Annals of Botany Page 1 of 10
doi:10.1093/aob/mcw274, available online at https://academic.oup.com/aob
How does climate influence xylem morphogenesis over the growing season?
Insights from long-term intra-ring anatomy in Picea abies
Daniele Castagneri1,*, Patrick Fonti2, Georg von Arx2 and Marco Carrer1
1
Universit
a degli Studi di Padova, Dept. TeSAF, Viale dell’Universit
a 16, 35020 Legnaro (PD), Italy and
2
Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Zürcherstrasse 111, 8903
Birmensdorf (ZH), Switzerland
*For correspondence. E-mail [email protected]
Received: 3 November 2016 Returned for revision: 15 November 2016 Editorial decision: 30 November 2016
Background and Aims During the growing season, the cambium of conifer trees produces successive rows of
xylem cells, the tracheids, that sequentially pass through the phases of enlargement and secondary wall thickening
before dying and becoming functional. Climate variability can strongly influence the kinetics of morphogenetic processes, eventually affecting tracheid shape and size. This study investigates xylem anatomical structure in the stem
of Picea abies to retrospectively infer how, in the long term, climate affects the processes of cell enlargement and
wall thickening.
Methods Tracheid anatomical traits related to the phases of enlargement (diameter) and wall thickening (wall
thickness) were innovatively inspected at the intra-ring level on 87-year-long tree-ring series in Picea abies trees
along a 900 m elevation gradient in the Italian Alps. Anatomical traits in ten successive tree-ring sectors were related to daily temperature and precipitation data using running correlations.
Key Results Close to the altitudinal tree limit, low early-summer temperature negatively affected cell enlargement.
At lower elevation, water availability in early summer was positively related to cell diameter. The timing of these relationships shifted forward by about 20 (high elevation) to 40 (low elevation) d from the first to the last tracheids in the
ring. Cell wall thickening was affected by climate in a different period in the season. In particular, wall thickness of
late-formed tracheids was strongly positively related to August–September temperature at high elevation.
Conclusions Morphogenesis of tracheids sequentially formed in the growing season is influenced by climate conditions in successive periods. The distinct climate impacts on cell enlargement and wall thickening indicate that different morphogenetic mechanisms are responsible for different tracheid traits. Our approach of long-term and highresolution analysis of xylem anatomy can support and extend short-term xylogenesis observations, and increase our
understanding of climate control of tree growth and functioning under different environmental conditions.
Key words: Cell size, cell-wall thickness, climate change, Norway spruce, quantitative wood anatomy, secondary
growth, tracheid, tree ring, xylogenesis.
INTRODUCTION
The anatomy of xylem determines the ability of a tree to transport water from soil to leaves and to support its own structure.
Recent studies linking tree physiology with wood anatomy have
demonstrated that the morphology of the xylem cells affects
functional properties such as hydraulic safety and efficiency
(Lachenbruch and McCulloh, 2014; Schuldt et al., 2016).
Therefore, modifications in xylem anatomy strongly determine
the performance and survival of trees, and consequently forests’
vulnerability to climate change and their capacity to fix carbon
(Anderegg, 2015; Sperry and Love, 2015; Pellizzari et al., 2016).
Secondary growth, i.e. the formation of xylem cells through
cambial activity, has been extensively investigated since the last
century, providing detailed insights into the physiology that regulates plant cell development (Heyn, 1940; Růzicka et al., 2015).
Experimental manipulations have additionally shown how internal factors, e.g. hormones (Aloni, 1980), and external factors,
e.g. temperature (Denne, 1971) and water availability (Hsiao,
1973), govern this process. However, the information gained was
mostly limited to observations in controlled settings (such as in a
greenhouse) performed on non-woody species or very young
trees, being only partially indicative of growth mechanisms occurring in adult trees in complex and variable natural systems
(Rathgeber et al., 2011; Wolkovich et al., 2012).
Wood formation (xylogenesis) is known to be influenced by
environmental conditions that vary in both space and time (Cuny
et al., 2015). Restricted water availability generally limits tree
growth in warm and dry areas, while low temperatures control
growth at high latitudes and elevations (Rossi et al., 2013).
However, the mechanisms behind the environmental influence
on meristematic processes are still rather poorly understood. This
knowledge is critical for a mechanistic understanding of the climate control of tree growth [e.g. carbon source-limitation versus
sink-limitation hypotheses (Körner, 2015)] and for assessing legacy effects on future xylem functioning under changing climatic
conditions (Sass-Klaassen et al., 2016).
Studies monitoring xylogenesis (i.e. weekly cytological observations of tree-ring formation) can provide important insights into environmental control of wood formation in adult
trees. They have evidenced site-specific adaptation of xylogenesis processes (Gricar et al., 2015) and latitude and elevation
C The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.
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Page 2 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy
Max. temperature (°C)
140
120
100
80
30
60
20
40
10
20
0
Precipitation (mm)
influences on the timing and duration of cambial activity
(e.g. Moser et al., 2010; Rossi et al., 2014). However, despite
the strong inter-annual variability of cambial phenology
(Treml et al., 2015), this process has usually been quantified
with just a few years (typically 1–5 in most studies) of continuous observations. In addition, studies monitoring xylogenesis
have provided clues on how climate before and during the
growing season affects the onset of cambial activity (Rossi
et al., 2013) and impacts the kinetics of cell formation,
influencing xylem cell morphological traits (Cuny et al., 2014).
However, these observations lack the necessary long-term perspective to thoroughly assess the responses of wood formation
and structure to inter-annual climate variability (Boulouf Lugo
et al., 2012). A long-term perspective can be obtained by the
retrospective quantitative analysis of xylem anatomy in treering series. Past investigations have shown that it is possible
to link cell anatomical features with inter-annual climate variability (Fonti et al., 2010). Methodological advances (von Arx
and Carrer, 2014; von Arx et al., 2016) now allow significantly longer (several centuries) and better replicated (two
to three orders of magnitude more cells) time series of xylem
anatomical features than in the recent past to be processed
and analysed efficiently, thus opening new investigation
prospects.
Based on the assumption that tracheids record the weather
conditions influencing their formation, we investigated the longterm tracheid anatomical variation in the stem of mature Picea
abies (Norway spruce) trees along a 900-m elevation gradient in
the Italian Alps. The most conspicuous effect of increasing elevation is an adiabatic decline of temperature, which further reduces
large-scale environmental (e.g. photoperiod, and seasonality in
precipitation and temperature) and genetic variability. In the
European Alps, this is generally coupled to increased mean precipitation (Körner, 2007). Elevation gradients are thus valuable
settings to assess responses to changing climate (Körner, 2007).
Previous analyses at the same location had already shown a climate influence on tree-ring width and potential hydraulic conductivity associated with the 900-m gradient (Castagneri et al.,
2015). Here we make use of this setting to evaluate how climatic
variability controls the morphogenesis of two functionally important anatomical traits: tracheid size, resulting from the process of
cell enlargement, and wall thickness, which results from the successive deposition of the cell wall. Tracheid anatomy was innovatively assessed at intra-ring level, with the aim at evaluating
how and when climate variability affects tracheid morphogenesis
within the growing season. Given that (1) tracheid enlargement
and wall thickening are two distinct processes (Vaganov et al.,
2006), (2) tracheid enlargement is more sensitive to water availability than other xylogenesis phases (Hsiao, 1973; Abe et al.,
2003), and (3) the carbon-demanding latewood wall thickening
is more limited at higher elevation by the temperature-sensitive
process of carbon fixation (Petit et al., 2011; Rossi et al., 2013;
Körner, 2015), we expect that these morphogenetic processes in
P. abies are differently influenced by climate across elevation.
Here we used an elevation gradient to evaluate the role of climate in shaping tracheid diameter and wall thickness, and to verify whether temporal shifts and duration of the influence of
climate on morphogenesis change according to the length of the
growing season, which is shorter at higher elevation (Körner,
2007).
0
0 30 60 90 120 150 180 210 240 270 300 330 360
Day of the year
FIG. 1. Annual course of maximum temperature and precipitation in the study
area. In red, daily maximum temperature mean over 31-d moving windows with
a daily step; in blue, precipitation sum over 31-d moving windows with a daily
step. Weather data were recorded from 1926 to 2012 at the meteorological station of Cortina d’Ampezzo, 1230 m a.s.l., located 3 km from the study sites.
MATERIALS AND METHODS
Study area
Samples were collected in the Eastern Italian Alps along a
northeast-facing slope at Croda da Lago (46 300 N, 12 070 E).
The slope, ranging from 1200 m a.s.l. up to the tree limit at
2150 m a.s.l., is covered by open, uneven-aged multi-layered
forest stands composed of Picea abies occurring exclusively or
mixed with other conifers (Larix decidua, Pinus cembra, Pinus
sylvestris and Abies alba). The stands have not been managed
for decades or affected by major disturbances. The soils are
shallow and calcareous and the climate of the region is relatively moist and cool. The average daily maximum (minimum)
temperature is 208 (89) C in July and 31 (58) C in
January (meteorological station of Cortina d’Ampezzo, 1230 m
a.s.l., 1926–2012, <3 km from the area). Mean annual precipitation, occurring as snow during winter, is 1080 mm, and is
most abundant from May to November (Fig. 1).
Sample collection and processing
Increment cores were collected from three stands located at
2100 (EL21), 1600 (EL16) and 1200 (EL12) m a.s.l. at a distance of 1–2 km from each other. According to the standard
sampling protocol for the analysis of tree-ring responses to climate (Schweingruber et al., 1990), we selected 15–20 dominant
and undamaged adult trees in each stand. For each tree, two
cores (515 mm in diameter) were collected with an increment
borer (Haglöf, Långsele, Sweden) perpendicularly to the slope
on opposite sides of the stem. To assign the annual rings to the
corresponding calendar year, the ring widths were measured to
the nearest 001 mm using TsapWin (Rinntech, Heidelberg,
Germany) for visual cross-dating, and cross-dating quality was
checked using COFECHA (Holmes, 1983). Subsequent cell anatomical measurements were then conducted for each site on a
selection of eight cores from eight trees (Table 1), avoiding
samples with visible defects such as nodes, reaction wood or
rotten and missing parts. The selected cores were divided into
pieces 4–5 cm long to prepare transverse sections (15–18 lm
thick) for anatomical measurements using standard protocols
Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 3 of 10
TABLE 1. Characteristics of trees selected for ring anatomical measurements
Plot
EL21
EL16
EL12
No. of samples
Height
(m)
Diameter
(cm)
Ring width
(mm)
No. of rings
Estimated age
(years)
8
8
8
198635
291631
366616
5536142
576679
703689
0906026
0896036
1866036
236646
289653
141612
263654
301642
149612
Values are mean 6 standard deviation of eight trees at site EL21 (2100 m a.s.l.), EL16 (1600 m a.s.l.) and EL12 (1200 m a.s.l.).
Stem diameter was measured 13 m above the ground.
Ring width is averaged over the period 1926–2012.
No. of rings is the total number of rings in the core. Estimated age includes estimation of rings missing to the stem pith.
(von Arx et al., 2016). In short, micro-sections were cut with a
rotary microtome (Leica, Heidelberg, Germany), stained with
safranin (1 % in distilled water) and fixed on permanent slides
with Eukitt (BiOptica, Milan, Italy). Overlapping digital images
were captured with a light microscope at 40 magnification
(Nikon Eclipse 80 with distortion-free lenses) with a resolution
of 0833 pixels mm1, and stitched together with PTGui software (New House Internet Service B.V., Rotterdam, The
Netherlands) to obtain a single image of 2–3 mm width from
each section. The images were then processed with the image
analysis software ROXAS v2.0 (von Arx and Carrer, 2014),
which provided the lumen size, wall thickness and relative position within the dated annual ring for each of the 4 million
measured tracheids. Analysis was restricted to the period covered by weather records, i.e. 1926–2012.
Intra-ring anatomical series and chronologies
Diameter and wall thickness were assessed for each tracheid
in the ring (Fig. 2). To analyse xylem anatomy at the sub-annual
time scale, each annual ring was divided into ten tangential sectors of equal width (Fig. 2). For each sector, we assessed the
90th percentile of the size distribution of cell diameter (CD) and
cell-wall thickness (CWT). For each core, we then built ten time
series for both CD and CWT, which represented the inter-annual
variations of tracheid anatomical features at intra-ring level.
To assess the climate influence on xylem traits, we sought to
emphasize high-frequency variation, related to inter-annual climate variability, through the removal of low-frequency variation not related to climate (Fritts, 1976). Indeed, anatomical
time series usually show long-term trends, due to tree height
growth during ontogenesis (Fig. 3A) (Carrer et al., 2015). We
therefore fitted a cubic smoothing spline with 50 % frequency
cut-off of 30 years to each individual anatomical series, and calculated the detrended index as the ratio between the observed
and fitted values for each annual ring (Cook and Kairiukstis,
1990) (Fig. 3B). Chronologies of the anatomical features (2
traits 3 sites 10 sectors ¼ 60 sector chronologies) were
built by calculating the bi-weight robust mean from the
detrended series, using the R package dplR (Bunn, 2008).
Statistical analyses and assessment of climate–anatomy
relationships
To evaluate the influence of elevation on xylem anatomical
traits, one-way analysis of variance (ANOVA) was applied to
test differences in CD and CWT in each sector among the three
sites. When elevation showed a significant effect, Tukey’s pairwise comparisons were used to determine which group was significantly different from the others. Besides, to assess
anatomical trait variations along the ring, one-way ANOVA
was applied between the ten sectors. Tukey’s pairwise comparisons were used to identify significantly different groups.
The mean correlation coefficients between all possible combinations of the eight series [r between trees (rbt)] was adopted
to assess the shared pattern across individual series building
each sector chronology (Fritts, 1976) with the R package dplR
(Bunn, 2008). Furthermore, the (in)dependence of information
provided by ring sectors separated by different distances was
evaluated for each anatomical feature and site by calculating
the shared variance (coefficient of determination, coefficient of
determination, R2100) among sector chronologies. All statistical analyses were performed on anatomical parameters from
1926 to 2012, corresponding to the period covered by the
weather records from Cortina d’Ampezzo meteorological station (Fig. 1).
Climate influence on cell anatomical features was quantified
by correlating each sector chronology with daily maximum
temperature and precipitation sum with Pearson’s correlations
using 31-d windows slid at 1-d steps from 1 July of the previous
year to 31 October of the current year. Climate time series were
obtained using daily-resolved weather records from the Cortina
d’Ampezzo meteorological station (Fig. 1). Besides this conventional approach with climatic data aligned to the calendar
date [day of year (DOY) approach], we also calculated the correlations aligning the climatic data to the date when a specific
degree-day temperature sum is reached (DD approach). The
DD temperature sums of each year (McMaster and Wilhelm,
1997) were calculated as:
m X
Tmax þ Tmin
j
2
5
where j ¼ 1, 2, . . . m are days with a mean temperature >5 C,
which is widely used as the lower limit for plant growth
(Grigorieva et al., 2010), and Tmax and Tmin are the daily maximum and minimum air temperatures ( C), respectively. As
EL16 and EL21 are located at higher elevations than the meteorological station, for these two sites temperature was adjusted
considering a lapse rate of 06 C/100 m of elevation (Auer
et al., 2005; Körner, 2007). For the alignment of the climatic
data, we considered seven DD thresholds (0, 20, 40, 60, 80, 100
Page 4 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy
50
5·0
40
4·0
30
3·0
20
2·0
10
1·0
0
B
6·0
1
2
3
4
5 6
Sector
7
8
9
10
CD
CWT
Growth direction
CWT (µm)
CD (µm)
A 60
Cell wall thickness
Cell diameter
0
FIG. 2. Example of a tree ring split into ten sectors (A) and representation of the anatomical features measured on each single tracheid (B). In (A), the overlay curves
show the intra-ring variation of the mean 90th percentile of cell diameter (CD) and cell-wall thickness (CWT) as obtained for the ten tree-ring sectors with equal
width. The coloured cells illustrate on a narrow strip how individual tracheids were assigned to the sectors.
A
B
60
CD (index)
Sector
1
2
50
5
6
CD (µm)
3
4
Sector 1
1·2
1·1
1·0
0·9
0·8
40
C
Sector 10
1·2
8
CD (index)
7
30
9
10
1·1
1·0
0·9
0·8
20
1800
1850
1900
Year
1950
2000
1940
1960 1980
Year
2000
FIG. 3. Cell diameter (CD) time series. (A) Variability of CD along a single core, as grouped by ring sector. The shaded area indicates the investigated period
(1926–2012). (B, C) Thirty-year spline-detrended CD series for sectors 1 (B) and 10 (C) for the eight trees measured at the 2100 m site over the period 1926–2012.
Thick lines indicate the mean ring-sector chronologies.
and 120 DD). For each of these cases with newly aligned climatic data, moving correlations were calculated with the 31-d
windows as for the DOY approach. For both DOY and DD
approaches, correlation P values were adjusted for multiple
comparisons using the false discovery rate correction
(Benjamini and Hochberg, 1995). Analyses were implemented
in R (version 3.1.0, R Development Core Team, 2014).
RESULTS
Sub-annual anatomical variability and common pattern
Variations in cell anatomical features along the ring sectors
were consistent among elevations. Within the ring, CD steadily
decreased from sector 1 to 10, with a pronounced reduction in
the last three sectors (Figs 2 and 3, Table 2). Cell-wall thickness
showed the opposite trend, with a sharp increase in the last sectors. In addition, the negative relationship between CD and
CWT along the sectors was consistent at the three sites (EL21,
r ¼ 097, P < 0001; EL16, r ¼ 098, P < 0001; EL12,
r ¼ 098, P < 0001; Fig. 4). For all sectors, both CD and
CWT were larger at EL12 than at the other two sites (Table 2).
The detrended anatomical series had a fairly strong common
pattern (rbt), revealing good synchronization of the inter-annual
variations in anatomical features within the same sector on different trees. In general, for both CD and CWT, rbt increased
across the annual ring (Table 2). The highest value (rbt ¼ 056)
occurred for CWT in the last sector at EL21.
Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 5 of 10
TABLE 2. Mean 90th percentile of cell diameter (CD) and cellwall thickness (CWT), and mean correlation between the
detrended time series (Rbt) of eight trees for tree-ring sector averaged over 1926–2012, at EL21 (at 2100 m a.s.l.), EL16
(1600 m a.s.l.) and EL12 (1200 m a.s.l.)
CD
EL21
1
2
3
4
5
6
7
8
9
10
A
1
2
3
4
5
6
7
8
9
10
021
023
023
022
024
025
035
043
044
043
CWT
EL16
Mean (lm)
506 c
504 c
A
498 c
B
484 c
C
469 c
D
453 b
E
434 b
F
401 b
G
332 b
H
228 b
A
A
525 b
517 b
B
509 b
C
492 b
D
475 b
E
458 b
F
440 b
G
407 b
H
335 b
I
225 b
Rbt
010
008
014
017
017
020
021
029
032
029
AB
EL12
A
EL21
EL16
EL12
Mean (lm)
592 a
587 a
B
571 a
C
556 a
D
545 a
E
531 a
F
513 a
G
476 a
H
400 a
I
283 a
37 b
37 b
G
38 b
FG
39 b
F
39 b
E
41 b
D
43 b
C
46 b
B
52 b
A
53 c
021
022
030
034
036
040
037
036
030
011
008
006
009
012
012
017
020
038
051
056
A
G
HI
G
H
34 c
33 c
HI
34 c
GH
35 c
FG
35 c
E
36 c
D
38 c
C
42 c
B
51 b
A
56 b
Rbt
002
007
006
010
009
010
015
027
031
026
HI
41 a
39 a
HI
40 a
GH
41 a
F
42 a
E
44 a
D
46 a
C
52 a
B
64 a
A
71 a
H
001
007
009
005
008
009
018
027
031
017
Different upper-case letters on the left of the value indicate significant differences between sectors for the same site (column), according to ANOVA
with Tukey’s pairwise comparisons. Different lower-case letters on the right
indicate significant differences between the three sites EL12, EL16 and EL21,
for the same sector (row).
60
EL12
EL16
EL21
CD (µm)
50
40
30
20
3
4
5
6
CWT (µm)
7
8
FIG. 4. Relationship between CD and CWT in the ten ring sectors. Colours represent the sector, from dark blue (sector 1) to dark purple (sector 10). Sites (see
key): EL21, 2100 m a.s.l.; EL16, 1600 m a.s.l.; EL12, 1200 m a.s.l.
Among the ten sectors, anatomical chronologies were relatively independent of each other. For both CD and CWT, the
proportion of shared variance in neighbouring sectors was high
(inter-sector distance ¼ 1, Fig. 5). However, it sharply decreased when considering more distant sectors (generally
<25 % at inter-sector distance >3), indicating that anatomical features of cells formed in different periods of the season (not consecutive sectors) were quite independent of each
other.
CD
B
CWT
75
Shared variance (%)
Sector
A
EL21
EL16
EL12
50
25
0
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
Inter-sector distance
FIG. 5. Proportion of shared variance (R2100) between ring-sector chronologies for (A) cell diameter (CD) and (B) cell-wall thickness (CWT) at equivalent
inter-sector distance (e.g. boxes at inter-sector distance 5 indicate the proportion
of shared variance between sectors 1 and 6, 2 and 7, 3 and 8, 4 and 9, and 5 and
10). Horizontal lines in the boxes indicate median values of shared variance,
boxes the first and third quartiles, and notches the 95 % confidence interval of
the median. Sites (see key): EL21, 2100 m a.s.l.; EL16, 1600 m a.s.l.; EL12,
1200 m a.s.l.
Climate influence on xylem anatomy
Correlation analyses between inter-annual climate variability
and sector chronologies revealed that different anatomical features showed distinct relationships with climate factors, mostly
during spring and summer. Moreover, we observed temporal
shifts in climatic responses along the ring, which reflect the seasonal changes in the timing of environmental constraints on cell
formation.
At EL21, a positive correlation between CD and maximum
temperature occurred from mid-June in the first ring sectors to
early July in the last ones (P < 005 in sectors 1, 2, 3 and 5;
P < 001 in sectors 6 and 10; P < 00001 in sectors 7–9; Fig.
6A). This correlation increased markedly when considering the
DD temperature sums. Specifically, when considering the 40DD threshold, which over the study period occurred on average
on 19 June (corresponding to DOY 170), correlations improved
for all the sectors (P < 0001 in all sectors; Fig. 7). During
August, there was a negative correlation with temperature
(P < 005 in nine sectors; Fig. 6A). At EL16, the negative correlation with temperature was observed 15–30 d earlier than
at EL21, specifically around DOY 170 (late June, sectors 3–7,
P < 005 to P < 001; Fig. 6C) to 215 (end of July, last sectors,
P < 005; Fig. 6C). Positive correlations with precipitation occurred a few days before, specifically around DOY 160 (midJune, sectors 3 and 4, P < 005; Fig. 6D) to 215 (July, last sectors, P < 005; Fig. 6D). At EL12, the negative correlation of
CD with temperature (P < 005 in sectors 4–0; P < 0001 in
sectors 6 and 7; Fig. 6E) and positive correlations with precipitation (significant in all sectors; P < 0001 in sectors 2, 6 and 7;
Fig. 6F) were slightly stronger than at EL16, and occurred
Page 6 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy
Temperature
Sector
Apr
May
Jun
Jul
Precipitation
Aug
Sep
Oct
Apr
May
Jun
Jul
Aug
Sep
Oct
1
2
3
4
5
6
7
8
9
10
A
EL21
B
EL21
1
2
3
4
5
6
7
8
9
10
C
EL16
D
EL16
1
2
3
4
5
6
7
8
9
10
E
EL12
F
EL12
100 120 140 160 180 200 220 240 260 280 300 100 120 140 160 180 200 220 240 260 280 300
Day of the year (DOY)
Day of the year (DOY)
Negative
<0·0001 <0·01
P value
<0·1 <0·1
<0·001 <0·05
n.s.
Positive
<0·01 <0·0001
<0·05 <0·001
FIG. 6. Correlations of the ten ring-sector chronologies of cell diameter (CD) with daily climate data. Mean daily maximum temperature and precipitation sums were
calculated over 31-d moving windows from DOY 100 (10 April) to DOY 300 (27 October) (x-axis) over the period 1926–2012 at the three sites: (A, B) EL21,
2100 m a.s.l.; (C, D) EL16, 1600 m a.s.l.; (E, F) EL12, 1200 m a.s.l. Results are aligned to the centre of the 31-d window. Colours represent P values adjusted for
the false discovery rate. Non-significant correlations are not represented (white).
10–15 d earlier. At this elevation, positive correlation with precipitation emerged in the first sectors and remained quite stable
across the whole ring, with a time lag of 40–50 d from the first
to the last sectors. At all three sites, CD was weakly connected
to climatic variability before the growing season, with generally
unclear patterns except for a positive relationship with late June
temperature at EL12 (P < 005 in sectors 8, 9 and 10), and
February precipitation at EL21 (P < 005 in sectors 1, 2, 3, 8
and 10) (Supplementary Data Fig. S1).
Cell-wall thickness showed completely different relationships with climate compared with CD (Fig. 8). Temperatures in
late spring and/or summer were positively related to this feature, while relationships with precipitation were weak at all elevations. May temperature was positively correlated with CWT
for the last three sectors at EL21 (P < 001 in sectors 8, 9 and
10; Fig. 8A) and the central sectors at EL12 (P < 0 01 in
sectors 4 and 5; Fig. 8E). Furthermore, a positive association
with late-summer temperature emerged for the last four
sectors, being stronger at EL21, particularly for the last three
sectors (r > 05, P < 00001; Fig. 8A), weaker at EL16 (r > 04,
P < 005; Fig. 8C), and not significant at EL12 (Fig. 8E).
Similarly to CD, this association increased at EL21 using the
DD approach (r > 06; Fig. 7 compared with Fig. 8). Climatic
conditions of the previous growing season and winter were
slightly related to CWT (Supplementary Data Fig. S1).
DISCUSSION
Intra-annual variability in xylem anatomy
This study demonstrated that the analysis of xylem anatomy
along tree-ring series can be used to assess the long-term climate influence on xylem morphogenesis. Compared with previous analyses of anatomical features considering the whole ring
or earlywood and latewood sections (Fonti et al., 2013), our approach aimed at improving the detail of results by increasing
the resolution at intra-seasonal level. Across the entire elevation
gradient, both CD and CWT showed a substantial synchronous
Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 7 of 10
Sector
Sector
Cell diameter
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Cell wall thickness
–60 –40 –20 0
20 40 60 80 100 120
Day aligned to temperature sums (DD)
Negative
<0·0001 <0·01
P value
<0·1
<0·1
<0·001 <0·05
n.s.
Positive
<0·01 <0·0001
<0·05 <0·001
FIG. 7. Correlations of the ten ring-sector chronologies of cell diameter (CD)
and cell-wall thickness (CWT) at EL21 (2100 m a.s.l.) with degree-day (DD)aligned temperature. Mean daily maximum temperature (1926–2012) was calculated over 31-d moving windows aligned according to the DD approach.
Temperature data span from 70 d before to 130 d after the day of the year when
DD temperature > 5 C reached 40 (x-axis). Results are aligned to the centre of
the 31-d window. Colours represent P values adjusted for the false discovery
rate. Non-significant correlations are not represented (white).
variability among trees (rbt; Table 2), although with differences
among the ring sectors. This suggests that cell anatomy, and
thus morphogenesis, is sensitive to environmental variability
(Fritts, 1976), but this sensitivity can also vary over the course
of the growing season (Sass and Eckstein, 1995). The different
patterns of rbt, with generally higher values for CWT towards
the last ring sectors and with increasing elevation, also indicate
that not all anatomical traits are equally sensitive to climate.
This is in line with previous observations showing different sensitivity to climate in different cell anatomical features, and between earlywood and latewood (Martin-Benito et al., 2013;
Szymczak et al., 2014).
The decreasing similarities with increasing distance between
ring sectors (Fig. 5) likely reflect changes in the environmental
influences that impact cell formation during the growing season. This suggests that successive tracheid rows along the ring
encode a distinct climate imprint throughout the growing season, demonstrating the potential of splitting the ring into sectors
for investigating the associations between cell morphogenesis
and intra-seasonal climate.
Temporal match of intra-ring climate correlations with cambial
activity observations
Sector-based correlations showed that cell anatomical features were significantly affected by seasonal climate variability.
The timing of cell enlargement and wall thickening, recorded in
previous investigations on cambial activity, matches well with
the timing of most of the observed significant correlations.
Indeed, significant correlations between CD and temperature at
high elevation started in early June for the first ring sectors,
lasting to mid-July in the last sectors (Fig. 6). This timing
matches the most intense phases of cambial activity and cell enlargement observed for P. abies at similar elevation, a few kilometres from the study site (Rossi et al., 2008). Information on
cambial activity in the same species under environmental conditions similar to those at the low-elevation sites are unfortunately not available. However, the observed lapse rate of 10–
15 d for both the temperature and precipitation associations
with CD within the same sector from intermediate to low elevation, and of 20–40 d for temperature associations in CD from
high to intermediate elevation, fits the lapse rate of 3–4 d per
100 m in elevation reported for xylogenesis phases of Larix decidua in the Swiss Alps (Moser et al., 2010). These observations indicate that the proposed approach properly captures
phenological shifts along elevation gradients.
Climate influence on CWT (Fig. 8) was evident in two distinct periods: a moderate positive temperature effect at the beginning of the season, and a second one at the end of the
summer for the last three or four sectors, lasting for up to
2 months. The latter, much stronger at high elevation, overlaps
with the wall-thickening phase in the last part of the ring observed in P. abies at high elevation (Gindl et al., 2001; Rossi
et al., 2008), and its duration is in line with current knowledge
on the duration of wall formation, which can last from 30 d
(mild conditions) to 70 d (cold conditions) in latewood cells
(Kirdyanov et al., 2003; Rossi et al., 2008, 2013; Cuny et al.,
2013).
Intra-ring anatomy reflects climate impact on cell morphogenesis
Our retrospective analysis of the climate influence on cell
features provided indirect indications on xylem morphogenesis
that are in line with current knowledge, mainly derived from
more direct approaches based on much shorter periods, such as
observations of cambial activity (e.g. Cuny et al., 2014; Rossi
et al., 2014) and diel stem radial variations (e.g. King et al.,
2013; Steppe et al., 2015). In particular, our results indicate that
climate drivers are diverse for different anatomical features, depending on the nature and timing of the underlying processes
during the growing season, and on the limiting conditions prevailing at different elevations.
Specifically, we interpret the positive influence of temperature in June to early July on CD at the highest elevation as an
indication that warm conditions promote the first phases of xylem formation (Lenz et al., 2013). In this context, temperature
at the onset of the growing season affects the production and
transport of hormones such as auxin and gibberellin (Schrader
et al., 2003; Aloni et al., 2015) and the translocation of nutrients. Correspondingly, the important role of temperature in cold
environments in the kinetics of cell differentiation and enlargement has already been observed both empirically (Gricar et al.,
2006; Rossi et al., 2008; Petit et al., 2011; Lenz et al., 2013)
and correlatively (Kirdyanov et al., 2003; Kalliokoski et al.,
2012; Fonti et al., 2013; Rossi et al., 2014). Stronger and more
Page 8 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy
Temperature
Sector
Apr
1
2
3
4
5
6
7
8
9
10
A
1
2
3
4
5
6
7
8
9
10
C
1
2
3
4
5
6
7
8
9
10
E
May
Jun
Jul
Precipitation
Aug
Sep
Oct
Apr
EL21
B
May
Jun
Jul
Aug
Sep
Oct
EL21
D
EL16
EL12
EL16
F
EL12
100 120 140 160 180 200 220 240 260 280 300 100 120 140 160 180 200 220 240 260 280 300
Day of the year (DOY)
Day of the year (DOY)
Negative
P value
Positive
<0·0001 <0·01 <0·1 <0·1 <0·01 <0·0001
<0·001 <0·05
n.s. <0·05 <0·001
FIG. 8. Correlations of the ten ring-sector chronologies of cell-wall thickness (CWT) with daily climate data. Mean daily maximum temperature and precipitation
sums were calculated over 31-d moving windows from DOY 100 (10 April) to DOY 300 (27 October) (x-axis) over the period 1926–2012 at the three sites: (A, B)
EL21, 2100 m a.s.l.; (C, D) EL16, 1600 m a.s.l.; (E, F) EL12, 1200 m a.s.l. Results are aligned to the centre of the 31-d window. Colours represent P values adjusted
for the false discovery rate. Non-significant correlations are not represented (white).
defined associations emerged when the climatic data were
aligned according to the DD temperature thresholds at high elevation (especially for CD in the first ring sectors; Figs 6 and 7),
but not at lower elevations (see Cuny et al., 2015). These add
additional evidence and further confirm that late-spring temperature controls the onset of cambial activity and the kinetics of
cell formation in cold environments (Kirdyanov et al., 2003;
Seo et al., 2008; Thibeault-Martel et al., 2008; Moser et al.,
2010; Olano et al., 2013). However, we also observed waterrelated constraints on tracheid morphogenesis. At high elevation, a negative temperature influence on CD emerged a few
weeks after the positive temperature stimulus. At lower elevation, the negative effect of temperature on CD was stronger,
better defined across the ring sectors, anticipated by a few
weeks, and associated with a positive effect of precipitation.
Apparently, at low elevation, early summer temperature does
not limit cell enlargement, resulting in generally larger tracheids compared with higher elevation. However, during dry
and warm years, low soil water content and high tree transpiration rates decrease water availability, reducing the cell turgor
pressure necessary to stretch the primary cell wall of the forming cells (Hsiao, 1973; Taiz and Zeiger, 2006).
While our results support the hypothesis that the climate effects on CD are directly connected to the processes shaping cell
size (i.e. cell enlargement), the responses encoded in CWT
seem to be primarily related to the amount of carbon assimilates
reaching the cambial zone (Wolf et al., 2012). Coherently with
studies on maximum latewood density, we found correlations
with climate for the last ring sectors in relation to the conditions
at the beginning and towards the end of the growing season
(Wimmer and Graber, 2000; Frank and Esper, 2005). The positive effect of April–May temperature on CWT might relate to
the improved carbon assimilation during spring (source activity) corresponding to an earlier onset of the growing season and
more intense assimilation activity. The late summer temperature association is instead probably linked to carbon mobilization and deposition rates at the time of wall thickening (sink
activity) (Hoch et al., 2002; Körner, 2015), which were apparently strongly related to temperature at high elevation. Our observations support previous findings that carbon fixation and
Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 9 of 10
carbohydrate mobilization are constrained by temperature at the
altitudinal limit of a species (Hoch and Korner, 2003; Simard
et al., 2013). Examples of this relationship are also given by observations that the long-lasting wall thickening process can be
slowed or interrupted by reduced temperatures at the end of
summer (Gindl, 1999; Vaganov et al., 2006; Xu et al., 2013), in
extreme cases resulting in rings characterized by thin walls
(known as light rings; Filion et al., 1986) or unlignified walls
(known as blue rings; Piermattei et al., 2014) in the last cells.
Concluding remarks and future prospects
Our study shows that xylem anatomy assessed through treering partitioning provides both high-resolution and long-term
indications on how, when and for how long climate drivers influence the processes of xylem morphogenesis. Under different
conditions along an elevation gradient, climate either affects
cell enlargement through regulation of the kinetics of the process (influenced by temperature) or by influencing the cell turgor pressure necessary for cell enlargement (related to water
availability). Similarly, we have shown that the deposition of
the cell wall in the second part of the ring is sensitive to the
temperature occurring both at the beginning of the growing season, which seemingly promotes carbon assimilation, and at the
end of the season, when deposition in the cell wall occurs.
Tree-ring anatomy provides a long-term perspective on tree
responses to climate, fundamental for soundly assessing forest
responses to ongoing climatic change (Reyer et al., 2015). This
can improve understanding of xylem formation, and help to assess changes over time in (1) growth phenology, (2) climatic
sensitivity, (3) xylem functioning and (4) xylem plasticity of
different species – all of them important topics for improving
our mechanistic understanding of how climate impacts xylem
hydraulic and structural properties and ultimately tree
performance.
SUPPLEMENTARY DATA
Supplementary data are available online at https://academic.
oup.com/aob and consist of the following. Figure S1: correlations of ring-sector chronologies with temperature and precipitation over all year round.
ACKNOWLEDGEMENTS
The study was supported by the University of Padova
(Research Project D320.PRGR13001, Senior Research Grants
2012). D.C. received a grant from PROFOUND COST action
for a short-term scientific mission at the WSL (ECOSTSTSM-FP1304-230215-053843). G.v.A. was supported by
grants from the Swiss State Secretariat for Education,
Research and Innovation SERI (SBFI C14.0104 and
C12.0100). This study profited from discussions within the
framework of the COST Action STReESS (COST-FP1106).
The authors are grateful to Enrico Marcolin, Francesco
Natalini, Giai Petit and Angela Luisa Prendin for their help in
laboratory work, and to two anonymous reviewers and the
handling editor, which helped to improve an earlier version of
this manuscript.
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