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

1Universit�a degli Studi di Padova, Dept. TeSAF, Viale dell’Universit�a 16, 35020 Legnaro (PD), Italy and2Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Zurcherstrasse 111, 8903

Birmensdorf (ZH), Switzerland*For correspondence. E-mail daniele.castagneri@unipd.it

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 ofxylem cells, the tracheids, that sequentially pass through the phases of enlargement and secondary wall thickeningbefore dying and becoming functional. Climate variability can strongly influence the kinetics of morphogenetic pro-cesses, eventually affecting tracheid shape and size. This study investigates xylem anatomical structure in the stemof Picea abies to retrospectively infer how, in the long term, climate affects the processes of cell enlargement andwall thickening.� Methods Tracheid anatomical traits related to the phases of enlargement (diameter) and wall thickening (wallthickness) were innovatively inspected at the intra-ring level on 87-year-long tree-ring series in Picea abies treesalong a 900 m elevation gradient in the Italian Alps. Anatomical traits in ten successive tree-ring sectors were re-lated 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 rela-tionships shifted forward by about 20 (high elevation) to 40 (low elevation) d from the first to the last tracheids in thering. Cell wall thickening was affected by climate in a different period in the season. In particular, wall thickness oflate-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 con-ditions in successive periods. The distinct climate impacts on cell enlargement and wall thickening indicate that dif-ferent morphogenetic mechanisms are responsible for different tracheid traits. Our approach of long-term and high-resolution analysis of xylem anatomy can support and extend short-term xylogenesis observations, and increase ourunderstanding 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, secondarygrowth, tracheid, tree ring, xylogenesis.

INTRODUCTION

The anatomy of xylem determines the ability of a tree to trans-port water from soil to leaves and to support its own structure.Recent studies linking tree physiology with wood anatomy havedemonstrated that the morphology of the xylem cells affectsfunctional properties such as hydraulic safety and efficiency(Lachenbruch and McCulloh, 2014; Schuldt et al., 2016).Therefore, modifications in xylem anatomy strongly determinethe 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 throughcambial activity, has been extensively investigated since the lastcentury, providing detailed insights into the physiology that regu-lates plant cell development (Heyn, 1940; Ru�zi�cka et al., 2015).Experimental manipulations have additionally shown how inter-nal 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 wasmostly limited to observations in controlled settings (such as in a

greenhouse) performed on non-woody species or very youngtrees, being only partially indicative of growth mechanisms oc-curring 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 byenvironmental conditions that vary in both space and time (Cunyet al., 2015). Restricted water availability generally limits treegrowth in warm and dry areas, while low temperatures controlgrowth at high latitudes and elevations (Rossi et al., 2013).However, the mechanisms behind the environmental influenceon meristematic processes are still rather poorly understood. Thisknowledge is critical for a mechanistic understanding of the cli-mate control of tree growth [e.g. carbon source-limitation versussink-limitation hypotheses (Korner, 2015)] and for assessing leg-acy effects on future xylem functioning under changing climaticconditions (Sass-Klaassen et al., 2016).

Studies monitoring xylogenesis (i.e. weekly cytological ob-servations of tree-ring formation) can provide important in-sights into environmental control of wood formation in adulttrees. They have evidenced site-specific adaptation of xylogen-esis processes (Gri�car et al., 2015) and latitude and elevation

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influences on the timing and duration of cambial activity(e.g. Moser et al., 2010; Rossi et al., 2014). However, despitethe strong inter-annual variability of cambial phenology(Treml et al., 2015), this process has usually been quantifiedwith just a few years (typically 1–5 in most studies) of continu-ous observations. In addition, studies monitoring xylogenesishave provided clues on how climate before and during thegrowing season affects the onset of cambial activity (Rossiet 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 per-spective to thoroughly assess the responses of wood formationand structure to inter-annual climate variability (Boulouf Lugoet al., 2012). A long-term perspective can be obtained by theretrospective quantitative analysis of xylem anatomy in tree-ring series. Past investigations have shown that it is possibleto link cell anatomical features with inter-annual climate vari-ability (Fonti et al., 2010). Methodological advances (von Arxand Carrer, 2014; von Arx et al., 2016) now allow signifi-cantly longer (several centuries) and better replicated (twoto three orders of magnitude more cells) time series of xylemanatomical features than in the recent past to be processedand analysed efficiently, thus opening new investigationprospects.

Based on the assumption that tracheids record the weatherconditions influencing their formation, we investigated the long-term tracheid anatomical variation in the stem of mature Piceaabies (Norway spruce) trees along a 900-m elevation gradient inthe Italian Alps. The most conspicuous effect of increasing eleva-tion is an adiabatic decline of temperature, which further reduceslarge-scale environmental (e.g. photoperiod, and seasonality inprecipitation and temperature) and genetic variability. In theEuropean Alps, this is generally coupled to increased mean pre-cipitation (Korner, 2007). Elevation gradients are thus valuablesettings to assess responses to changing climate (Korner, 2007).Previous analyses at the same location had already shown a cli-mate influence on tree-ring width and potential hydraulic con-ductivity associated with the 900-m gradient (Castagneri et al.,2015). Here we make use of this setting to evaluate how climaticvariability controls the morphogenesis of two functionally impor-tant anatomical traits: tracheid size, resulting from the process ofcell enlargement, and wall thickness, which results from the suc-cessive deposition of the cell wall. Tracheid anatomy was inno-vatively assessed at intra-ring level, with the aim at evaluatinghow and when climate variability affects tracheid morphogenesiswithin the growing season. Given that (1) tracheid enlargementand wall thickening are two distinct processes (Vaganov et al.,2006), (2) tracheid enlargement is more sensitive to water avail-ability than other xylogenesis phases (Hsiao, 1973; Abe et al.,2003), and (3) the carbon-demanding latewood wall thickeningis more limited at higher elevation by the temperature-sensitiveprocess of carbon fixation (Petit et al., 2011; Rossi et al., 2013;Korner, 2015), we expect that these morphogenetic processes inP. abies are differently influenced by climate across elevation.Here we used an elevation gradient to evaluate the role of cli-mate in shaping tracheid diameter and wall thickness, and to ver-ify whether temporal shifts and duration of the influence ofclimate on morphogenesis change according to the length of thegrowing season, which is shorter at higher elevation (Korner,2007).

MATERIALS AND METHODS

Study area

Samples were collected in the Eastern Italian Alps along anortheast-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 at2150 m a.s.l., is covered by open, uneven-aged multi-layeredforest stands composed of Picea abies occurring exclusively ormixed with other conifers (Larix decidua, Pinus cembra, Pinussylvestris and Abies alba). The stands have not been managedfor decades or affected by major disturbances. The soils areshallow and calcareous and the climate of the region is rela-tively moist and cool. The average daily maximum (minimum)temperature is 20�8 (8�9) �C in July and 3�1 (�5�8) �C inJanuary (meteorological station of Cortina d’Ampezzo, 1230 ma.s.l., 1926–2012, <3 km from the area). Mean annual precipi-tation, occurring as snow during winter, is 1080 mm, and ismost abundant from May to November (Fig. 1).

Sample collection and processing

Increment cores were collected from three stands located at2100 (EL21), 1600 (EL16) and 1200 (EL12) m a.s.l. at a dis-tance of 1–2 km from each other. According to the standardsampling protocol for the analysis of tree-ring responses to cli-mate (Schweingruber et al., 1990), we selected 15–20 dominantand undamaged adult trees in each stand. For each tree, twocores (5�15 mm in diameter) were collected with an incrementborer (Haglof, Langsele, Sweden) perpendicularly to the slopeon opposite sides of the stem. To assign the annual rings to thecorresponding calendar year, the ring widths were measured tothe nearest 0�01 mm using TsapWin (Rinntech, Heidelberg,Germany) for visual cross-dating, and cross-dating quality waschecked using COFECHA (Holmes, 1983). Subsequent cell an-atomical measurements were then conducted for each site on aselection of eight cores from eight trees (Table 1), avoidingsamples with visible defects such as nodes, reaction wood orrotten and missing parts. The selected cores were divided intopieces 4–5 cm long to prepare transverse sections (15–18 lmthick) for anatomical measurements using standard protocols

30

140

120

100

80

60

40

20

0

20

Max

. tem

pera

ture

(°C

) Precipitation (m

m)

10

00 30 60 90 120 150 180

Day of the year210 240 270 300 330 360

FIG. 1. Annual course of maximum temperature and precipitation in the studyarea. In red, daily maximum temperature mean over 31-d moving windows witha daily step; in blue, precipitation sum over 31-d moving windows with a dailystep. Weather data were recorded from 1926 to 2012 at the meteorological sta-

tion of Cortina d’Ampezzo, 1230 m a.s.l., located �3 km from the study sites.

Page 2 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy

(von Arx et al., 2016). In short, micro-sections were cut with arotary microtome (Leica, Heidelberg, Germany), stained withsafranin (1 % in distilled water) and fixed on permanent slideswith Eukitt (BiOptica, Milan, Italy). Overlapping digital imageswere captured with a light microscope at 40� magnification(Nikon Eclipse 80 with distortion-free lenses) with a resolutionof 0�833 pixels mm�1, and stitched together with PTGui soft-ware (New House Internet Service B.V., Rotterdam, TheNetherlands) to obtain a single image of 2–3 mm width fromeach section. The images were then processed with the imageanalysis software ROXAS v2.0 (von Arx and Carrer, 2014),which provided the lumen size, wall thickness and relative posi-tion within the dated annual ring for each of the �4 millionmeasured tracheids. Analysis was restricted to the period cov-ered by weather records, i.e. 1926–2012.

Intra-ring anatomical series and chronologies

Diameter and wall thickness were assessed for each tracheidin the ring (Fig. 2). To analyse xylem anatomy at the sub-annualtime scale, each annual ring was divided into ten tangential sec-tors of equal width (Fig. 2). For each sector, we assessed the90th percentile of the size distribution of cell diameter (CD) andcell-wall thickness (CWT). For each core, we then built ten timeseries for both CD and CWT, which represented the inter-annualvariations of tracheid anatomical features at intra-ring level.

To assess the climate influence on xylem traits, we sought toemphasize high-frequency variation, related to inter-annual cli-mate variability, through the removal of low-frequency varia-tion not related to climate (Fritts, 1976). Indeed, anatomicaltime series usually show long-term trends, due to tree heightgrowth during ontogenesis (Fig. 3A) (Carrer et al., 2015). Wetherefore fitted a cubic smoothing spline with 50 % frequencycut-off of 30 years to each individual anatomical series, and cal-culated the detrended index as the ratio between the observedand fitted values for each annual ring (Cook and Kairiukstis,1990) (Fig. 3B). Chronologies of the anatomical features (2traits � 3 sites � 10 sectors ¼ 60 sector chronologies) werebuilt by calculating the bi-weight robust mean from thedetrended series, using the R package dplR (Bunn, 2008).

Statistical analyses and assessment of climate–anatomyrelationships

To evaluate the influence of elevation on xylem anatomicaltraits, one-way analysis of variance (ANOVA) was applied to

test differences in CD and CWT in each sector among the threesites. When elevation showed a significant effect, Tukey’s pair-wise comparisons were used to determine which group was sig-nificantly different from the others. Besides, to assessanatomical trait variations along the ring, one-way ANOVAwas applied between the ten sectors. Tukey’s pairwise compari-sons were used to identify significantly different groups.

The mean correlation coefficients between all possible com-binations of the eight series [r between trees (rbt)] was adoptedto assess the shared pattern across individual series buildingeach sector chronology (Fritts, 1976) with the R package dplR(Bunn, 2008). Furthermore, the (in)dependence of informationprovided by ring sectors separated by different distances wasevaluated for each anatomical feature and site by calculatingthe shared variance (coefficient of determination, coefficient ofdetermination, R2�100) among sector chronologies. All statisti-cal analyses were performed on anatomical parameters from1926 to 2012, corresponding to the period covered by theweather records from Cortina d’Ampezzo meteorological sta-tion (Fig. 1).

Climate influence on cell anatomical features was quantifiedby correlating each sector chronology with daily maximumtemperature and precipitation sum with Pearson’s correlationsusing 31-d windows slid at 1-d steps from 1 July of the previousyear to 31 October of the current year. Climate time series wereobtained using daily-resolved weather records from the Cortinad’Ampezzo meteorological station (Fig. 1). Besides this con-ventional approach with climatic data aligned to the calendardate [day of year (DOY) approach], we also calculated the cor-relations aligning the climatic data to the date when a specificdegree-day temperature sum is reached (DD approach). TheDD temperature sums of each year (McMaster and Wilhelm,1997) were calculated as:

Xm

j

Tmax þ Tmin

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 maxi-mum and minimum air temperatures (�C), respectively. AsEL16 and EL21 are located at higher elevations than the meteo-rological station, for these two sites temperature was adjustedconsidering a lapse rate of 0�6 �C/100 m of elevation (Aueret al., 2005; Korner, 2007). For the alignment of the climaticdata, we considered seven DD thresholds (0, 20, 40, 60, 80, 100

TABLE 1. Characteristics of trees selected for ring anatomical measurements

Plot No. of samples Height Diameter Ring width No. of rings Estimated age(m) (cm) (mm) (years)

EL21 8 19�863�5 55�3614�2 0�9060�26 236646 263654EL16 8 29�163�1 57�667�9 0�8960�36 289653 301642EL12 8 36�661�6 70�368�9 1�8660�36 141612 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 1�3 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.

Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 3 of 10

and 120 DD). For each of these cases with newly aligned cli-matic data, moving correlations were calculated with the 31-dwindows as for the DOY approach. For both DOY and DDapproaches, correlation P values were adjusted for multiplecomparisons using the false discovery rate correction(Benjamini and Hochberg, 1995). Analyses were implementedin 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 sectorswere consistent among elevations. Within the ring, CD steadily

decreased from sector 1 to 10, with a pronounced reduction inthe last three sectors (Figs 2 and 3, Table 2). Cell-wall thicknessshowed the opposite trend, with a sharp increase in the last sec-tors. In addition, the negative relationship between CD andCWT along the sectors was consistent at the three sites (EL21,r¼� 0�97, P< 0�001; EL16, r¼� 0�98, P< 0�001; EL12,r¼� 0�98, P< 0�001; Fig. 4). For all sectors, both CD andCWT were larger at EL12 than at the other two sites (Table 2).

The detrended anatomical series had a fairly strong commonpattern (rbt), revealing good synchronization of the inter-annualvariations in anatomical features within the same sector on dif-ferent trees. In general, for both CD and CWT, rbt increasedacross the annual ring (Table 2). The highest value (rbt¼ 0�56)occurred for CWT in the last sector at EL21.

A B6·0

5·0

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T (µm

)CD

(µm

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1·0

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Sector7 8 9 10

60

50

40

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20

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0

CDCWT

Cell wall thickness

Cell diameter

Growth direction

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 curvesshow 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.

1·2Sector 1

1·1

60A B

C

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(µm

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1800 1850 1900Year

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(in

dex)

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1940 1960 1980Year

2000

Sector1

2

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4

5

6

7

8

9

10

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.

Page 4 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy

Among the ten sectors, anatomical chronologies were rela-tively independent of each other. For both CD and CWT, theproportion of shared variance in neighbouring sectors was high(inter-sector distance ¼ 1, Fig. 5). However, it sharply de-creased when considering more distant sectors (generally<25 % at inter-sector distance >3), indicating that anatomi-cal features of cells formed in different periods of the sea-son (not consecutive sectors) were quite independent of eachother.

Climate influence on xylem anatomy

Correlation analyses between inter-annual climate variabilityand sector chronologies revealed that different anatomical fea-tures showed distinct relationships with climate factors, mostlyduring spring and summer. Moreover, we observed temporalshifts in climatic responses along the ring, which reflect the sea-sonal changes in the timing of environmental constraints on cellformation.

At EL21, a positive correlation between CD and maximumtemperature occurred from mid-June in the first ring sectors toearly July in the last ones (P< 0�05 in sectors 1, 2, 3 and 5;P< 0�01 in sectors 6 and 10; P< 0�0001 in sectors 7–9; Fig.6A). This correlation increased markedly when considering theDD temperature sums. Specifically, when considering the 40-DD threshold, which over the study period occurred on averageon 19 June (corresponding to DOY 170), correlations improvedfor all the sectors (P< 0�001 in all sectors; Fig. 7). DuringAugust, there was a negative correlation with temperature(P< 0�05 in nine sectors; Fig. 6A). At EL16, the negative cor-relation with temperature was observed �15–30 d earlier thanat EL21, specifically around DOY 170 (late June, sectors 3–7,P< 0�05 to P< 0�01; Fig. 6C) to 215 (end of July, last sectors,P< 0�05; Fig. 6C). Positive correlations with precipitation oc-curred a few days before, specifically around DOY 160 (mid-June, sectors 3 and 4, P< 0�05; Fig. 6D) to 215 (July, last sec-tors, P< 0�05; Fig. 6D). At EL12, the negative correlation ofCD with temperature (P< 0�05 in sectors 4–0; P< 0�001 insectors 6 and 7; Fig. 6E) and positive correlations with precipi-tation (significant in all sectors; P< 0�001 in sectors 2, 6 and 7;Fig. 6F) were slightly stronger than at EL16, and occurred

60

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EL12EL16EL21

CD

(µm

)

40

30

203 4 5

CWT (µm)6 7 8

FIG. 4. Relationship between CD and CWT in the ten ring sectors. Colours rep-resent 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.

TABLE 2. Mean 90th percentile of cell diameter (CD) and cell-wall thickness (CWT), and mean correlation between thedetrended time series (Rbt) of eight trees for tree-ring sector av-eraged over 1926–2012, at EL21 (at 2100 m a.s.l.), EL16

(1600 m a.s.l.) and EL12 (1200 m a.s.l.)

Sector CD CWT

EL21 EL16 EL12 EL21 EL16 EL12Mean (lm) Mean (lm)

1 A 50�6 c A 52�5 b A 59�2 a G 3�7 b HI 3�4 c HI 4�1 a

2 A 50�4 c AB 51�7 b A 58�7 a G 3�7 b H 3�3 c H 3�9 a

3 A 49�8 c B 50�9 b B 57�1 a G 3�8 b HI 3�4 c HI 4�0 a

4 B 48�4 c C 49�2 b C 55�6 a FG 3�9 b GH 3�5 c GH 4�1 a

5 C 46�9 c D 47�5 b D 54�5 a F 3�9 b FG 3�5 c F 4�2 a

6 D 45�3 b E 45�8 b E 53�1 a E 4�1 b E 3�6 c E 4�4 a

7 E 43�4 b F 44�0 b F 51�3 a D 4�3 b D 3�8 c D 4�6 a

8 F 40�1 b G 40�7 b G 47�6 a C 4�6 b C 4�2 c C 5�2 a

9 G 33�2 b H 33�5 b H 40�0 a B 5�2 b B 5�1 b B 6�4 a

10 H 22�8 b I 22�5 b I 28�3 a A 5�3 c A 5�6 b A 7�1 a

Rbt Rbt1 0�21 0�10 0�21 0�08 0�02 0�012 0�23 0�08 0�22 0�06 0�07 0�073 0�23 0�14 0�30 0�09 0�06 0�094 0�22 0�17 0�34 0�12 0�10 0�055 0�24 0�17 0�36 0�12 0�09 0�086 0�25 0�20 0�40 0�17 0�10 0�097 0�35 0�21 0�37 0�20 0�15 0�188 0�43 0�29 0�36 0�38 0�27 0�279 0�44 0�32 0�30 0�51 0�31 0�3110 0�43 0�29 0�11 0�56 0�26 0�17

Different upper-case letters on the left of the value indicate significant dif-ferences between sectors for the same site (column), according to ANOVAwith Tukey’s pairwise comparisons. Different lower-case letters on the rightindicate significant differences between the three sites EL12, EL16 and EL21,for the same sector (row).

75

A BCD CWT

50

Sha

red

varia

nce

(%)

25

01 2 3 4 5 6 7 8 9

Inter-sector distance1 2 3 4 5 6 7 8 9

EL21EL16EL12

FIG. 5. Proportion of shared variance (R2�100) between ring-sector chronolo-gies for (A) cell diameter (CD) and (B) cell-wall thickness (CWT) at equivalentinter-sector distance (e.g. boxes at inter-sector distance 5 indicate the proportionof shared variance between sectors 1 and 6, 2 and 7, 3 and 8, 4 and 9, and 5 and10). Horizontal lines in the boxes indicate median values of shared variance,boxes the first and third quartiles, and notches the 95 % confidence interval ofthe median. Sites (see key): EL21, 2100 m a.s.l.; EL16, 1600 m a.s.l.; EL12,

1200 m a.s.l.

Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 5 of 10

10–15 d earlier. At this elevation, positive correlation with pre-cipitation emerged in the first sectors and remained quite stableacross the whole ring, with a time lag of 40–50 d from the firstto the last sectors. At all three sites, CD was weakly connectedto climatic variability before the growing season, with generallyunclear patterns except for a positive relationship with late Junetemperature at EL12 (P< 0�05 in sectors 8, 9 and 10), andFebruary precipitation at EL21 (P< 0�05 in sectors 1, 2, 3, 8and 10) (Supplementary Data Fig. S1).

Cell-wall thickness showed completely different relation-ships with climate compared with CD (Fig. 8). Temperatures inlate spring and/or summer were positively related to this fea-ture, while relationships with precipitation were weak at all ele-vations. May temperature was positively correlated with CWTfor the last three sectors at EL21 (P< 0�01 in sectors 8, 9 and10; Fig. 8A) and the central sectors at EL12 (P< 0 �01 insectors 4 and 5; Fig. 8E). Furthermore, a positive associationwith late-summer temperature emerged for the last foursectors, being stronger at EL21, particularly for the last three

sectors (r> 0�5, P< 0�0001; Fig. 8A), weaker at EL16 (r> 0�4,P< 0�05; Fig. 8C), and not significant at EL12 (Fig. 8E).Similarly to CD, this association increased at EL21 using theDD approach (r> 0�6; Fig. 7 compared with Fig. 8). Climaticconditions of the previous growing season and winter wereslightly related to CWT (Supplementary Data Fig. S1).

DISCUSSION

Intra-annual variability in xylem anatomy

This study demonstrated that the analysis of xylem anatomyalong tree-ring series can be used to assess the long-term cli-mate influence on xylem morphogenesis. Compared with previ-ous analyses of anatomical features considering the whole ringor earlywood and latewood sections (Fonti et al., 2013), our ap-proach aimed at improving the detail of results by increasingthe resolution at intra-seasonal level. Across the entire elevationgradient, both CD and CWT showed a substantial synchronous

Sec

tor

123456789

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10

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Day of the year (DOY)

Negative P value

<0·001 <0·05 n.s.

<0·0001 <0·01 <0·1 <0·1

<0·05 <0·001

Positive<0·01 <0·0001

100 120 140 160 180 200 220 240 260 280 300Day of the year (DOY)

100 120 140 160 180 200 220 240 260 280 300

EL21 EL21

EL16 EL16

EL12 EL12

Apr May

Temperature Precipitation

Jun Jul Aug Sep Oct Apr May Jun Jul Aug Sep Oct

A B

C D

E F

FIG. 6. Correlations of the ten ring-sector chronologies of cell diameter (CD) with daily climate data. Mean daily maximum temperature and precipitation sums werecalculated 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).

Page 6 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy

variability among trees (rbt; Table 2), although with differencesamong the ring sectors. This suggests that cell anatomy, andthus morphogenesis, is sensitive to environmental variability(Fritts, 1976), but this sensitivity can also vary over the courseof the growing season (Sass and Eckstein, 1995). The differentpatterns of rbt, with generally higher values for CWT towardsthe last ring sectors and with increasing elevation, also indicatethat not all anatomical traits are equally sensitive to climate.This is in line with previous observations showing different sen-sitivity to climate in different cell anatomical features, and be-tween earlywood and latewood (Martin-Benito et al., 2013;Szymczak et al., 2014).

The decreasing similarities with increasing distance betweenring sectors (Fig. 5) likely reflect changes in the environmentalinfluences that impact cell formation during the growing sea-son. This suggests that successive tracheid rows along the ringencode a distinct climate imprint throughout the growing sea-son, demonstrating the potential of splitting the ring into sectorsfor investigating the associations between cell morphogenesisand intra-seasonal climate.

Temporal match of intra-ring climate correlations with cambialactivity observations

Sector-based correlations showed that cell anatomical fea-tures were significantly affected by seasonal climate variability.

The timing of cell enlargement and wall thickening, recorded inprevious investigations on cambial activity, matches well withthe timing of most of the observed significant correlations.Indeed, significant correlations between CD and temperature athigh elevation started in early June for the first ring sectors,lasting to mid-July in the last sectors (Fig. 6). This timingmatches the most intense phases of cambial activity and cell en-largement observed for P. abies at similar elevation, a few kilo-metres from the study site (Rossi et al., 2008). Information oncambial activity in the same species under environmental con-ditions similar to those at the low-elevation sites are unfortu-nately not available. However, the observed lapse rate of 10–15 d for both the temperature and precipitation associationswith CD within the same sector from intermediate to low eleva-tion, and of 20–40 d for temperature associations in CD fromhigh to intermediate elevation, fits the lapse rate of �3–4 d per100 m in elevation reported for xylogenesis phases of Larix de-cidua in the Swiss Alps (Moser et al., 2010). These observa-tions indicate that the proposed approach properly capturesphenological shifts along elevation gradients.

Climate influence on CWT (Fig. 8) was evident in two dis-tinct periods: a moderate positive temperature effect at the be-ginning of the season, and a second one at the end of thesummer for the last three or four sectors, lasting for up to2 months. The latter, much stronger at high elevation, overlapswith the wall-thickening phase in the last part of the ring ob-served in P. abies at high elevation (Gindl et al., 2001; Rossiet al., 2008), and its duration is in line with current knowledgeon 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 cellfeatures provided indirect indications on xylem morphogenesisthat are in line with current knowledge, mainly derived frommore direct approaches based on much shorter periods, such asobservations of cambial activity (e.g. Cuny et al., 2014; Rossiet al., 2014) and diel stem radial variations (e.g. King et al.,2013; Steppe et al., 2015). In particular, our results indicate thatclimate drivers are diverse for different anatomical features, de-pending on the nature and timing of the underlying processesduring the growing season, and on the limiting conditions pre-vailing at different elevations.

Specifically, we interpret the positive influence of tempera-ture in June to early July on CD at the highest elevation as anindication that warm conditions promote the first phases of xy-lem formation (Lenz et al., 2013). In this context, temperatureat the onset of the growing season affects the production andtransport of hormones such as auxin and gibberellin (Schraderet al., 2003; Aloni et al., 2015) and the translocation of nutri-ents. Correspondingly, the important role of temperature in coldenvironments in the kinetics of cell differentiation and enlarge-ment has already been observed both empirically (Gri�car 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

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Cell wall thickness

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Day aligned to temperature sums (DD)–60 –40 –20 0 20 40 60 80 100 120

Negative P value

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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 calcu-lated 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 whenDD temperature > 5 �C reached 40 (x-axis). Results are aligned to the centre ofthe 31-d window. Colours represent P values adjusted for the false discovery

rate. Non-significant correlations are not represented (white).

Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy Page 7 of 10

defined associations emerged when the climatic data werealigned according to the DD temperature thresholds at high ele-vation (especially for CD in the first ring sectors; Figs 6 and 7),but not at lower elevations (see Cuny et al., 2015). These addadditional evidence and further confirm that late-spring temper-ature controls the onset of cambial activity and the kinetics ofcell 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 water-related constraints on tracheid morphogenesis. At high eleva-tion, a negative temperature influence on CD emerged a fewweeks after the positive temperature stimulus. At lower eleva-tion, the negative effect of temperature on CD was stronger,better defined across the ring sectors, anticipated by a fewweeks, and associated with a positive effect of precipitation.Apparently, at low elevation, early summer temperature doesnot limit cell enlargement, resulting in generally larger tra-cheids compared with higher elevation. However, during dryand warm years, low soil water content and high tree transpira-tion rates decrease water availability, reducing the cell turgor

pressure necessary to stretch the primary cell wall of the form-ing cells (Hsiao, 1973; Taiz and Zeiger, 2006).

While our results support the hypothesis that the climate ef-fects on CD are directly connected to the processes shaping cellsize (i.e. cell enlargement), the responses encoded in CWTseem to be primarily related to the amount of carbon assimilatesreaching the cambial zone (Wolf et al., 2012). Coherently withstudies on maximum latewood density, we found correlationswith climate for the last ring sectors in relation to the conditionsat the beginning and towards the end of the growing season(Wimmer and Graber, 2000; Frank and Esper, 2005). The posi-tive effect of April–May temperature on CWT might relate tothe improved carbon assimilation during spring (source activ-ity) corresponding to an earlier onset of the growing season andmore intense assimilation activity. The late summer tempera-ture association is instead probably linked to carbon mobiliza-tion and deposition rates at the time of wall thickening (sinkactivity) (Hoch et al., 2002; Korner, 2015), which were appar-ently strongly related to temperature at high elevation. Our ob-servations support previous findings that carbon fixation and

Apr

A B

C D

E F

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Temperature Precipitation

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EL21 EL21

EL16 EL16

EL12 EL12

Sep Oct Apr May Jun Jul Aug Sep OctS

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Day of the year (DOY)Negative P value

<0·001 <0·05 n.s.

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<0·05 <0·001

Positive<0·01 <0·0001

100 120 140 160 180 200 220 240 260 280 300Day of the year (DOY)

100 120 140 160 180 200 220 240 260 280 300

FIG. 8. Correlations of the ten ring-sector chronologies of cell-wall thickness (CWT) with daily climate data. Mean daily maximum temperature and precipitationsums 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).

Page 8 of 10 Castagneri et al. — Climate influence on xylem morphogenesis as evidenced by intra-ring anatomy

carbohydrate mobilization are constrained by temperature at thealtitudinal limit of a species (Hoch and Korner, 2003; Simardet al., 2013). Examples of this relationship are also given by ob-servations that the long-lasting wall thickening process can beslowed or interrupted by reduced temperatures at the end ofsummer (Gindl, 1999; Vaganov et al., 2006; Xu et al., 2013), inextreme 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 tree-ring partitioning provides both high-resolution and long-termindications on how, when and for how long climate drivers in-fluence the processes of xylem morphogenesis. Under differentconditions along an elevation gradient, climate either affectscell enlargement through regulation of the kinetics of the pro-cess (influenced by temperature) or by influencing the cell tur-gor pressure necessary for cell enlargement (related to wateravailability). Similarly, we have shown that the deposition ofthe cell wall in the second part of the ring is sensitive to thetemperature occurring both at the beginning of the growing sea-son, which seemingly promotes carbon assimilation, and at theend of the season, when deposition in the cell wall occurs.

Tree-ring anatomy provides a long-term perspective on treeresponses to climate, fundamental for soundly assessing forestresponses to ongoing climatic change (Reyer et al., 2015). Thiscan improve understanding of xylem formation, and help to as-sess changes over time in (1) growth phenology, (2) climaticsensitivity, (3) xylem functioning and (4) xylem plasticity ofdifferent species – all of them important topics for improvingour mechanistic understanding of how climate impacts xylemhydraulic and structural properties and ultimately treeperformance.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Figure S1: correla-tions of ring-sector chronologies with temperature and precipi-tation over all year round.

ACKNOWLEDGEMENTS

The study was supported by the University of Padova(Research Project D320.PRGR13001, Senior Research Grants2012). D.C. received a grant from PROFOUND COST actionfor a short-term scientific mission at the WSL (ECOST-STSM-FP1304-230215-053843). G.v.A. was supported bygrants from the Swiss State Secretariat for Education,Research and Innovation SERI (SBFI C14.0104 andC12.0100). This study profited from discussions within theframework of the COST Action STReESS (COST-FP1106).The authors are grateful to Enrico Marcolin, FrancescoNatalini, Giai Petit and Angela Luisa Prendin for their help inlaboratory work, and to two anonymous reviewers and thehandling editor, which helped to improve an earlier version ofthis manuscript.

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