Evidence of threshold temperatures for xylogenesis in conifers at high altitudes

ECOPHYSIOLOGY

Evidence of threshold temperatures for xylogenesis in conifersat high altitudes

Sergio Rossi Æ Annie Deslauriers Æ Tommaso Anfodillo ÆVinicio Carraro

Received: 23 June 2006 / Accepted: 17 November 2006 / Published online: 13 December 2006� Springer-Verlag 2006

Abstract Temperature is the most important factor

affecting growth at high altitudes. As trees use much of

the allocated carbon gained from photosynthesis to

produce branches and stems, information on the timing

and dynamics of secondary wood growth is crucial to

assessing temperature thresholds for xylogenesis. We

have carried out histological analyses to determine

cambial activity and xylem cell differentiation in

conifers growing at the treeline on the eastern Alps in

two sites during 2002–2004 with the aim of linking the

growth process with temperature and, consequently, of

defining thresholds for xylogenesis. Cambial activity

occurred from May to July–August and cell differen-

tiation from May–June to September–October. The

earliest start of radial enlargement was observed in

stone pine in mid-May, while Norway spruce was the

last species to begin tracheid differentiation. The

duration of wood formation varied from 90 to

137 days, depending on year and site, with no differ-

ence between species. Longer durations were observed

in trees on the south-facing site because of the earlier

onset and later ending of cell production and differ-

entiation. The threshold temperatures at which xylo-

genesis had a 0.5 probability of being active were

calculated by logistic regressions. Xylogenesis was

active when the mean daily air temperature was

5.6–8.5�C and mean stem temperature was 7.2–9�C.

The similar thresholds among all trees suggested the

existence of thermal limits in wood formation that

correspond with temperatures of 6–8�C that are sup-

posed to limit growth at the treeline. Different soil

temperature thresholds between sites indicated that

soil temperature may not be the main factor limiting

xylogenesis. This study represents the first attempt

to define a threshold through comparative assessment

of xylem growth and tissue temperatures in stem

meristems at high altitudes.

Keywords Alps � Cambial activity �Cell differentiation � Treeline � Tree ring

Introduction

High-altitude forests have been studied extensively in

order to gain an understanding of why trees cannot

grow above a certain altitude (Korner 2003). Given

that these ecotones are strongly temperature-limited,

they have recently assumed additional relevance as

potential indicators of climate change (Beniston et al.

1997; Theurillat and Guisan 2001; Pisaric et al. 2003).

The physiological determinant of treeline position at a

global scale is still uncertain despite several hypotheses

having been put forward (Tranquillini 1979;

Sveinbjornsson 2000; Smith et al. 2003). The hypothesis

best supported by experimental data asserts that low

temperatures limit the production of new cells by

meristems irrespective of photoassimilate abundance

(growth limitation hypothesis; Korner 1998). Deslau-

riers and Morin (2005) found that tracheid production

rate varies with the seasonal dynamics of minimum

temperature. Cambial activity becomes particularly

Communicated by Hermann Heilmeier.

S. Rossi (&) � A. Deslauriers � T. Anfodillo �V. CarraroTreeline Ecology Research Unit, Dipartimento TeSAF,Universita degli Studi di Padova, viale dell’Universita 16,35020 Legnaro, PD, Italye-mail: [email protected]

123

Oecologia (2007) 152:1–12

DOI 10.1007/s00442-006-0625-7

limited after the young plants have emerged from the

herbaceous-shrub layer where the generally warmer

temperatures are more favourable to growth (Grace

and Norton 1990; Hattenschwiler and Korner 1995).

This leads to a progressive decrease in longitudinal

annual growth when a plant approaches the ‘‘tree

habitus’’, i.e., more that 3 m in height. Because trees

growing at the treeline appear to be limited by pro-

cesses involving tissue formation, a basic knowledge of

intra-annual stem growth is fundamental to determin-

ing a reliable temperature threshold directly based on

cambial activity processes.

Within a global perspective, treeline position seems

to coincide with a mean temperature in the growing

season ranging between 6 and 7�C (Korner 2003),

suggesting that growth processes might be strongly

limited below this threshold. In situ measurements at

the northern treeline revealed that growth occurred

when the average daily temperature was above 5�C

(Holtmeier 1997; Schmitt et al. 2004), and no cam-

bial activity was observed at a temperature of 2�C

(Philipson et al. 1971) or with soil temperatures below

3–5�C (Turner and Streule 1983; Shonenberger and

Frey 1988; Korner 2003). Grace (1989) proposed stem

growth at the polar treeline with temperatures above

7�C, while Kramer and Kozlowski (1979) reported that

plants tended to become dormant at temperatures

below 10�C. Malyseve (1993) found that temperatures

of between 0 and 5�C during the growing season were

more effective than temperatures exceeding 10�C in

delimiting the arctic forest boundary in northern Asia .

Comparative studies on temperature limits to cell

growth processes therefore suggest that the critical

temperature range is between 0 and 10�C (Korner

1998).

Relationships between climate and wood produc-

tion, in terms of tree-ring width or density, have been

widely studied at the treeline in the Alps (Gindl et al.

2000; Carrer and Urbinati 2001; Motta and Nola 2001).

A number of studies have revealed positive effects of

June–July temperature on tree-ring width in Pinus

cembra and Larix decidua (Urbinati et al. 1998; Carrer

and Urbinati 2004; Oberhuber 2004), but no reliable

conclusion could be reached about the period of wood

production at the treeline. Carrer et al. (1998) mea-

sured stem growth with band dendrometers and found

that intra-annual variations in stem size indicated that

tree-ring formation in L. decidua and Picea abies at the

treeline lasts 50–60 days, from mid-June to the begin-

ning of August. These results provided experimental

support for the proposed relationships between sum-

mer temperature and tree-ring width. However, the

radius changes recorded by dendrometers also take

into account variations in stem water content (Zweifel

et al. 2000; Deslauriers et al. 2006) and, consequently,

are only an indirect assessment of the xylogenetic

activity. Tranquillini (1979) reported cell production in

the cambium occurring between mid-June and mid-

July and cell wall formation persisting until October at

1950 m a.s.l. in the Tyrol (Austria) in L. decidua

seedlings. However, studies of xylem cell formation are

still required on tree-shaped plants in order gain a

better understanding of xylogenesis at the upper forest

limits.

The few analyses carried out to date which take cell

production and xylogenesis in high-altitude forests into

account have provided very little information on

cambial activity, duration of xylem cell development

and the effect of climate on treeline trees over a short

time scale. Data are therefore needed on periods of

intra-annual xylem cell formation and threshold tem-

peratures for cambial activity. The aim of the investi-

gation reported here was to assess the timing and

dynamics of cell production and differentiation in the

stems of high-altitude conifer species in order to link

the growth processes with air, soil and meristem tem-

peratures and, consequently, define thresholds of

cambial activity. Histological analyses of developing

xylem cells were carried out on samples collected

within a very short time scale (7 days), thereby pro-

viding a precise definition of intra-annual tree-ring

formation during 2002–2004 at two sites in the eastern

Italian Alps.

Materials and methods

Study sites

The study area was located near the Cinque Torri

mountain group (Cortina d’Ampezzo, Belluno prov-

ince), at a high altitude above the upper limit of the

closed forest, in the eastern Italian Alps (46�27¢N,

12�08¢E). Two sites (5T-S and 5T-N) with uneven-aged

trees were selected on the two opposite slopes of the

mountains. 5T-S was located at 2080 m a.s.l. on a

south-facing slope, within a mixed open forest con-

sisting of groups of 5–15 trees of larch (Larix decidua

Mill.) and stone pine (Pinus cembra L.) and the occa-

sional Norway spruce [Picea abies (L.) Karst.]; as such,

this site corresponds to the timberline ecotone defini-

tion (Korner 1998). The forest originated from a col-

onisation of abandoned pastures and areas felled

during the first World War. 5T-N was located at a

distance of 500 m from 5T-S, on the north-facing slope

at 2130 m a.s.l. This site corresponds to a treeline

2 Oecologia (2007) 152:1–12

123

ecotone, as trees were very sparse and tree height

above the site decreased abruptly, with only isolated

larches and pines.

Temperature monitoring

Two standard weather stations were installed to mea-

sure air, soil and stem temperatures as a means of

monitoring microclimate variations at the sites. These

stations were positioned between the groups of trees at

site 5T-S and between the sparse trees at site 5T-N.

Temperatures were measured at intervals of 1 min and

recorded as averages every 15 min by means of CR10X

dataloggers (Campbell Scientific Corporation). Air

temperature was measured at 2 m. Soil temperature

was measured by six sensors per site which were placed

in the soil at a depth of about 15 cm. Stem temperature

was measured on two selected trees per species at 5T-S

and on three pines and one spruce at 5T-N. Stem

sensors were placed facing south, at a height of 1.3 m,

beneath the bark close to the cambial zone and pro-

tected by insulating shields. Daily mean, minimum and

maximum air, stem and soil temperatures were later

calculated with the time series obtained from the 96

(four values per hour) measurements (Fig. 1).

Tree selection

Forty-one dominant adult trees with upright stem and

similar growth patterns were selected for the study.

Trees with polycormic stems, partially dead crowns,

reaction wood or evident damage due to parasites were

excluded. The homogeneity of growth rate among

plants was assessed by extracting two wood cores from

each stem and counting the number of tracheids in the

tree rings of the three preceding growing seasons

(Deslauriers et al. 2003). Following a first preliminary

selection, 26 trees were chosen among the larches,

stone pines and Norway spruces at the sites, five trees

per species at each site, with the exception of spruce at

5T-N where only one tree was growing (Tables 1, 2).

Xylem sampling and preparation

Tree-ring formation was studied during 2002–2004.

Wood microcores (diameter: 2.4 mm) were collected

weekly between April and October from the stem

[between 30 cm below to 30 cm above breast height

(1.3 m)] using an increment puncher in 2002 and 2003

(Forster et al. 2000) and Trephor in 2004 (Rossi et al.

2006a). The very small wounds inflicted by the thin

piercing tubes of the tools and the consequent narrow

traumatised tissues around the sampling points allowed

repeated samplings by microcore extraction (Forster

et al. 2000). Samples usually contained the previous

four or five tree rings and the developing annual layer

with the cambial zone and adjacent phloem. Dead

outer bark was removed prior to collecting the sam-

ples. A mean of 624 microcores were collected each

year and examined under a microscope, with only an

2002raeY

51-

01-

5-

0

5

01

51

02

3002raeY 4002raeY

053003052002051001050

Tem

pera

ture

(°C

)

51-

01-

5-

0

5

01

51

02

raeyehtfoyaD

053003052002051001050

5T-S

5T-N

053003052002051001050

erutarepmetmetSerutarepmetriAerutarepmetlioS

Fig. 1 Mean daily air, stem and soil temperatures recorded at sites 5T-S and 5T-N during 2002–2004

Oecologia (2007) 152:1–12 3

123

infrequent detection of isolated resin ducts. Wood

samples were always taken at least 5 cm apart to avoid

getting resin ducts on adjacent cores, which is a com-

mon reaction to disturbance in conifers (Deslauriers

et al. 2003). As only 13 sections in 3 years showed

tangentially oriented clusters of resin ducts in the

developing tree ring, there was clearly only incidental

spreading of the disturbance reaction of the xylem to

the wound to adjacent samples.

The microcores were placed in Eppendorf microtu-

bes with an ethanol solution (50% in water) and stored

at 5�C to avoid tissue deterioration. Microcores were

oriented by marking the transversal side with a pencil

under a stereo-microscope at a magnification of 10–

20·, dehydrated through a successive series of ethanol

and D-limonene and embedded in paraffin (Rossi et al.

2006a). Transverse sections were cut (6–10 lm in

thickness) from the samples with a rotary microtome.

Xylem analysis

Sections were stained for 10 min with cresyl violet

acetate (0.16% in water) and observed within 20 min

under visible and polarized light at a magnification of

400–500· to differentiate the developing xylem cells. In

each sample, the radial number of cells in the cambial

zone, radial enlargement phase, cell-wall thickening

phase and mature cells were counted along three radial

files. In cross-section, cambial cells were characterized

by thin cell walls and small radial diameters (Rossi

et al. 2006b). During cell enlargement, the tracheids

were composed of a protoplast still enclosed in the thin

primary wall but with a radial diameter at least twice

that of a cambial cell. Deformed rows of tracheids were

frequently observed in this phase due to the enlarge-

ment process occurring despite strong compression

between xylem tissues and bark. Observations under

polarized light discriminated between enlarging and

cell-wall thickening tracheids. Because of the

arrangement of cellulose microfibrils, the developing

secondary walls glistened when observed under polar-

ized light. However, no glistening was observed in the

enlargement zones where the cells still only consisted

of a primary wall (Abe et al. 1997). The progress of

cell-wall lignification was followed with cresyl violet

acetate stain, which reacted with the lignin (Rossi et al.

2006b), changing from violet to blue with increasing

lignification. The colour change over the whole cell

wall revealed the end of lignification and maturation of

the tracheid (Gricar et al. 2005).

The cell number in the three files was averaged for

each tree and used to assess onset, duration and ending

of xylem growth. Xylem formation was considered to

have begun in the spring when at least one horizontal

row of cells was observed in the enlarging phase. In

late summer, when no further cell was observed to be

undergoing wall thickening and lignification, xylem

formation was considered to be complete.

Statistical analyses

Analysis of variance (ANOVA) was used to compare

results (onset, duration and ending of xylogenesis, final

number of cells produced) between sites and species.

Responses among years were considered as within-

subject effects in repeated measures ANOVA [GLM

Table 1 Characteristics of the sampled trees at sites 5T-S and 5T-N in terms of mean and standard deviation of tree heights, diametersand ages

Species Height (m) Diameter (cm) Age (year)

5T-S 5T-N 5T-S 5T-N 5T-S 5T-N

Larix decidua 11.6 ± 1.0 6.4 ± 0.7 26.8 ± 4.8 15.4 ± 1.6 60.6 ± 18.7 63.8 ± 9.2Pinus cembra 10.1 ± 0.7 6.7 ± 1.5 29.2 ± 8.6 20.4 ± 5.2 60.4 ± 7.9 56.0 ± 13.4Picea abies 10.4 ± 0.6 8.0 22.4 ± 6.2 22.8 45.0 ± 8.7 74.0

Table 2 June–September mean air, stem and soil temperatures during 2002–2004 at sites 5T-S and 5T-N

Year Air temperature (�C) Stem temperature (�C) Soil temperature (�C)

5T-S 5T-N 5T-S 5T-N 5T-S 5T-N

2002 8.9 (2.8)a 9.1 (2.7) 10.2 (4.5) 9.6 (3.3) 9.3 (4.2) 7.3 (1.8)2003 11.4 (2.9) 11.6 (2.8) 12.8 (4.8) 11.7 (3.3) 11.1 (4.9) 9.3 (2.4)2004 9.0 (2.0) 9.2 (1.9) 11.1 (4.3) 9.7 (1.7) 9.8 (4.6) 7.0 (2.6)

a Annual mean temperatures are reported in parentheses. All values were calculated from daily means

4 Oecologia (2007) 152:1–12

123

procedure in SAS (1999)]. The hypothesis of com-

pound symmetry of the variance-covariance matrix

(Mauchly’s criterion) and sphericity (Huynh-Feldt

condition) were tested to check the assumptions for

using repeated measures ANOVA (Potvin et al. 1990).

The ANOVA model residuals were also examined

graphically and by performing the Shapiro-Wilk sta-

tistic [UNIVARIATE procedure in SAS (1999)] for

outliers and evidence of non-normality (Quinn and

Keough 2002).

Logistic regressions [LOGISTIC procedure in SAS

(1999)] were used to calculate the probability of xylo-

genesis being active at a given temperature where

binary responses were coded as non-active (value zero)

or active (value 1). The logistic regression takes the

general form:

LogitðpxÞ ¼ lnpx

1� px

� �¼ b0 þ b1xj

where px is the probability of xylogenesis being active,

xj is the temperature on a given day j, b0 and b1 are the

intercept and slope of the logit regression (Quinn and

Keough 2002). Temperatures thresholds (x) were cal-

culated when the probability of xylogenesis being ac-

tive was 0.5, i.e. when Logit(p) = 0 and x = –b0/b1.

Therefore, for a temperature above x, the wood for-

mation was more likely to be active than non-active.

Model verification included v2 of the likelihood ratio,

Wald’s v2 for regression parameter and goodness of fit

and Hosmer-Lemeshow’s C for eventual lack of fit

(Quinn and Keough 2002). For each tree, site and year,

the model was fitted with the respective temperature

series (mean, minimum and maximum air, stem and

soil temperatures). Only ten of 702 models were ex-

cluded because of a lack of fit. Temperature thresholds

were then compared between sites and species using

univariate ANOVA models [GLM procedure in SAS

(1999)].

Results

Air, stem and soil temperature

The area around the Cinque Torri has a typical alpine

climate of cold winters and cool summers, with mean

daily air temperatures reaching 17–20�C during 2002–

2004. In the three study years, the mean annual air

temperature was between 1.9 and 2.9�C and the mean

June–September air temperature was between 8.9 and

11.6�C, with higher values recorded in 2003 (Table 2).

The mean daily air temperature was very similar at

sites 5T-S and 5T-N, with differences restricted to

0.2�C during the warmest months (Fig. 1). However,

the main differences between sites were observed in

the day/night temperatures. Warmer daytime air tem-

peratures and colder temperatures during the night

were recorded at site 5T-S; consequently, daily tem-

perature excursions were higher at the south-facing

site. Stem temperature was coupled to air temperature,

with higher values collected on trees at site 5T-S.

However, stem temperature was closer to air temper-

ature from April to September while, in the colder

months, air temperature data with wider amplitudes

and higher temperature excursions were recorded.

Major differences were observed between the two sites

for annual and June–September soil temperatures

(Table 2). During sampling in May, snowmelts were

observed to occur earlier at site 5T-S. Soil tempera-

tures recorded in late spring also confirmed these

observations: during the snowmelt at site 5T-N, the

soil temperature fluctuated around 0�C and the soil

only began to warm when the snow had disappeared,

10–15 days after site 5T-S (Fig. 1).

Cambial activity

Similar annual dynamics were observed in the cambial

zones of the three tree species (Fig. 2). In spring and

autumn, when there was no cell production, the dor-

mant cambium consisted of six to eight cells in close

proximity to each other, as shown by the horizontal

dotted line in Fig. 2. In May, the number of cells in the

cambial zone had increased to 12–14, indicating the

onset of cell division in the cambium. Some variations

among trees were observed in terms of cambial cell

numbers, especially during the spring and early sum-

mer. Cambial activity was considered to have begun

when the number of cambial cells exceeded the mini-

mum number of dormant cells and the standard devi-

ation did not cross the horizontal dotted line (Fig. 2).

The earliest increases in the number of cambial cells

were observed in pine at the beginning of May; this was

followed 1–2 weeks later in larch and spruce. At site

5T-N, wide cambial zones were always observed

1–2 weeks later than at site 5T-S, with the exception of

pine in 2002 and spruce in 2004. However, the first

samplings in 2002 began in mid-May when pine was

already showing wide cambial zones, thereby pre-

venting a precise assessment of the differences

between the two sites for this species. The earliest

cambial activity onset was observed for the three

species in 2003, when very high temperatures were also

recorded in April–May (Fig. 1).

Oecologia (2007) 152:1–12 5

123

Once annual activity had finished and the cambium

had stopped dividing, the number of cells in the cam-

bial zone fell to a minimum value that corresponded to

quiescence conditions of the meristems. High vari-

ability was also observed at the end of cell production.

Halts in cambial activity were observed from mid-July

L. decidua

468

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P. abies P. cembra

468

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468

1012141618

sllecl

aib

mac

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re

bm

uN

468

1012141618

468

1012141618

Day of the year

100 150 200 250 300 100 150 200 250 300100 150 200 250 300468

1012141618

20

02

ra

eY

30

02

ra

eY

40

02

ra

eY

Fig. 2 Numbers of cells in the cambial zones of Larix decidua,Picea abies and Pinus cembra during 2002–2004 at sites 5T-S(black dots) and 5T-N (white dots). Error bars and horizontaldotted line indicate the standard deviations among trees in terms

of the number of dormant cambial cells. Periods of cambialactivity, when error bars do not cross the horizontal dotted line,are highlighted in grey

6 Oecologia (2007) 152:1–12

123

for pine in 2004 (5T-N) to the end of August for spruce

in 5T-S. Cell division ended earlier in trees at site 5T-N

than in trees at site 5T-S, with the exception of larch in

2002. Delays in the onset and ending of cambial

activity led to an average reduction of 30% in the

overall period for cell production between sites 5T-S

and 5T-N. However, a very short period of cambial

activity was observed in 2002 for pines growing at site

5T-S in which there was a rapid decline in the number

of cambial cells, from 14.2 on 28 May (day 148 of the

year) to 10.6 on 25 June (day 176).

Cell differentiation

Onset, duration and ending of cell differentiation were

computed in days of the year for each tree. The aver-

ages are reported in Fig. 3, where the bars correspond

to the mean value and error bars to the standard

deviation between the five trees.

The onset of radial enlargement occurred between

mid-May and mid-June and was significantly different

between the three species (ANOVA, F = 23.20,

P < 0.0001) (Fig. 3a–c). The earliest start of radial

enlargement was observed every year in pine tracheids

at both sites. Larch tracheids began cell differentiation

4–14 days after pine and 2–11 days before spruce, with

the exception of 2002 when tracheid enlargement was

observed to occur earlier in spruce than in larch. In all

species, the onset of cell differentiation occurred 3–

10 days earlier at the south-facing site (ANOVA,

F = 14.90, P < 0.01). Significant differences among

years were detected by repeated measurements

(ANOVA, F = 16.57, P < 0.0001). The first tracheids

were observed in cell enlargement 3–13 days later in

2002 and 2004 than in 2003.

Xylogenesis was considered to be concluded when

cells were no longer observed in the process of radial

enlargement, wall thickening or lignification. Cell dif-

ferentiation ended from early September to mid-Octo-

ber (Fig. 3d–f). The range of variation for the

completion of xylogenesis was greater than that for

onset of cell differentiation (44 days for end of cell dif-

ferentiation versus 30 days for the onset of cell

enlargement). Significant variations in the ending of cell

differentiation were observed among species (ANOVA,

F =14.79, P < 0.001), with average differences of 8 days

between pine and larch and 2 weeks between pine and

spruce. Spruce were the last to complete lignification,

except in 2003 when immature cells were observed in

larch until the end of September. Cell differentiation

ended earliest at site 5T-N for all species (ANOVA,

F = 17.03, P < 0.001), with larches showing the greatest

differences between sites 5T-S and 5T-N. The repeated

measurements revealed a significant difference among

the 3 years (ANOVA, F = 66.14, P < 0.0001), with xy-

logenesis ending much later in 2004 – after 1 October

and 22 September in 5T-S and 5T-N, respectively.

The duration of xylogenesis – i.e., the time required

to complete cell differentiation for all the tracheids

forming the tree ring – varied between 90 and 137 days

(Fig. 3g–i), with no difference detected between species

(ANOVA, F = 0.16, P > 0.05). Conversely, there were

significant differences between sites (ANOVA,

F = 23.03, P < 0.001), with longer durations of tree-ring

formation estimated for site 5T-S, where 123.5 days

were required to complete cell differentiation. At site

5T-N, the average period between the onset of cell

enlargement and ending of lignification was 107 days.

Larger reductions were observed for larch in all 3 years

and for pine in 2002. Significant variations among the

3 years were detected by repeated measurements

(ANOVA, F = 23.49, P < 0.0001). In 2004, the average

duration of tree-ring formation was 124 days versus 113

estimated in 2003. Shorter periods were calculated in

2002 for completion of the tree ring, with 108 days of

cell differentiation.

Interaction effects between sites and species were

also tested, but the results were not significant

(P > 0.05), thus indicating that site effects on onset,

duration and ending of xylogenesis were independent

of species.

Xylem cells in the tree ring

At the end of the growing season, we found different

numbers of cells in the tree ring of the three species

(ANOVA, F = 8.82, P < 0.01), as reported in Fig. 4.

The highest numbers of tracheids were observed in

spruce, with values ranging from 45 (in 2002, 5T-N) to

76 (in 2002, 5T-S), while the number of xylem cells in

larch varied between 32 (in 2002, 5T-N) and 51 (in 2003,

5T-S). Fewer cells were produced in trees at site 5T-N

than at 5T-S (ANOVA, F = 6.58, P < 0.05), with

15–30% fewer tracheids in the tree ring (57 cells pro-

duced in 5T-S versus 45 cells in 5T-N overall for the

three species). The final number of cells varied signifi-

cantly between years (ANOVA, F = 16.77, P < 0.0001),

with the fewest tracheids observed in 2002. There

was no evident pattern in 2003 and 2004. The largest

numbers of cells were produced by spruce in 2004 and

by larch and pine in 2003.

Threshold temperatures

The threshold temperature at which there was a 0.5

probability of active xylogenesis was calculated and

Oecologia (2007) 152:1–12 7

123

reported as an average for each species and site

(Fig. 5). For air temperature, the ranges of thresholds

when both sites were considered were 1.7–4.8, 5.6–8.5

and 10.9–13.3�C for the minimum, mean and maximum

temperatures, respectively. The minimum, mean and

maximum stem temperatures at which there was a 0.5

probability of active xylogenesis were higher than the

air temperature threshold, being 2.6–4.2, 7.2–9 and

17.2–22�C (Fig. 5). The standard deviations associated

with the mean values of stem temperature thresholds

were low, indicating a very slight variation between

trees, especially for the minimum and mean stem

temperature. Lower thresholds were found for mini-

mum (0.2–4.5�C), mean (2.6–7.5�C) and maximum

(8.3–12.6�C) soil temperatures. No difference was

found between species and sites (ANOVA, P > 0.05),

with the exception of minimum air temperature

(ANOVA, F = 1.84, P < 0.05) and all soil temperature

series (ANOVA, P < 0.01).

Discussion

The growth limitation hypothesis attempted to explain

the existence of cold treelines throughout the world by

suggesting that cell formation (i.e. cell division and

differentiation) could not occur below a minimum

temperature threshold; if it did occur, however, there

would be an abrupt slow down (Korner 1998).

According to several authors (Korner 1998, 2003), this

minimum temperature should range between 0 and

10�C. According to Korner (2003), mean air and stem

threshold temperatures of 6–8�C define treeline posi-

tions worldwide . However, these thresholds were only

P. abies

noitamrof

gnir-eertfognidn

E)r aey

ehtfosyad(

200

220

240

260

280

300

2002 2003 2004

fonoitaru

D)syad(

noitamrof

gnir-eert 60

80

100

120

140

160

Year

2002 2003 2004 2002 2003 2004

L. decidua

gnir-eertfotesnO

)raeyehtfo

syad(noita

mrof 100

120

140

160

180P. cembra

5T-S5T-N

a b c

d e f

g h i

Fig. 3 Onset (a–c), ending (d–f) and overall duration (g–i) of tree-ring formation for L. decidua, P. abies and P. cembra at sites 5T-Sand 5T-N during 2002–2004. Error bars indicate standard deviation among trees

8 Oecologia (2007) 152:1–12

123

calculated on an annual basis. Our histological analyses

performed on at a weekly scale showed that xylogen-

esis was active when the minimum daily air tempera-

ture was above 2–4�C and the minimum stem

temperature was higher than 4�C. The converging

temperature thresholds among sites and species in the

three study years confirmed the existence of thermal

limits to stem growth, thus also explaining the shorter

periods of xylogenesis observed at the north-facing

site.

Cambium is a sink for non-structural carbohydrates,

and cambial activity requires a continuous supply of

energy in the form of sucrose which, for the first cells to

be formed, is extracted from the storage tissues or

L. decidua

0

20

40

60

80

100

5T-S

5T-N

P. abies

sllec fo rebmu

Ngnir eert eht ni

0

20

40

60

80

100

P. cembra

Year

2002 2003 20040

20

40

60

80

100

Fig. 4 Final number of xylem cells produced by L. decidua, P.abies and P. cembra during 2002–2004 at sites 5T-S and 5T-N.Error bars indicate the standard deviation among trees

L. decidua

)C°(

erutarepmet

riA

0

2

4

6

8

10

12

14

5T-S 5T-N

)C°(

erutarepmetlio

S

0

2

4

6

8

10

)C°(

erutarepmet

metS

0

4

8

12

16

20

P. abies

Site

5T-S 5T-N

P. cembra

5T-S 5T-N

Fig. 5 Threshold minimum (black dots), mean (white dots) andmaximum (grey dots) temperatures corresponding with the 0.5-probability of active xylogenesis for L. decidua, P. abies and P.cembra estimated during 2002–2004 at sites 5T-S and 5T-N.Error bars indicate the standard deviation among trees

Oecologia (2007) 152:1–12 9

123

produced by photosynthesis (Hansen and Beck 1990,

1994; Oribe et al. 2003). During cell maturation, trees

assign a large amount of carbon obtained from pho-

tosynthesis to the deposition of cellulose microfibrils in

order to provide the developing cells with secondary

walls (Hansen et al. 1997). The estimated mean tem-

perature of 6–8�C seems to be the threshold limiting

the demand for photo-assimilates by the metabolic

processes involved in cell growth. Since shoot exten-

sion was also inhibited by air temperatures lower than

6–8�C (James et al. 1994), and 5% of maximum rate of

root growth occurred at a soil temperature of 6�C

(Turner and Streule 1983 in Shonenberger and Frey

1988), a critical mean temperature of between 6 and

8�C does exist, affecting growth processes in all parts of

the tree (shoots, stem and roots). The temperature-

limited growth mechanisms at the cell level are,

therefore, also expected to be the same in all plant

meristems.

The onset of cell division occurred early in the

season, when air and stem temperatures were still low,

and it ended at the end of July or, at the latest, in

August, when stem temperatures achieved maximum

values (12–15�C; compare Figs. 1 and 2). The high

spring temperatures in 2003 induced an earlier

resumption of cell production in the cambium and a

consequent earlier onset of xylem cell differentiation.

Shorter durations of cambial activity were observed in

trees at the north-facing site (5T-N), where cell pro-

duction lasted for 6–11 weeks. These results are con-

sistent with the 6-week-long active period required to

maintain long-term growth by Pinus sylvestris near the

treeline in northern Finland (Schmitt et al. 2004). Al-

though the conclusion of cell division indicated the end

of tracheid production, wood formation continued

until the autumn through enlargement, wall formation

and lignification of the newly produced tracheids. The

differentiation processes are dynamic phases with

durations in earlywood and latewood (Rossi et al.

2006b). The large amount of woody material deposi-

tion that initiates the formation of thicker cell walls in

latewood tracheids is related to a longer duration ra-

ther than higher rate of secondary wall formation

(Uggla et al. 2001). In the species studied here, the

latest latewood tracheids remained in the maturation

phase for up to 40–60 days, as reported for the same

sites by Rossi et al. (2006b); by subtracting the Julian

days, this corresponded to the end of differentiation

(Fig. 3d–f) and conclusion of cambial activity (Fig. 2).

Trees living in cold climates concentrate xylem cell

formation early in the season, synchronizing cambial

activity with photoperiod and culminating wood pro-

duction at the end of June when the day length is

maximum (Rossi et al. 2006c). By avoiding high cell

production rates during late summer, plants guarantee

newly formed tracheids enough time before winter to

complete differentiation, particularly cell-wall forma-

tion and lignification in latewood.

In larch, stone pine and Norway spruce, overall

wood formation lasted for a period varying from 100 to

140 days between mid-May and the beginning of

October. Korner and Paulsen (2004) also estimated an

(unspecified) growing period of 129–139 days for the

eastern Alps by counting the days when the soil tem-

perature at a depth of 10 cm exceeded 3.2�C, which in

the spring corresponded to a weekly mean canopy

temperature of 0�C. Between the south and north-

facing sites, Differences of 1–3 weeks in the active

period of cambium have been estimated between the

south- and north-facing sites, based on rapid declines in

height and root seasonal growth patterns observed

across altitudinal gradients in treeline ecotones (Han-

sen-Bristow 1986; Stevens and Fox 1991; Sveinbjorns-

son 2000). This reduction led to differences of 5–

25 days in the duration of wood formation, and these

differenceswere in turn related to delays of 3–10 days

in the onset of differentiation and advances of 3–

20 days in the ending of cell-wall lignification. A de-

layed onset of cell enlargement found in trees at the

north-facing site (5T-N) could not be related to dif-

ferences in air temperatures given the similarity be-

tween sites. On the contrary, greater differences

between sites were observed in the stems, with tem-

peratures being up to 1.4�C warmer at the southern site

(5T-S), and in the soil during late spring. Other studies

have reported that where temperature strongly limits

tree radial growth, the thawing of the upper soil layer

must have started before radial growth is able to

commence (Graumlich and Brubaker 1986; Cairns and

Malanson 1998; Vaganov et al. 1999). Cold soils inhibit

root activity and water uptake, thus delaying turgor

increase in the vacuoles and enlargement of the newly-

produced tracheids in the stem. However, our results

showed different soil threshold temperatures between

sites, thus indicating that soil temperature could not be

the main limiting factor affecting xylogenesis. In terms

of stem temperature, converging thresholds were ob-

served for the two sites, demonstrating that a minimum

threshold of 3–4�C has to be reached to sustain xylo-

genesis. As stem sensors measure cambium tempera-

ture, where the xylogenesis processes occur, this result

demonstrates the direct effect of temperature on the

metabolic activities of wood formation (Grace 1988;

James et al. 1994).

In the three study years, differences of 15–18 days

were estimated in the duration of secondary stem

10 Oecologia (2007) 152:1–12

123

growth, with a variability of up to 15% in complete cell

maturation. Delays in the onset of radial growth did

not necessarily correspond to corresponding delays in

the ending of growth. Higher variability was observed

at the conclusion of differentiation: about 10 ± 8 days

at the start of xylogenesis versus 22 ± 14 days at the

end of lignification at both sites. Wood formation is a

complex process with several differentiation phases.

Cells produced in spring and early summer must pass

through several of these phases before reaching phys-

iological maturity. Moreover, a higher cell production

during cambial activity leads to an increased number of

developing tracheids (Ford et al. 1978) and, conse-

quently, prolonged cell maturation later in the season

(Gricar et al. 2005).

Conclusion

The results of our study reveal that in larch, stone

pine and Norway spruce, wood formation occurred

when certain threshold temperatures were reached.

Although the timing and duration of xylogenesis

varied among these species, sites and years, air and

stem temperature thresholds were stable for all of the

trees studied, ranging from 5.6 to 8.5�C and from 7.2

to 9�C, respectively. These results correspond to the

supposed temperatures limiting growth at the treeline

and thus provide strong evidence that temperature is

a critical factor controlling xylem cell production and

differentiation at high altitudes. This study represents

the first attempt to define a threshold through com-

parative assessment of xylem growth and tissue tem-

peratures in stem meristems of trees growing at high

altitudes.

Acknowledgments This work was funded by the MAXY 2004(CPDA045152) and MIUR-PRIN 2005 (2005072877). The au-thors wish to thank C. Filoso, F. Fontanella, M. Gardin, L. Ma-rini, M. Mazzaro and R. Menardi for their technical support andthe Regole of Cortina d’Ampezzo for permitting the study ontheir property. Special thanks are extended to M. Carrer and C.Korner for their recommendations on the manuscript.

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