 2008 were detected, whereas during the long-term period 1998,

2005 and 2011 were dry years while 2003 was very warm (Fig. 1).

Seasonal development

In spring 2006 before budburst, four mature individuals per species were randomly selected,

tagged and the size (diameter at 1.3 m, height) and number of stems per tree were registered

(Table 1). Within each multi-stemmed tree, ten current-year shoots were randomly selected

and collected monthly from the southern side and upper third of the crown during three

consecutive years (2006, 2007 and 2008). We assumed that all stems of each tree were

equivalent in architectural terms. Current-year shoots were frozen until measures were taken

in the laboratory. To minimize shoot length variability, the average length of the shoots

selected for analyses was similar among sampling dates for both species within each tree and

year. Shoot length and diameter (the average of two perpendicular measures taken in the

middle of the stem without nodes) were measured in each shoot with a centesimal calliper

(Mitutoyo, Kawasaki, Japan). Diameter was transformed to stem cross-sectional area

assuming a circular shape. Buds were oven-dried at 60 °C to a constant weight before being

individually weighted to the nearest 0.001 g to obtain the apical bud mass.

In order to assess the relationships between bud mass, size and composition (number

and type of primordia contained within the bud) we performed bud dissection in selected

dates of bud development, namely: budburst (April-May), formation of the scaled bud (June-

July) and enlargement of the bud (August). Dissection of buds is a destructive method and, in

most cases, we cannot know the exact number of leaves produced by the shoot derived from

dissected buds. There are at least two methods to solve this problem. First, we may select a

8

bud of similar size and position on a shoot of similar characteristics as the dissected bud and

follow its development (Puntieri et al., 2002a). Second, we can assume that the dissected

apical bud will produce a similar shoot as the bearing shoot. We followed the second

approach which renders a reliable approximation to the actual fate of the analysed buds. The

apical buds of 35 shoots of Q. ilex and 40 shoots of Q. faginea were removed and dissected at

10-100x magnification under a stereomicroscope (Leica MZ6, Leica Microsystems,

Heerbrugg, Switzerland). Then, the components of the bud (hypsophylls, scales, cataphylls,

and leaf primordia) were visually distinguished, according to their different size, shape, and

extension growth at budburst, and counted following the method proposed by Nitta and

Ohsawa (1998, 1999).

To quantify changes in stem secondary growth the radial increment of those trees

sampled for bud enlargement was measured using manual band dendrometers (Agriculture

Electronics Corporation, Tucson, USA) during the three years of study. In March 2006,

dendrometers were placed at 1.3 m on the thickest stem of the selected multi-stemmed

individuals. Bark was carefully brushed off before installing the dendrometers, which were

read at 24-day intervals on average. Displacement measurements were taken with a precision

of 0.1 mm and readings after rainy days were discarded. Radial growth was transformed to

basal area considering the initial diameter of the trees.

Inter-annual measurements

Samplings for the inter-annual assessment of bud size and shoot growth were done at mid-

October from 1997 to 2011, i.e. during 15 years. Each year 20 mature trees were randomly

selected for each species. Annually, we collected 15 shoots per tree from the southern side

and upper third of the crown (n = 300 shoots). Then, we randomly selected 30 shoots per

species, excluding very big and very small shoots and also lammas shoots, to get the annual

9

mean values of shoot length and stem cross-sectional area and bud size. October was selected

as the most adequate time for annual sampling because shoot development is completed and

buds have almost reached their final size by that time of the year. Shoot length and stem

cross-sectional area were measured and apical buds were removed. After being oven-dried the

apical buds were individually weighted as previously described.

To estimate the radial growth patterns of the sampled trees during the period 1997-

2011, the main trunk was cored at 1.3 m using an increment Pressler borer. All wood samples

were prepared following standard dendrochronological methods, they were air dried, sanded

and visually cross-dated (Cook and Kairiukstis, 1990). Tree-ring widths were then measured

at a precision of 0.001 mm using a LINTAB measuring system (Rinntech, Heidelberg,

Germany). Lastly, they were standardized and transformed into tree-ring width indices by

subtracting the raw width by the mean value and then retaining the residuals, i.e. the

differences between observed and fitted values.

Climatic data and water balance

Daily climate data (maximum temperature, Tmax; minimum temperature, Tmin; and total

precipitation, P) for the period 1997-2011 were obtained from the weather station of Luna

(42º 10’ N, 0º 56’ W, 409 m a.s.l.), situated at ca. 18 km from the study site. Daily air

temperature were obtained from January 2007 up to December 2011 at the study site using a

Hobo®

data logger which recorded temperatures every 30 minutes (Onset Computer Co.,

Bourne, USA) (Fig. 1). To estimate local temperatures for the previous years (1997-2006), a

linear regression between local data (y) and data from the weather station (x) was calculated (y

= 0.2131 + 0.9067x; r = 0.97, P < 0.0001). Daily cumulative water balance (hereafter

abbreviated as WB) was calculated as explained in Montserrat-Martí et al. (2009). Briefly, the

soil WB was estimated by using a modified Thornthwaite water-budget procedure based on

10

mean temperature and total precipitation data. Following this procedure, soil–water balance is

modelled by estimating soil-water withdrawal (actual evapotranspiration), recharge, and

surplus. Actual evapotranspiration is related to the percentage of the current soil at the

available water capacity, and to potential evapotranspiration, which is estimated from mean

temperature and day length.

Data analyses

To compare tree features (diameter at 1.3 m, height, number of stems per tree) and studied

variables (basal area, stem cross-sectional area and apical bud mass) between species and

among years we performed non-parametric Mann-Whitney U tests. We also used this test to

compare shoot length between species and among years by performing pairwise comparisons.

To compare basal area, stem cross-sectional area and apical bud mass values among years and

sampling dates we performed linear mixed-effects models considering years and dates as

fixed factors and individuals as random factors.

To estimate the growth rates of measured variables (basal area, stem cross-sectional

area, apical bud mass) we fitted the following logistic model to cumulative growth data (y) as

a function of time (x) using the CurveExpert software (Hyams, 2010):

( )) (1)

where a is the maximum or asymptotic value, b is the maximum daily rate and c is the Julian

day when the maximum growth rate was attained.

Correlation analyses were performed using the Pearson correlation coefficient to

evaluate the relationships between growth (tree-ring width indices, shoot length, stem cross-

sectional area, apical bud mass) and climatic variables (Tmax, Tmin, P, WB). Correlations

were calculated for the 15-year (1997-2011) study period considering the growing season

from March to early October. To detect potential lags or cumulative effects between climatic

11

and growth variables, climatic data were averaged (Tmax, Tmin) or summed (P, WB) for

periods of increasing time span (5, 10, 15 and 30 days). To simplify, we only present the

maximum correlations, in absolute terms, and the period when it was observed.

Figure 2. Intra-annual trends observed in cumulative stem basal area (BA), current-year stem

cross-sectional area (SCA) and apical bud mass (ABM) of Q. ilex and Q. faginea. The

different symbols and lines correspond to data (means ± SE) and fitted logistic models,

respectively, for the three years of study. Time is given as days (bottom axis) and as months

(top axis).

0

1

2

3

4

5

6

7

8

9

J F M A M J J A S O N D J F M A

BA

(cm

2)

0

1

2

3

4

5

6

J F M A M J J A S O N D J F M A

SC

A (

mm

2)

0.0

0.5

1.0

1.5

Day

0 60 120 180 240 300 360 420 480

AB

M (

mg)

0

1

2

3

42006

2007

2008

0.0

0.5

1.0

1.5

0 60 120 180 240 300 360 420 480

0

2

4

6

8

10

12

Q. fagineaQ. ilex

12

Results

Seasonal dynamics of basal area, stem cross-sectional area and bud size

Considering all data, we found significant differences between species for both apical bud

mass (F = 96.91, P < 0.0001) and basal area (F = 11.82, P < 0.001), but not for stem cross-

sectional area (F = 0.01, P = 0.95). As expected all variables showed significantly different

values among sampling dates and among years, excepting apical bud mass which did not

differ among years in both species (Fig. 2; Table 2).

Table 2. Main statistics (F and P between parentheses) of linear mixed-effects models fitted

to compare growth variables among the three study years for each oak species. Individual

trees were considered random factors.

Variable Factor Q. ilex Q. faginea

Stem basal area Year 13.67 (<0.001) 16.38 (<0.001)

Date 17.03 (<0.001) 85.69 (<0.001)

Stem cross-sectional area Year 40.82 (<0.001) 12.61 (<0.001)

Date 116.45 (<0.001) 137.04 (<0.001)

Apical bud mass Year 2.22 (0.14) 2.26 (0.13)

Date 184.37 (<0.001) 502.01 (<0.001)

Basal area presented the lowest values in 2006 and the highest ones in 2008. In the case of

stem cross-sectional area both species showed increasing values from 2006 to 2008. In Q.

ilex, apical bud mass showed an alternate yearly pattern with the highest values in 2006 and

the lowest ones in 2007, whereas in Q. faginea apical bud mass behaved as the stem cross-

sectional area did, i.e. showing minimum and maximum mean values in 2006 and 2008,

respectively (Fig. 2, Table 3).

Growth of all measured variables, except the ending of apical bud mass development,

started and finished earlier in Q. faginea than in Q. ilex during the three years of study (Figs. 2

and 3). Q. faginea showed a larger final apical bud mass than Q. ilex, while basal area and

stem cross-sectional area did not differ between species (Table 3).

13

Table 3. Statistical analyses based in the logistic model ( ( )) as a function

of time (x, in Julian days) for cumulative values of stem basal area, current-year stem cross-

sectional area and apical bud mass of Q. ilex (Qi) and Q. faginea (Qf). Models were highly

significant (P < 0.001, R2 > 0.96) in all cases.

a b c

Qi Qf Qi Qf Qi Qf

Basal area

2006 1.93 ± 0.61b 3.70 ± 1.58 0.019 ± 0.008 0.051 ± 0.020 9 July ± 7abA 3 June ± 2B

2007 3.32 ± 1.34ab 5.41 ± 2.52 0.035 ± 0.015 0.066 ± 0.029 16 July ± 2aA 4 June ± 4B

2008 4.31 ± 1.40a 5.55 ± 2.00 0.033 ± 0.013 0.047 ± 0.020 2 July ± 5bA 8 June ± 3B

Stem cross-sectional area

2006 1.02 ± 0.06b 1.11 ± 0.15b 0.010 ± 0.001 0.009 ± 0.001 29 May ± 3bA 15 May ± 5B

2007 1.16 ± 0.09ab 1.22 ± 0.18ab 0.010 ± 0.001 0.010 ± 0.001 3 June ± 3bA 12 May ± 4B

2008 1.33 ± 0.10a 1.48 ± 0.17a 0.012 ± 0.002 0.009 ± 0.001 21 June ± 1aA 22 May ± 9B

Apical bud mass

2006 3.38 ± 0.67B 8.73 ± 1.44A 0.023 ± 0.004B 0.069 ± 0.009bA 13 August ± 7 4 August ± 5

2007 2.62 ± 0.50B 9.40 ± 0.79A 0.023 ± 0.007B 0.074 ± 0.009bA 7 August ± 6 3 August ± 6

2008 2.82 ± 0.52B 10.94 ± 1.80A 0.027 ± 0.005B 0.095 ± 0.009aA 12 August ± 4 9 August ± 9

Abbreviations: a, maximum value (units: basal area, cm2; stem cross-sectional area, mm

2;

apical bud mass, mg), b, maximum rate (units: basal area, cm2 d

-1; stem cross-sectional area,

mm2 d

-1; apical bud mass, mg d

-1), c, day of maximum rate. Small letters correspond to

significant differences among years and capital letters indicate significant differences between

species (P < 0.05, Mann-Whitney U tests).

Considering all analysed variables and years, maximum growth rates were significantly

higher for Q. faginea than for Q. ilex only in the case of apical bud mass. The day of the

maximum growth rate was always observed earlier in Q. faginea than in Q. ilex for basal area

and stem cross-sectional area, and such date showed a higher year-to-year variability in Q.

ilex than in Q. faginea. The stem cross-sectional area of Q. faginea was the variable showing

the earliest day of maximum growth rate, which was on average the 16th

May, whereas the

apical bud mass of Q. ilex was the variable with the corresponding latest day of maximum

enlargement rate being on average the 11th

August (Table 3, Fig. 3). The maximum

enlargement rate of apical bud mass was attained in the first two weeks of August in both

species and for the three years of study (Table 3).

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Figure 3. Intra-annual growth rates of basal area (BA), current-year stem cross-sectional area

(SCA) and apical bud mass (ABM) during the three years of study based on the fitted logistic

models (see Fig. 2). Black areas correspond to Q. ilex rates, grey areas represent Q. faginea

rates and dark grey areas indicate the intersections between both species. Time is given as

days (bottom axis) and as months (top axis).

BA

rate

(m

m2 d

-1)

0.00

0.02

0.04

0.06

0.08

Month

J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J

Q. ilex

Q. faginea S

CA

rate

(m

m2 d

-1)

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

2006 2007 2008

Day

30 90 150 210 270 330 390 450 510 570 630 690 750 810 870 930 990 1050

AB

M r

ate

(m

g d

-1)

0.00

0.02

0.04

0.06

0.08

15

Bud dissection revelated that all leaf primordia were initiated before the period of maximum

growth rate of apical bud mass was reached (Table 4). In early August Q. ilex was initiating

the hypsophylls of the following apical bud, while Q. faginea probably had all hypsophylls

already initiated. Anyway, if any new primordia were formed during August it would be the

last elements of the heteroblastic series of the year, i.e. the hypsophylls. Scales are initiated at

the beginning of the year of growth when buds are at the swellins stage, just before the onset

of budburst.

Table 4. Number of pairs (minimum-maximum values) of primordia or formed structures

(hypsophylls, scales, cataphylls and stipules) contained within the buds of the two studied oak

species along the period of bud development.

Q. ilex Q. faginea

Event Hypsophylls Scales Cataphylls Stipules Hypsophylls Scales Cataphylls Stipules

Budburst

(April-May) 1-2 1-2 0 0 2-3 2-4 0 0

Formation of

baled buds

(June-July)

1-2 1-3 2-3 5-11 2-3 3-7 3-6 4-7

Intense bud

enlargement

(August)

1-2 1-3 2-3 8-11 2-3 3-7 3-6 8-12

Climatic drivers of growth and bud enlargement

Most significant correlations between climate and growth parameters were observed for time

spans ranging between 5 and 15 days (Table 5). Maximum (Tmax) and minimum (Tmin)

spring and early summer temperatures correlated negatively with tree-ring width indices,

shoot length and stem cross-sectional area, whereas late July to early August temperatures,

particularly Tmin, showed positive relationships with apical bud mass for both species. Spring

precipitation and cumulative water balance were positively correlated with all growth

variables, except in the case of apical bud mass of Q. ilex and Q. faginea, which were more

related to early summer water availability and water balance in late summer, respectively.

Furthermore, apical bud mass responded positively to early July and negatively to late August

16

precipitation and water availability in Q. ilex and Q. faginea, respectively (Table 5). apical

bud mass was mainly and positively related to early August Tmin considering 5- to 15-day

spans, but also to 5-day Tmax (Table 5, Fig. 4).

Discussion

The maximum enlargement rate of the buds of both oak species is reached in mid-summer,

between the 3rd

and the 13th

August, although the peak enlargement rate of Q. faginea was on

average 6 days earlier than that of Q. ilex. In spite of this moderate temporal lag in maximum

bud enlargement rates, both oak species present very similar patterns of apical bud

enlargement. This brief period in early August probably corresponds to the relatively least

favourable period for vegetative growth since secondary growth is constrained by low soil

water availability (Montserrat-Martí et al. 2009). During summer, low precipitation amounts

and elevated air temperatures, causing high evapotranspiration rates, lead to soil water deficit

(Alla et al. 2012). However, despite the fact that both studied species present a very low

activity in shoot elongation or stem thickening during this period their buds enlarge

intensively as previously described by Montserrat-Martí et al. (2009). Contrastingly, the mid-

summer water shortage experienced by plants under Mediterranean climatic conditions limits

the hydration levels required by some meristems to grow (Palacio et al., 2008). Our analyses

on bud composition evidence that the final number of leaf primordia is reached in both

species before the maximum rate of bud enlargement occurred. Since fresh and dry bud mass

are highly related in both studied species (Q. ilex, R2 = 0.95, n = 120; Q. faginea, R

2 = 0.98, n

= 134, P < 0.001 in both cases) we assume that bud mass is a reliable estimate of the number

of leaf primordia formed within the bud. Finally, our findings suggest that conditions suitable

for bud growth may be provided inside enclosed structures which are highly protected by an

envelope of scale-like structures, namely bud scales and cataphylls protecting shoot primordia

17

Table 5. Significant (P < 0.05) Pearson correlation coefficients obtained between growth (tree-ring width index, current-year shoot length,

current-year stem cross-sectional area and apical bud mass) of Q. ilex (Qi) and Q. faginea (Qf) and related climatic variables (Tmax, Tmin, mean

maximum and minimum temperatures, respectively; P, precipitation; WB, cumulative water balance).

Variable

Time span

(days)

Tree-ring width index Shoot length Stem cross-sectional area Apical bud mass

Qi Qf Qi Qf Qi Qf Qi Qf

Tmax

5 -0.55 (11-15 May) -0.55 (11-15 May) -0.62 (11-15 April) -0.81 (1-5 May) -0.60 (26-30 March) -0.67 (26-30 March) 0.65 (30 July-3 August) 0.61 (30 July-3 August)

10 -0.57 (20-29 June) -0.57 (10-19 June) -0.51 (1-10 April) -0.70 (1-10 May) −−− -0.71 (21-30 May) −−− −−−

15 -0.60 (15-29 June) -0.62 (15-29 June) -0.61 (1-15 April) -0.70 (1-15 May) -0.63 (16-30 March) -0.64 (16-30 March) −−− −−−

30 -0.69 (June) -0.61 (June) -0.63 (April) -0.63 (April) -0.52 (May) -0.66 (May) −−− −−−

Tmin

5 -0.51 (25-29 June) -0.77 (20-24 June) -0.64 (10-14 June) -0.70 (1-5 May) -0.61 (26-30 March) −−− 0.66 (30 July-3 August) 0.57 (30 July-3 August)

10 -0.54 (10-19 July) -0.72 (20-29 June) −−− -0.63 (1-10 May) -0.55 (21-30 March) −−− 0.70 (30 July-8 August) 0.72 (30 July-8 August)

15 -0.81 (15-29 June) -0.52 (15-29 June) −−− −−− -0.72 (16-30 March) −−− 0.50 (30 July-13 August) 0.74 (30 July-13 August)

30 −−− -0.65 (June) −−− −−− −−− −−− −−− −−−

P

5 −−− −−− −−− 0.70 (26-30 April) 0.60 (20-24 May) 0.53 (26-30 March) 0.74 (30 June-4 July) -0.59 (24-28 August)

10 −−− 0.50 (21-30 May) −−− 0.71 (21-30 April) −−− −−− 0.68 (30 June-9 July) −−−

15 0.68 (16-30 April) −−− −−− −−− −−− −−− 0.70 (1-15 July) −−−

30 −−− −−− −−− −−− −−− −−− −−− −−−

WB

5 −−− −−− −−− 0.70 (26-30 April) −−− 0.57 (26-30 March) 0.69 (30 June-4 July) -0.65 (24-28 August)

10 −−− −−− −−− 0.67 (21-30 April) −−− −−− 0.64 (30 June-9 July) −−−

15 0.72 (16-30 April) −−− −−− −−− −−− −−− −−− −−−

30 −−− −−− −−− −−− −−− −−− −−− −−−

The dates of the displayed correlations are indicated between parentheses. Correlations were calculated for the period 1997-2011 considering the

growing season from March to early October. Climatic data were averaged (Tmax, Tmin) or summed (P, WB) for periods of increasing time span

(5, 10, 15 and 30 days).

18

and scaly cupules enclosing developing acorns. Contrastingly, primary and secondary growths

are mostly arrested at mid-summer in the studied species (Montserrat-Martí et al., 2009;

Gutiérrez et al., 2011).

The variables of primary growth were influenced by climate when growth started and

when it was fully performed. Similarly, the radial growth on both studied species were

negatively influenced by temperature and positively by water availability in spring and early

summer, when secondary growth attains its maximum rate, in agreement with other published

results (Corcuera et al., 2004a, 2004b; Gutiérrez et al., 2011). Moreover, a higher availability

of water in early summer seems to favour the earlier accomplishment of vegetative shoot

growth and the start of bud enlargement in Q. ilex. The same correlation was not found in Q.

faginea, as it finishes its shoot development at mid spring, 4-7 weeks earlier than Q. ilex. On

the other hand, precipitation at the end of August probably favours the enlargement of acorns

over that of buds in Q. faginea but not in Q. ilex, reflecting the early acorn development of the

former species (Montserrat-Martí et al., 2009).

The most unexpected and interesting result was the positive association between apical

bud mass and temperature in summer, particularly the minimum, coinciding with the period of

maximum bud enlargement rates between late July and mid-August. High air temperatures in

summer restrict primary and secondary growth through enhanced evapotranspiration rates

since they preclude tissue hydration during the driest period of the growing season

(Montserrat-Martí et al. 2009). Nevertheless, the growth of scale-protected structures such as

buds seems to be enhanced by warm summer nights, i.e. high minimum temperatures, as

growing tissues are actively enlarging at night (Boyer, 1968; Ortega, 2010). Our results

suggest that buds are more responsive to warm night temperatures during summer, when the

studied oak species may access soil water reserves (Villar-Salvador et al., 1997). However,

19

other meristems as the cambium are more controlled by the tree water status (Gutiérrez et al.,

2011).

Figure 4. Inter-annual variations of the apical bud mass of Q. ilex and Q. faginea as related to

minimum temperatures of the ten-day interval 30 July-8 August along the 15 years of study

(1997-2011). We found for this time interval the highest Pearson correlation coefficients

between temperature and apical bud mass.

Our results aid to explain the alternating year-to-year pattern of long shoots and small buds

with short shoots and big buds formed the following year observed in Q. ilex. The idea that

the production of numerous long shoots would constrain bud development, must be

complemented by the effect of the early to mid-August minimum temperatures on bud

enlargement. In the studied climatic series we observed a regular alternating pattern of

minimum summer temperatures during the period 1997-2004 (Fig. 4). In this period, the

minimum temperatures from late July to mid-August strongly correlated with bud size in Q.

ilex, explaining the pattern described by Montserrat-Martí et al. (2009). We did not observe

the same zigzag pattern in the deciduous Q. faginea, despite showing a similar temperature-

Year

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Apic

al-bud m

ass (

mg)

0

2

4

6

8

10

12

Mean m

inim

um

tem

pera

ture

(3

0 J

uly

-8 A

ugust)

(ºC

)

12

14

16

18

20

22Q. ilex

Q. faginea

Tmin

20

bud correlation or even stronger than in the case of Q. ilex. This could be explained by the

longer period of bud enlargement of Q. faginea as compared with Q. ilex, which would

mitigate the effects of temperature in this short summer period on the final bud size.

During the last 60 years the minimum temperatures of the considered summer period

have significantly increased at a mean rate of +0.03 ºC yr-1

(r = 0.30, P = 0.017). Such

observation is consistent with the increase of air temperatures associated to ongoing climate

warming (IPCC, 2007). This trend might result in the increase of the apical bud size in the

forthcoming years, which could have consequences for the development of tree crowns.

However, we do not know how plastic bud enlargement would be and to what extent would it

allow trees to adjust their phenological patterns to the forecasted warmer conditions. For

example, the severe summer droughts recorded in 2005 and 2011 led to the formation of very

small buds in both oak species, despite their contrasting leaf phenologies, probably because

plants were not able to adequately hydrate bud tissues at mid-summer (Alla et al., unpubl.

res.). The involvement of more than one driving factor on the final size of buds makes it

difficult to predict the variations of bud size associated to climate change.

We consider this study a relevant example showing the usefulness of phenological

analyses of tree species as a tool to interpret their responses to environmental factors and to

facilitate the understanding of their ecological adaptations. The study of the temporal

arrangement of different phenological events including the developmental pattern of buds

allows analysing how these events overlap with each other, and how they are affected by

different climatic stressors. Taking into account that growth in seasonally dry and cold

climates is limited by temperature and water availability (Chapin et al., 1987), and that

different plant structures show distinct climatic thresholds, the accurate knowledge of the

temporal arrangement of phenophases is key for interpreting the functional responses of

plants.

21

To conclude, Q. faginea started bud enlargement earlier than Q. ilex, but both species

reached maximum bud enlargement rates in the first two weeks of August, with a few days of

advanced peak in the deciduous species. Warm night temperatures in this short summer

period enhance bud enlargement. These findings suggest that the rise in air temperature

forecasted by climatic models will enhanced bud size in both studied Mediterranean oak

species.

Acknowledgements

We thank the comments provided by two anonymous reviewers and the handling editor (N.

Chaffey) for improving a previous version of the manuscript. A.Q.A. and J.J.C. acknowledge

the support of MAEC-AECID and ARAID, respectively. This work was supported by the

Spanish Ministerio de Ciencia e Innovación and FEDER (grant numbers CGL2008-04847-

C02-01, CGL2010-16880 BOS, CGL2011-26654 BOS). We thank the support of the

Globimed network. We thank the “Oficina del Regante de Aragón” and “Agencia Estatal de

Meterorología (AEMET)” for providing climatic data. We acknowledge Sara Palacio for

revising a previous version of this work. We thank J. Albuixech and E. Lahoz for their help in

the field and the laboratory, respectively.

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