Temporal and spatial variation of flowering among Pinus nigra Arn. clones under changing climatic conditions

Forest Ecology and Management 259 (2010) 786–797

Contents lists available at ScienceDirect

Forest Ecology and Management journal homepage: www.elsevier.com/locate/foreco

Temporal and spatial variation of flowering among Pinus nigra Arn. clones under changing climatic conditions P.G. Alizoti *, K. Kilimis, P. Gallios Aristotle University of Thessaloniki, Faculty of Forestry and Natural Environment, Laboratory of Forest Genetics and Tree Improvement, P.O. Box 238, 54124 Thessaloniki, Greece

A R T I C L E I N F O

A B S T R A C T

Keywords: Climate change Floral phenology Flowering variation Flowering receptivity Flowering asynchrony Pollen shedding Synchronization Panmixia Seed orchard Quantitative traits Clonal heritability Pinus nigra Arn

Until recently, the most important factors affecting the economics and genetics of the seed crop from seed orchards were considered to be the timing and duration of flowering, variation in fertility and the total number of clones used to establish the seed orchards. Change in climatic conditions however is an emerging factor that could prove crucial regarding the timing of flowering and synchronization among clones and thus, the quality and quantity of seed production. The temporal and spatial variation in flowering phenology and the duration of flowering were studied in consecutive years in a Pinus nigra Arn. seed orchard. Sixty plus trees representing the distribution of the species in Northern Greece were used to establish the seed orchard, and nineteen ramets per clone were planted in a honeycomb experimental design in order to avoid kinship. Temporal variation among clones, as well as spatial variation among ramets within clones growing at different sites of the orchard were recorded, for initiation and duration of male and female flowering. The majority of clones were synchronized in dates of flowering during the year with weather conditions close to the long-term climatic conditions, except for a limited number of clones that were precocious or late flowering. The pronounced variability in climatic conditions over the 2 years strongly affected the flowering and synchronization among clones, resulting in almost complete asynchrony during the xerothermic year, which was characterized by a prolonged mean monthly temperature increase of 2.3 8C and a water deficit of 53% in a 7-month-period (November to May). These results suggest that one of the effects of a warmer and drier climate may be the lack of flowering synchronization, as pollen shedding might be completed before female conelets reach the phase of receptivity. The restriction of male parentage to a limited number of clones severely violates the panmixia assumption and could result in fertilization failure. The projected climate change for the Mediterranean region could potentially prove detrimental for fertility and flowering synchronization of forest trees, having consequences on the quantity and genetic diversity of the seed crop in seed orchards, and the natural regeneration of forest trees in forest ecosystems due to the reduced percentage of sound seed. ß 2009 Elsevier B.V. All rights reserved.

1. Introduction Pinus nigra Arn. (black pine) is one of the most important pines for high elevation sites in southern Europe and especially the Mediterranean basin. Its distribution ranges from Morocco to Asia Minor, southern France, northern Italy, Austria, the Balkan Peninsula, Crimea, and Cyprus, while isolated occurrences have been reported on the Caucasian coast of the Black Sea and on Mediterranean islands (Mirov, 1967; Grossoni, 2008). In Greece the species covers an area extending from the northern border of the country down to the southern part of the Peloponnesos peninsula and the islands of Samos, Lesvos, Thassos and Euboia, forming many isolated populations. It is considered one of the most

* Corresponding author. Tel.: +30 2310 992769. E-mail address: [email protected] (P.G. Alizoti). 0378-1127/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2009.06.029

important coniferous species regarding its ecological value, wood production and silvicultural significance and is thus extensively used for reforestation purposes throughout the country. The need for improved black pine seed is satisfied by the existing breeding programme for black pine in Greece, which is structured in three different populations (Southern, Central and Northern Greece). The three 1st generation seed orchards are in production phase and cover the demand for black pine seed (Matziris, 2005). The main objective of a seed orchard, which is the production of genetically improved seed, can be met if the criteria for panmictic equilibrium are fulfilled. The main prerequisites of ‘‘panmixia’’ are the following (Eriksson et al., 1973; Woessner and Franklin, 1973; Weir and Zobel, 1975): (a) completely random fertilization, i.e. the time of flowering is completely synchronous, the sperm nuclei of each clone have the same probability of reaching the ovules of each clone, and there is no incompatibility, (b) equal number of male

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

gametes/clone for all clones, (c) equal number of female gametes for all clones, (d) lack of genetic barriers that may affect embryo viability, (e) lack of fertilization from alien pollen, and (f) equal self-fertility for all clones in the seed orchard. Flowering phenology in a seed orchard is a crucial factor affecting the gene exchange among clones and thus the genetic composition of the seed crop (El-Kassaby et al., 1988; Erickson and Adams, 1989; Burczyk and Chalupka, 1997). According to Weir and Zobel (1975) the knowledge of flowering phenology is a fundamental need for the successful operation of a seed orchard. Failure of synchronization in female flower receptivity and pollen shedding would negatively affect the panmictic equilibrium that is expected in an idealized seed orchard (El-Kassaby et al., 1988, 1984), as it can result in non-random cross-fertilization, higher percentage of empty seeds and increased selfing (Bhumibhamon, 1978). Notable variation in flowering phenology, synchronization and crop yield has been reported for Scots pine (Savolainen et al., 1993), Douglas fir (El-Kassaby et al., 1984; Copes and Sniezko, 1991), Norway spruce (Eriksson et al., 1973; Danusevicius, 1987) and black pine (Matziris, 1994). Initiation and development of flowers, and consequently flowering phenology can be severely affected by several environmental factors, e.g. temperature, light, water availability and nutrition (Owens and Blake, 1985; Kinnaird, 1992; Nikkanen, 2001). The effects of these factors might be due to changes in water or mineral availability, rate and amount of assimilates, or changes in the production of and reaction to endogenous growth regulators (Lyndon, 1992). Application of irrigation, fertilization and thinning can positively affect female flowering, pollen quantity and shedding, as well as seed production (Sweet, 1995; Healy et al., 1999). In general, environments favoring growth enhance production of female flowers. According to Meagher (1988) female flowering is more energy costly than the male one, due to the additional cost of seed and fruit production. The cost during the early stages of female pine cones development is compensated by increasing the photosynthetic rates of the foliage near the conelets (McDowell et al., 2000). However, as the cones mature till the second fall of their initial development, the cost may be at least partially compensated by increasing photosynthetic rates or by the photosynthesis of the reproductive structure itself (Obeso, 2002). Climate change is an overarching pressure that may aggravate many threats (as forest fires, water abstraction and pollution, desertification) to forest ecosystems and biodiversity in Europe and especially the Mediterranean region; a biodiversity hotspot harbouring more than four times as many vascular plants than the rest of Europe (EEA, 2007). The Mediterranean region climate is characterized by pronounced, climatic bi-seasonality with dry and hot summers and moist and cool autumns and winters; also occasional, violent precipitation events and a large year-to-year variability of total rainfall (Scarascia-Mugnozza et al., 2000). According though to the European Environmental Agency fourth assessment of Europe’s environment (EEA, 2007), Europe has warmed up more quickly (1.4 8C) than the global average (0.74 8C) between 1906 and 2005. Particularly significant warming has been observed over the southern and south-eastern Europe, the Iberian Peninsula, the north-western Russia and the Baltic states. Furthermore, southern and south-eastern Europe became also up to 20% drier in the 20th century (EEA, 2007). Climate change scenarios predict an increase of the global average temperature among 1.8 and 4.0 8C by 2100 (EEA, 2007), with some studies suggesting a wider range of 1.1–6.4 8C (IPCC, 2007). Europe is likely to warm 2.1–4.4 8C or possibly 2.0–6.3 8C (EEA, 2007; Schro¨ter et al., 2005) by 2100. In southern Europe, climate change is projected to worsen conditions (high temperature and drought) in a region already vulnerable to climate variability (IPCC, 2007), with a temperature increase up to 6 8C during the summer (Gianna-

787

kopoulos, 2005) and less winter precipitation (Giorgi et al., 2004). Climate change is expected to become the main driver of biodiversity loss in the future, affecting physiology, phenology and species distribution in the Mediterranean region, and it is expected that by 2100 almost 25% of the plant species growing in southern Europe may have disappeared (EEA, 2007; Thomas et al., 2004; Bakkenes et al., 2006). Climate change could also prove detrimental for fertility and flowering synchronization of forest trees in the Mediterranean region, having consequences on the quantity and quality of seed production in seed orchards, and the natural regeneration of forest trees in forest ecosystems due to the reduced percentage of sound seed. Aims of the present study were to: (a) determine the variation in female and male phenology in a P. nigra Arn. clonal seed orchard, (b) evaluate the synchronization in receptivity and pollen shedding, (c) evaluate the level of genetic variation among clones regarding the female and male phenological stages of development and (d) determine to what extent pronounced climatic differences can affect both flowering phenology and synchronization. 2. Materials and methods 2.1. Genetic material The study was carried out in a black pine clonal seed orchard located in the Aristotle University Forest of Taxiarchis, Chalkidiki, Greece (Fig. 1). The orchard was established in November 1980 and is comprised of sixty clones derived from intensively selected plus trees in the natural forests of five Northern Greece populations, namely Mt. Olympus, Mt. Vermion, Mt. Pieria, Chalkidiki peninsula and Thassos island (Fig. 1). Each clone was represented in the seed orchard by nineteen ramets. Grafts were 2 years old at the time of establishment and were planted at a spacing of 8.6 m  5.0 m in a honeycomb experimental design to avoid kinship. The honeycomb

Fig. 1. Origin of the clones and location of the seed orchard.

788

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

selection methodology (Fasoulas, 1973, 1980, 1988; Fasoula and Fasoula, 2000) and the honeycomb designs (Fasoulas, 1973, 1993; Fasoulas and Fasoula, 1995; Batzios and Roupakias, 1997), are based on the hexagonal arrangement of the plots in the field and on the principle of the moving replicate (Fasoulas and Fasoula, 1995). The moving hexagonal replicate ensures that every entry occupies the center of a complete hexagon shaped replicate, while the number of different entries tested in the moving replicates can range from 3 to 243 (Fasoulas, 1973, 1988; Fasoulas and Fasoula, 1995; Batzios and Roupakias, 1997). In this way, it was ensured that each ramet of a certain clone of the seed orchard occupied the center of a hexagonal moving replicate and was surrounded by the rest 59 different genotypes (ramets per clone), minimizing in this way the risk of selfing and its detrimental effect on the seed crop. Two ramets per clone were randomly selected in the orchard in order to monitor the female and male flower development in 2 successive years (2006, 2007). Two whorls (3rd and 6th) per ramet and clone, and four branches per whorl, each one with different orientation (North, South, East, West), were marked. In this way the representative monitoring of the whole crown could be ensured, as studies have shown that variation may exist in flowering dates within the same tree (Jonsson et al., 1976). Between late April and early June the eight marked branches per tree were observed three times a week to determine the phenological stage of female and male strobili. Observations of flowering of all selected branches were made until all pollen was released and the female conelets were no longer receptive. Four female stages of flowering development were distinguished, as described by Matziris (1994): stage 1, the female bud is increasing in size and becomes cylindrical, but is still completely covered by the bud scales (receptivity 0%); stage 2, the apex of the enlarged cylindrical bud is opened and the first ovuliferous scales appear. At this stage the ovules are not receptive, but the pollen grains may get into the bud scales and if they survive they may be able to take part in fertilization (receptivity 20%); stage 3, the scales of the female conelet are gradually separated and almost form right angles with the conelet axis (receptivity 100%); stage 4, the ovuliferous scales increase in size and thickness so that the conelet is no longer receptive (receptivity 0%). The male stages were described as follows: stage 1, male strobili are developing, but are still closed in integuments; stage 2, microsporangia are visible but tightly packed; stage 3, microsporangia are not tightly packed and yellow liquid emerges from the strobili when under pressure; stage 4, yellow strobili start shedding their pollen (100% pollen shedding); stage 5, brown and dry strobili (end of pollen shedding).

Fig. 2. Range of the monthly mean, average maximum and minimum temperatures, for the period January 2005 to May 2007, and of the grand mean (1974–2007) temperature.

Compared to grand mean temperatures, the mean monthly temperature rose on average by almost 2.3 8C. The area of the study receives on average annually 769 mm of precipitation; 234 mm in winter, 177.5 mm in spring, 149.7 in summer and 207.7 in fall. The wettest month of the year is February with an average monthly precipitation of 71 mm and the driest month is August with an average monthly precipitation of 42 mm. Fig. 3 presents the precipitation (in mm) per month, starting from January 2005 to May 2007, the average precipitation per month (1974–2007), and the deviation of precipitation per month from the average value. The dramatic negative deviation of precipitation from the average values, starting from November 2006 to May 2007, equals to a loss of 230 mm of rain out of the 430 mm that the site receives on average during the above period. The prolonged drought and the warm winter and spring (November 2006 to May 2007) represent a significant anomaly from the mean climate, as they occurred during the normally moist and cool period of the year. 2.3. Statistical analyses The successive stages of male and female flowering development were expressed in number of days required from January 1st

2.2. Climatic conditions The climate in the area of research is characterized by climatic bi-seasonality with dry and hot summers and moist, cool autumns and winters. The annual grand mean temperature is 11 8C. The colder month is January with a grand mean temperature down to 1.9 8C and the warmest month is July with a grand mean temperature up to 21 8C. The climatic data for the study period were obtained from the meteorological station at the Aristotle University Forest of Taxiarchis located close to the black pine clonal seed orchard. Fig. 2 presents the monthly mean, mean maximum and mean minimum monthly temperatures from January 2005 to May 2007, as well as the grand mean monthly temperature calculated for the period 1974–2007. The temperature (monthly mean, mean monthly maximum, mean monthly minimum) increase starting from November 2006 till May 2007 is evident, and especially the increase of the mean monthly minimum temperature which is the most dramatic one.

Fig. 3. Range of monthly precipitation (mm), grand mean precipitation (mm) (1974–2007) and deviation of the monthly precipitation from the grand mean, for the period January 2005 to May 2007.

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

to reach these stages, while the duration of stages was estimated by subtraction. The variables of male onset, male ending, male duration, onset of female stage 2, duration of female stage 2, onset of female stage 3, ending of female stage 3, female duration of stage 3 and total female duration (stages 2 and 3) were subjected to statistical analysis. 2.3.1. Combined analysis The linear model followed for the combined analysis for both years for each trait was: Y i jk ¼ m þ yri þ C j þ yrC i j þ ei jk where Yijk is the individual tree value of the kth ramet of the ith clone in the ith year, m is the grand mean, yri is the ith year fixed effect (i = 1, . . ., 2), Cj is the jth clone random effect, NID (0, s 2C ) (i = 1, . . ., 60), yrCij is the ith year by the jth clone random effect, NID (0, s 2yrC ), eijk is the random error effect of the kth ramet of the jth clone in the ith year (k = 1, 2), NID (0, s 2e ). It should be mentioned that during 2007, the two ramets of five clones, as well as one ramet from six other clones of the seed orchard did not produce any female conelets. The estimates of broad sense heritability were obtained on clonal mean basis ðHC2 Þ and individual tree basis ðHi2 Þ over the 2 years according to Matziris (1994), Lush (1940), Shelbourne and Thulin (1974), St. Clair and Kleinschmidt (1986), and Leinekugel le Cocq et al. (2005) as following: HC2 ¼

s 2C s 2C þ ðs 2yrC =yrÞ þ ðs 2e =n  yrÞ

  Hi2 ¼ s 2C = s 2C þ s 2yrC þ s 2e where s 2C , variance due to clone; s 2yrC , variance due to year by clone interaction; s 2e , error variance; yr, number of years; n, number of ramets per clone. The standard errors of heritability estimates were calculated according to Becker (1984). The individual tree heritability thus represents the relative magnitude of all genetic effects, including additive and interaction effects within and between loci, to total phenotypic variation. The individual tree heritability is used to predict genetic gains from clonal ramet mass selection. The clone mean heritability estimate is larger than the individual tree heritability, indicating the genetic gain that may be obtained through selection and deployment of clones from the specific population (i.e. the seed orchard), or from roguing the existing seed orchard (Matziris, 1994; Leinekugel le Cocq et al., 2005). As the clonal seed orchard was not replicated in space, the estimates of the genetic variance ðs 2C Þ are biased upwards as they include the clone  environment interaction component of variance. However, the variance and heritability estimates are valid and applicable to the specific environmental conditions of the seed orchard. 2.3.2. Analysis for each year The linear model followed for the analysis per year for each trait was: Y jk ¼ m þ C j þ e jk where Yjk is the individual tree value of the kth ramet of the ith clone, m is the grand mean, Cj is the jth clone random effect, NID (0, s 2C ) (i = 1, . . ., 60), eijk is the random error effect of the kth ramet of the jth clone (k = 1, 2), NID (0, s 2e ). The clonal mean heritability (HC2 ) and the individual tree heritability (Hi2 ) for each year were estimated as: HC2 ¼

s 2C s 2C þ ðs 2e =nÞ

Hi2 ¼ s 2C = s 2C þ s 2e

789




where s 2C , variance due to clone; s 2e , error variance; n, number of ramets per clone. Analyses were conducted using Proc GLM and Proc Mixed procedures in SAS (SAS Institute, 1996). Variance components of random effects were estimated by restricted maximum likelihood (REML) (Khuri and Sahai, 1985; Searle et al., 1992). The estimated means per variable and year were compared according to the Type III test of fixed effects (SAS-Proc Mixed). The index of overlapping phenologies (PO) that quantifies the similarity of any pair of male and female phenograms in terms of symmetry in gamete contribution proportions was estimated, as described by Askew and Blush (1990), for each one of the sixty clones. The PO index is a quantitative measure of the proportional symmetry of the female and male phenograms and is estimated by the ratio of the common area to the maximum area between the female and male phenograms summed across all registered days ¯ ) were and for each pair of clones. The average index values (PO estimated according to Askew and Blush (1990), by considering each specific clone: (a) as a female parent for all the outcross seed orchard pollen parents and (b) as a male parent for all the outcross ¯ clonal values are seed orchard seed parents. Thus, the average PO indicators of a clone’s: (a) female synchronization with the remaining clones of the orchard that act as male parents, and consequently the clone’s potential to be fertilized by rest clones of the orchard, and (b) male synchronization with the remaining clones of the orchard that act as female parents and thus, the clone’s potential to fertilize the all the rest orchard clones. For the evaluation of spatial variation in the seed orchard the variables of male flowering onset and duration, as well as, those of female onset and duration were subjected to spatial analysis following Proc Variogram and Proc krige2d (Gaussian model) in SAS (SAS Institute, 1996). 3. Results 3.1. Significance of effects The effect of the year was significant (p < 0.0001) for almost all female and male variables, except for initiation of pollen shedding and onset of female stage 2. The large variation among clones in the phenological stages of male and female flowering is evident from the results of the analysis (Table 1). The clonal differences were significant for almost all traits except for onset of female stage 2, onset of female stage 3 and total duration of female flowering. The clone-by-year interaction effect was significant only for onset and duration of male flowering and female total duration (duration of stages 2 and 3) (Table 1). Significant variation among clones was recorded for all the male and female characteristics, except for the female duration of stages 2 and 3 and the onset of female stage 3, in 2006, while in 2007 clones differed significantly only for the initiation of pollen shedding. 3.1.1. Flowering differences among years The overall initiation of male flowering occured almost the same time in the 2 years of study. However, the overall termination and thus, duration of male flowering differed significantly, as in 2007 pollen shedding was terminated on average 8.6 days earlier and the duration of pollen shedding lasted on average 8.8 days less than in 2006 (Fig. 4). The difference in meteorological conditions among the 2 years affected significantly all the female flowering traits. The overall initiation of female bud break stage (2) and receptivity stage (3) was significantly delayed in 2007, by 11.2 and 11.7 days,

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

790

Table 1 Mean squares (MS) and significance testing (Pr > F) derived from the combined and per year analysis of variance (Ons, Term, dur = onset, termination, duration of male flowering, respectively. Ons-2, Dur-2, Ons-3, Term-3, Dur-3, T-dur = onset of stage 2, duration of stage 2, onset of stage 3, termination of stage 3, duration of stage 3 and total duration of female flowering, respectively). Combined over the years Male Sov yr

a

b

df 1

C

59

C  yr

59

Error

120

2006 C Error 2007 C Error

59

MS Pr > F MS Pr > F MS Pr > F

MS Pr > F

60

59

MS Pr > F

60

Female

Ons 0.84 0.43 5.08 0.0003 4.59 0.002 2.36

Term 3873.72 <.0001 9.69 0.011 7.36 0.15 5.83

Dur 3988.9 <.0001 11.59 0.016 13.14 0.004 7.19

6.41 0.0018 2.98

13.72 0.007 7.16

19.75 0.002 9.40

3.19 0.0084 1.61

3.75 0.66 4.21

4.66 0.45 4.48

b

df 1

59 54 109

59 60

54 49

Ons-2 6648.98 <.0001 16.36 0.0002 8.97 0.20 7.41

Dur-2 0.85 0.64 8.70 0.575 8.685 0.573 9.13

Ons-3 6930.28 <.0001 8.84 0.0432 3.85 0.97 6.04

Term-3 3538.09 <.0001 10.44 0.0065 7.76 0.13 6.01

Dur-3 528.02 <.0001 11.38 0.13 6.71 0.87 8.88

T-dur 486.62 <.0001 11.79 0.23 15.76 0.02 10.00

19.39 0.0096 10.50

11.49 0.631 12.54

7.57 0.3918 7.05

12.84 0.0143 7.23

11.84 0.431 11.32

18.03 0.076 12.40

5.56 0.07 3.63

5.99 0.25 4.96

5.51 0.31 4.80

5.33 0.28 4.51

6.63 0.34 5.89

8.95 0.20 7.06

a

Sov = source of variation, yr = year, C = clone, and C  yr = clone-by-year interaction. b Difference in degrees of freedom among male and female flowering traits, is due to the fact that both ramets of 5 clones and 1 ramet of six other clones did not produce any female flowers during 2007.

respectively. Ending of female flowering stage 3 occurred also significantly later during the second year of the study. The duration of receptivity stage (3) and the total female duration were reduced significantly, by almost 3 days, during 2007 (Fig. 4).

delayed and shrunk. Only part of the bud break stage 2, which only has a receptivity of 20%, partially coincided with the pollen shedding period, and the peak of bud break stage (2) was reached almost 8 days later than the peak of pollen shedding (Fig. 6).

3.2. Clonal synchronization Flowering pattern differed for the 2 years of the study and is depicted in the phenograms of the 2 years (Figs. 5 and 6). In 2006, the clones of the seed orchard exhibited phenologic synchronicity, as the peaks of pollen shedding clones and fully receptive clones coincided. Pollen was released earlier than the commencement of the receptive time (stage 3) and lasted for a longer period of time (Fig. 5). In 2007, asynchrony was recorded among the pollen shedding period and the receptive period of the clones. Initiation of pollen shedding coincided with that of 2006, but its duration was significantly reduced, and the receptive period was significantly

Fig. 5. Overall phenological overlap for the year 2006. The percentage of receptive and pollen shedding clones is shown (f-st2 = female flowering at stage 2, f-st3 = female flowering at stage 3, f-total = female stages 2 and 3, pl. sh = pollen shedding).

Fig. 4. Average number of days starting from May 1st for the onset, termination and duration of male (onset = onset, term = termination, dur = duration) and female flowering stages (onset-2 = onset of stage 2, onset-3 = onset of stage 3, dur2 = duration of stage 2, dur-3 = duration of stage 3, term-3 = termination of stage 3, tot dur = total duration of stages 2 and 3), for the years 2006 and 2007 (significant for *p < .05, **p < 0.1, and ***p < .001 and ns = no significance).

Fig. 6. Overall phenological overlap for the year 2007. The percentage of receptive and pollen shedding clones is shown (f-st2 = female flowering at stage 2, f-st3 = female flowering at stage 3, f-total = female stages 2 and 3, pl. sh = pollen shedding).

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

791

Table 2 ¯ ) for all the seed orchard clones in 2006 (Cl = clone, PO ¯ male = the specific clone serves as a male parent for all the outcross seed Average index of phenological overlap (PO ¯ female: the specific clone serves as female parent for all the outcross seed orchard pollen parents.). orchard seed parents, PO Cl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Male

Female

Cl

¯ PO

(min–max)

¯ PO

(min–max)

0.813 0.847 0.691 0.522 0.548 0.662 0.549 0.499 0.662 0.722 0.663 0.575 0.548 0.522 0.841 0.662 0.664 0.548 0.575 0.575 0.576 0.575 0.575 0.575 0.574 0.665 0.575 0.663 0.665 0.222

0.30–1.00 0.35–1.00 0.40–1.00 0.33–1.00 0.35–0.80 0.44–1.00 0.12–0.92 0.32–0.73 0.44–1.00 0.47–1.00 0.44–1.00 0.37–0.84 0.35–0.80 0.33–0.76 0.35–1.00 0.44–1.00 0.44–1.00 0.35–0.80 0.36–0.85 0.37–0.84 0.36–0.85 0.37–0.85 0.37–0.85 0.37–0.85 0.12–1.00 0.44–1.00 0.37–0.85 0.44–1.00 0.43–1.00 0.08–0.50

0.976 0.968 0.910 0.584 0.509 0.761 0.537 0.509 0.761 0.965 0.761 0.762 0.765 0.429 0.676 0.509 0.508 0.641 0.761 0.807 0.761 0.765 0.641 0.984 0.785 0.509 0.807 0.509 0.811 0.785

0.35–1.00 0.35–1.00 0.40–1.00 0.28–0.76 0.15–0.75 0.35–1.00 0.11–1.00 0.15–0.75 0.35–1.00 0.35–1.00 0.35–1.00 0.35–1.00 0.35–1.00 0.13–0.63 0.15–1.00 0.15–0.75 0.15–0.75 0.30–0.84 0.35–1.00 0.36–1.00 0.35–1.00 0.37–1.00 0.30–0.84 0.55–1.00 0.16–1.00 0.15–0.75 0.36–1.00 0.15–0.75 0.37–1.00 0.15–1.00

Due to the complete asynchrony of pollen shedding and female receptivity period in 2007, the average index of phenological ¯ ) was estimated for all clones, serving as both female overlap (PO and male parents in the seed orchard only for 2006. The average ¯ ) for the clones values of the index of phenological overlap (PO serving as female parents, ranged from 0.43 for clone 17 to 1.00 for clone 50 (Table 2). The indices varied for each female parent’s pollinators. For example, clone 52 had pollinator indices ranging from 0.15 to 1.00. For individual clones acting as pollinators (males), the average overlap with the female receptive phase ranged from 0.22 for clone 30 to 0.85 for clone 2. The indices for the male parents were also variable, ranging for instance from 0.13 to 1.00 for clone 43 (Table 2).

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Male

Female

¯ PO

(min–max)

¯ PO

(min–max)

0.702 0.575 0.664 0.522 0.572 0.671 0.572 0.522 0.664 0.520 0.545 0.627 0.574 0.443 0.572 0.545 0.624 0.475 0.545 0.522 0.545 0.520 0.661 0.664 0.658 0.520 0.539 0.548 0.662 0.576

0.30–1.00 0.36–0.80 0.43–1.00 0.33–0.76 0.39–0.87 0.43–1.00 0.36–0.80 0.33–0.77 0.40–1.00 0.30–0.77 0.36–0.80 0.28–0.94 0.13–1.00 0.28–0.64 0.37–0.87 0.32–0.85 0.28–0.95 0.31–0.69 0.34–0.81 0.33–0.75 0.35–0.80 0.33–0.77 0.43–1.00 0.45–1.00 0.43–1.00 0.33–0.74 0.34–0.80 0.36–0.81 0.44–1.00 0.36–0.84

0.910 0.716 0.965 0.509 0.761 0.676 0.408 0.765 0.910 0.508 0.763 0.675 0.785 0.512 0.508 0.991 0.992 0.807 0.761 1.000 0.762 0.507 0.509 0.508 0.609 0.761 0.614 0.851 0.641 0.644

0.41–1.00 0.35–1.00 0.35–1.00 0.15–0.75 0.35–1.00 0.15–1.00 0.12–0.60 0.35–1.00 0.41–1.00 0.15–0.75 0.35–1.00 0.15–1.00 0.15–1.00 0.29–0.67 0.15–0.75 0.65–1.00 0.47–1.00 0.36–1.00 0.35–1.00 – 0.35–1.00 0.15–0.75 0.15–0.75 0.15–0.75 0.29–0.80 0.35–1.00 0.35–0.80 0.48–1.00 0.30–0.84 0.30–0.84

termination of the receptivity stage (3) were under the stronger genetic control than all other female flowering traits. In 2007, bud break stage (2) and total female duration were more genetically controlled than the rest of the female flowering traits (Fig. 8). Heritability values were low for the receptivity stage (3) in 2006, but were considerably higher in 2007. It is also noteworthy that all female flowering parameters were low to moderately heritable in 2007, while in 2006 there were parameters like the duration of bud break stage (2) that appeared to be under no genetic control (Fig. 8). 3.4. Spatial variation

3.3. Clonal variation

The spatial pattern was more pronounced in the case of onset of pollen shedding in 2007, when compared to the respective spatial

Based on the combined analysis, it appeared that the male flowering parameters were all under low or no genetic control (Fig. 7). From the analysis per year, it was demonstrated that the onset of pollen shedding was under the strong genetic control for both years, while the clonal mean and individual tree heritability estimates for duration and termination of pollen shedding were severely reduced in 2007 (Fig. 7). The clonal mean heritability estimates were much higher than the individual tree heritability estimates and ranged among 0.47 and 0.54 for 2006 and among 0.06 and 0.48 for 2007. Regarding the female flowering, the combined analysis showed that the onset of bud break stage (2) and receptivity stage (3) were under stronger genetic control than the duration of the receptivity stage (Fig. 8). From the analysis by year it was shown that the individual tree heritability estimates were moderate to low for all the female flowering variables for both years. In 2006, bud break stage (2) and

Fig. 7. Clonal mean and individual tree heritability estimates and their standard errors for the male flowering (ons = onset, term = termination, and dur = duration), derived from the combined and the per year (2006, 2007) analysis.

792

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

Fig. 8. Clonal mean and individual tree heritability estimates and their standard errors for the female flowering variables (ons-2 = onset of stage 2, ons-3 = onset of stage 3, term-3 = termination of stage 3, dur-3 = duration of stage 3, tot dur = total duration of stages 2 and 3), derived from the combined and the per year (2006, 2007) analysis.

pattern in 2006. No clear North–South or East–West pattern was identified, but there were still clusters indicating delay in pollen shedding (Fig. 9). No spatial pattern in male duration was revealed from the analysis for either years of the study (Fig. 10). The onset of female receptivity stage (3) (Fig. 11) as well as the duration of receptivity, showed no spatial pattern for 2007, while clusters with significantly longer duration of female receptivity were observed for 2006, even if no clear pattern related to distance was identified (Fig. 12). 4. Discussion The assessment of flowering phenology in 2 successive years characterized by different annual climatic conditions suggests that the timing and duration of flowering may depend to a large extent on temperature increase, but mainly on water availability. The meteorological conditions during the first year of the study were close to the long-term average temperature and precipitation values. The second year was characterized by mean monthly temperatures of 2.3 8C greater than the long-term average temperatures and a water deficit of 230 mm of rain, experienced from November 2006 till May 2007. This accounted for a deficit of 53% when compared to the normal precipitation that the site should have received during the above period. It is worth mentioning that the site receives annually 768.9 mm of rain on average, out of which the 436.5 mm (43% of the annual precipitation) during the above mentioned period. The climate change is projected to warm southern Europe more than the 2.1– 4.4 8C or possibly the 2.0–6.3 8C estimated for the rest of Europe by 2100 (EEA, 2007; Schro¨ter et al., 2005; Giannakopoulos, 2005). Moreover, southern Europe already became 20% drier during the 20th century, and is expected to become more severely affected by drought till 2100 (EEA, 2007; Giorgi et al., 2004). It was also estimated that the projected average temperature increase of 3– 4 8C in the Mediterranean basin could lead to an increase in potential evapotranspiration of 400 mm/year (Naveh, 1997). This combined with a changing frequency and magnitude of rainfall events and soil degradation processes decreasing its water-holding capacity, could lead to an increase in aridity, even if the annual amount of precipitation were unchanged (Boer and De Groot, 1990). Thus, it can be suggested that the meteorological conditions experienced by the seed orchard clones in 2007 could represent the conditions projected by the climate change scenarios. The reproductive structures of forest trees and their development is mediated by environmental cues (photoperiod, temperature, soil moisture). Conifer apices can be determined as seed

conelets, vegetative buds or pollen strobili and the developmental sequence they undergo is largely determined by their position on the stem and in the crown and the physiological state of the tree. Temperature and moisture conditions that put the trees into stress during periods of initiation and differentiation are the principal environmental factors determining the fate and development of apices (Webber, 2004). Most conifer species are monoecious and pollen conelets are normally differentiated before the seed conelets. The development of female conelets, the development of ovules, pollen uptake and fertilization are negatively affected by seed orchard environments prone to drought and high temperatures (Webber, 2004). The cost of development of reproductive structures in terms of carbon may be at least partially compensated by increasing photosynthetic rates or by the photosynthesis of the reproductive structure itself (Obeso, 2002). An increase in photosynthetic activity should be accompanied by an increase in resource uptake in order the plant to function as a balanced system in terms of resource uptake and use (Bazzaz, 1997). In Pseudotsuga menziesii var. glauca female cones had maximum instantaneous refixation rates of 54% which integrated over the season, offset 6% of their total carbon requirements, while male conelets were completely dependent on vegetative tissues for carbon (McDowell et al., 2000). Foliage near female cones had elevated photosynthesis during the early stages of cone development and consistently lower nitrogen concentration than foliage far from cones (McDowell et al., 2000). Reznick et al. (2000) suggested that the occurrence of costs would be most apparent in habitats with low resource availability or other stress conditions. However, much less attention has been given so far to roots and the plant ability to adjust its below-ground activities, especially in xerothermic conditions. In the Mediterranean region, dehydration stress coincides with high light and high temperatures. Under these conditions, leaves are the most exposed and vulnerable plant organs. These environmental conditions accentuate the negative effects of drought on the photosynthetic machinery, usually as a result of the inability of leaves to utilize and dissipate excess intercepted energy when stomata are closed (Pereira et al., 2007). Drought and low nutrient availability are also common to Mediterranean-type ecosystems, and the two interact strongly. Water deficits inhibit uptake and transport of several ions in the transpiratory stream to a certain extent (Gollan et al., 1992), while at the same time ‘‘nitrogen use efficiency’’ (i.e. the rate of photosynthesis per unit of leaf nitrogen) decreases (Pereira et al., 2007). The differences in meteorological conditions recorded during the years of the study resulted in pronounced differences in all male and female flowering stages. The prolonged period (more than 7 months) of high temperatures (2.3 8C mean monthly temperature rise) and drought (53% less precipitation than normally expected), may have affected negatively photosynthesis, nutrient uptake and transport in the transpiratory stream and thus the coverage of reproduction cost, that should be partially compensated by increased photosynthetic rates (Obeso, 2002). The P. nigra male conelets, that have a considerably larger size (length 3–4.5 cm) than the female conelets at the reception phase (length 0.8–1.5 cm), initiated their flowering earlier than female conelets and eventhough the timing of male flowering did not differ significantly among the years of the study, the duration of pollen shedding was significantly reduced by 9 days during the xerothermic 2007. This might be due to the reduced photosynthesis during 2007 and the complete dependency of male strobili on the vegetative tissues for resources. The stressful conditions caused significant delay of the female flowering onset by 11 days and a decrease of 3 days in the duration of female receptivity. This could be attributed to the fact that the development of female conelets required elevated photosynthesis during the early stages

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

793

Fig. 9. (a and b) Spatial pattern of male flowering onset in 2006 and 2007 (on male = onset pollen shedding in number of days starting from January 1st).

of cone development (McDowell et al., 2000), but those requirements could not be readily met, due to the reduced photosynthesis and the prior flowering cost of the male strobili. The average duration of receptivity ranged among 9 days in 2006 and 6 days in 2007. The results are in agreement with the results of Matziris (1994), who studied the flowering of black pine clones in the Peloponnesos seed orchard. Vidakovic (1974) though reports a receptive period of 1–3 days for the same species, which is shorter than the period (2–13 days) reported by Lill and Sweet (1977) for Pinus radiata. The findings indicate that the duration of pollen shedding is affected more than the duration of female receptivity from the adverse climatic conditions. The above results are in agreement with the findings of Matziris (1994). Sarvas (1962), Jonsson et al. (1976) and Gullenberg et al. (1982) reported

significant differences for the duration of pollen shedding in Scots pine and stressed the strong influence of climatic conditions on pollen shedding duration. The differences in timing of female and male flowering and duration between years resulted in the complete asynchrony of clones during the stressful year. According to Reynolds et al. (2000) asynchrony of biological activities, such as different phenologies or different growth responses to temperature can be adopted by plants in order to avoid competition for water. Clonal flowering was synchronized to a large extent during 2006, ¯ indices. As the as indicated by the respective phenogram and the PO average index value of a given male parent measured for all possible female parents indicates the contribution of that pollen parent to the seed crop, it can be concluded that almost all clones contributed

794

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

Fig. 10. (a and b) Spatial pattern of male flowering duration in 2006 and 2007 (dur male = duration of pollen shedding in number of days).

moderately to high to the seed crop of the specific year (Askew and Blush, 1990). The average index of all male parents that contribute to a specific female parent can serve as a measure for potential alien pollen to contaminate that parent’s seed crop (Askew and Blush, 1990). High index values were estimated for most of the female parents and thus, the possibility for contamination of the produced seed by foreign pollen (pollen originating from genotypes other than the clones participating in the seed orchard) was minimized. One ¯ index value (0.22) for its clone was identified with a low PO contribution as pollinator, while the lower value for a seed parent was 0.43, meaning that it could receive pollen from almost half of the clones of the orchard. Variation in synchronization was found to occur in nearly all first generation seed orchards (Sarvas, 1962; Jonsson et al., 1976; Bhumibhamon, 1978; El-Kassaby et al., 1988;

Matziris, 1994). Thus, the ideal population model, which implies that effective population number is equal to actual number of clones in the seed orchard, is not valid. Also, because the flowering variation of clones in a seed orchard is not random but genetically controlled (Matziris, 1993), the assumptions of the equal contribution of clones and panmixia are violated. Significant variation for all male and female flowering parameters was recorded during the normal year, while in the stressful year significant differences were found only for the onset of male flowering. The detected spatial micro-environmental differences in the site regarding male and female flowering parameters, might have resulted to spatial variation among ramets within clones growing at different sites of the orchard. Erickson and Adams (1989)

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

795

Fig. 11. (a and b) Spatial pattern of female flowering in 2006 and 2007 (on female = onset of female receptive stage 3 in number of days starting from January 1st).

reported significant flowering variation among ramets in a clone that could be explained by height differences among ramets. The genetic control of flowering traits varied depending by trait and year. Strong genetic control was evident for onset of pollen shedding in both years, when compared to termination and duration of pollen shedding. Genetic control of pollen shedding duration was dramatically reduced in the stressful year, which was anticipated given the dependency of duration on environmental conditions (Matziris, 1994; Jonsson et al., 1976; Sarvas, 1962). Initiation of flowering at the bud break stage was under strong genetic control in both years, while initiation of the receptive stage was under low to moderate genetic control. It is noteworthy that for the stressful year low to moderate genetic control was recorded

for all female flowering parameters, while this was not the case for the normal year. The temperature and water stress might have pushed the clones to their limits resulting in greater expression of differences in flowering traits. In conclusion, climate changes towards greater aridity may decrease water and nutrient availability due to enhanced temporal heterogeneity and increased asynchrony of water availability. The pronounced variability in climatic conditions over the 2 years of the present study strongly affected flowering dates and synchronization among clones, resulting in almost complete asynchrony during the more stressful year. These results may suggest that one of the effects of a warmer and drier climate may be the lack of flowering synchronization, as pollen shedding might be completed

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797

796

Fig. 12. (a and b) Spatial pattern of the female receptive phase duration in 2006 and 2007 (fem dur = duration of female receptive stage 3 in number of days).

before female conelets reached the phase of receptivity, leading to the restriction of male parentage to a limited number of clones and thus, to the severe violation of the panmixia assumption, or even to fertilization failure. Climate change could also prove detrimental for fertility and flowering synchronization of forest trees in the Mediterranean region, having consequences on the quantity and quality of seed production in seed orchards, and the natural regeneration of forest trees in forest ecosystems due to the reduced percentage of sound seed. Acknowledgements The authors sincerely thank all the people involved in the selection of genetic material and establishment of the seed

orchard, especially Professor Emeritus K. Panetsos and Assoc. Prof. A. Scaltsoyiannes. We also thank the Aristotle University of Thessaloniki Forest Administration for providing the meteorological data. References Askew, G.R., Blush, D., 1990. Short note: an index of phonological overlap in flowering for clonal conifer seed orchards. Silv. Gen. 39 (3–4), 168–171. Bakkenes, M., Eickhout, B., Alkemade, J.R.M., 2006. Impacts of climate stabilization scenarios on plant species in Europe. Global Environ. Change 16, 19–28. Batzios, D.P., Roupakias, D.G., 1997. HONEY: a microcomputer program for plant selection and analysis of the honeycomb designs. Crop Sci. 37, 744–747. Bazzaz, F.A., 1997. Allocation of resources in plants: state of the science and critical questions. In: Bazzaz, F.A., Grace, J. (Eds.), Seeds. The Ecology of Regeneration in Plant Communities. CAB International, Oxford, UK, pp. 1–26.

P.G. Alizoti et al. / Forest Ecology and Management 259 (2010) 786–797 Becker, W.A., 1984. Manual of Quantitative Genetics, 4th ed. Academic Enterprises, Pullman, Washington, p. 188. Bhumibhamon, S., 1978. Studies on Scots pine seed orchards in Finland with special emphasis on the gametic composition of the seed. Commun. Inst. For. Fenn. 94 (4), 1–118. Boer, M.M., De Groot, R.S., 1990. Lanscape-ecological impact of climatic change. In: Proc. Europ. Conf. Lunteren, The Netherlands. Burczyk, J., Chalupka, W., 1997. Flowering and cone production variability and its effect on parental balance in a Scots pine clonal seed orchard. Ann. Sci. For. 54, 129–144. Copes, D.L., Sniezko, R.A., 1991. The influence of floral bud phenology on the potential mating system of a wind-pollinated Douglas-fir orchard. Can. J. For. Res. 21, 813–820. Danusevicius, J., 1987. Flowering and seed production of clones and their stimulation in seed orchards. For. Ecol. Manag. For. Ecol. Manage. 19, 233–240. EEA, 2007. Europe’s Environment. 4th Assessment. Copenhagen. El-Kassaby, Fashler, A.M.K., Sziklai, O., 1984. Reproductive phenology and its impact on genetically improved seed production in a Douglas-fir seed orchard. Silv. Gen. 33, 120–125. El-Kassaby, Y., Ritland, L., Fashler, A.M.K., Devitt, D., 1988. The role of reproductive phenology, parental balance and supplemental mass pollination in a Sitka spruce seed orchard. Silv. Gen. 37 (2), 76–82. Erickson, V.J., Adams, W.T., 1989. Mating success in a coastal Douglas fir seed orchard as affected by distance and floral phenology. Can. J. For. Res. 19, 1248– 1255. Eriksson, V.J., Jonsson, A., Lindgren, D., 1973. Flowering in a clonal trial of Picea Abies Karst. Stud. For. Suc. 110, 5–45. Fasoula, D.A., Fasoula, V.A., 2000. Honeycomb breeding: principles and applications. Plant Breed. Rev. 18, 177–250. Fasoulas, A.C., 1973. A new approach of plant breeding. Publ. No. 3. Dept. Genet. Plant Breed., Aristotle University of Thessaloniki. Fasoulas, A.C., 1980. Principles and methods of plant breeding. Publ. No. 10. Dept. Genet. Plant Breed., Aristotle University of Thessaloniki. Fasoulas, A.C., 1988. The honeycomb methodology of plant breeding. P.O. Box 1555, GR 54124 Thessaloniki, Greece. Fasoulas, A.C., Principles of crop breeding. P.O. Box 1555, GR 54124 Thessaloniki, Greece, p. 127. Fasoulas, A.C., Fasoula, V.A., 1995. Honeycomb selection designs. Plant Breed. Rev. 13, 87–139. Giannakopoulos, C., Bindi, M., Moriondo, M., Tin, T., 2005. Climate change impacts in the Mediterranean resulting from a 2 8C global temperature rise. WWF Report. Gland, Switzerland. Giorgi, F., Bi, X., Pal, J., 2004. Mean, interannual variability and trends in a regional climate change experiment over Europe. II. Climate change scenarios (2071– 2100). Clim. Dynam. 23, 839–858. Gollan, T., Schurr, U., Schulze, E.D., 1992. Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. I. The concentration of cations, anions, amino-acids, in and pH of, the xylem sap. Plant Cell Environ. 15, 551–559. Grossoni, P., 2008. Pinus nigra Arnold. In: Roloff, A., Weisgerber, H., Lang, U., Stimm, B. (Eds.), Enzyklopa¨die der Holzgewa¨chse: Handbuch und Atlas der Dendrologie. Wiley Verlag. Gullenberg, V., Yarzdani, R., Rudin, D., 1982. Genetic differentiation between adjacent populations of Pinus sylvestris. Silvae Fennica 16 (2), 205–214. Healy, W.M., Lewis, A.M., Boose, E.F., 1999. Variation of red oak acorn production. For. Ecol. Manage. 116, 1–11. IPCC, Climate Change, 2007. The physical science basis. In: Summary for Policy Makers, IPCC Secr., Geneva. Jonsson, A., Ekberg, I., Eriksson, G., 1976. Flowering in a seed orchard of Pinus sylvestris. Stud. For. Suc. 135, 1–38. Khuri, A.I., Sahai, H., 1985. Variance components analysis: a selective literature survey. Intern. Stat. Rev. 53, 279–300. Kinnaird, M.F., 1992. Phenology of flowering and fruiting of an east African riverine forest ecosystem. Biotropica 24, 187–194. Leinekugel le Cocq, T., Quiring, D., Verrez, A., Park, Y.S., 2005. Genetically based resistance of black spruce (Picea mariana) to the yellowheaded spruce sawfly (Pikonema alaskensis). For. Ecol. Manage. 215, 84–90. Lill, B., Sweet, G., 1977. Pollination in Pinus radiate. N.Z.J. For. Sci. 7 (1), 21–34. Lush, J.L., 1940. Intra-Sire correlations or regressions of offspring on dam as a method of estimating heritability of characteristics. J. Anim. Sci. 293– 301. Lyndon, R.F., 1992. The environment control of reproductive development. In: Marshall, C., Grace, J. (Eds.), Fruit and Seed Production. Aspects of Development,

797

Environment, Physiology and Ecology. Cambridge University Press, Cambridge, pp. 9–32. Matziris, D., 1993. Variation in cone production in a clonal seed orchard of black pine. Silv. Gen. 42 (2–3), 136–141. Matziris, D., 1994. Genetic variation in the phenology of flowering in black pine. Silv. Gen. 43 (5–6), 321–328. Matziris, D., 2005. Genetic variation and realized genetic gain from black pine tree improvement. Silv. Gen. 54 (3), 96–104. McDowell, S.C.L., McDowell, N.G., Marshall, J.D., Hultine, K., 2000. Carbon and nitrogen allocation to male and female reproduction in Rocky Mountain Douglas-fir (Pseudotsuga menziesii var. glauca, Pinaceae). Am. J. Bot. 87, 539–546. Meagher, T.R., 1988. Sex determination in plants. In: Lovett, D.J., Lovett, D.L. (Eds.), Plant Reproductive Ecology: Pattern and Strategies. Oxford University Press, New York, pp. 125–138. Mirov, N.T., 1967. The Genus Pinus. The Ronald Press Company, New York. Naveh, Z., 1997. Conservation, restoration, and research priorities for Mediterranean uplands threatened by global climate change. In: Moreno, J., Oechel, W.C. (Eds.), Global Change and Mediterranean-type Ecosystems. Springer-Verlag, NY, pp. 483–507. Nikkanen, T., 2001. Reproductive phenology in a Norway spruce seed orchard. Silvae Fennica 35 (1), 35–53. Obeso, J.R., 2002. The costs of reproduction in plants. New Phytol. 155, 321–348. Owens, N.J., Blake, M.D., 1985. Forest Tree Seed Production. Canadian Forest Service, Inf. Rep. PI-X-53, Victoria, BC, 161 pp. Pereira, J.S., Chaves, M.M., Caldeira, M.C., Correia, A., 2007. Water availability and productivity. In: Morison, J., Morecroft, M. (Eds.), Plant Growth and Climate Change. Blackwell Publication, pp. 119–145. Reynolds, J.F., Kemp, P.R., Tenhunen, J.D., 2000. Effects of long-term rainfall variability on evapotranspiration and soil water distribution in the Chihuahuan Desert: a modelling analysis. Plant Ecol. 150, 145–159. Reznick, D., Nunney, L., Tessier, A., 2000. Big houses, big cars, superfleas and the cost of reproduction. Trends Ecol. Evol. 15, 421–425. Sarvas, R., 1962. Investigations on the flowering and seed crop in Pinus sylvestris. Com. Inst. For. Fenn. 53 (4), 188. SAS Institute, Inc., 1996. SAS/STAT1 Software: Changes and Enhancements, Release 6.11, SAS Inst. Inc. Cary, NC, p. 1094. Savolainen, O., Ka¨rkka¨inen, K., Harju, A., Nikkanen, T., Rusanen, M., 1993. Fertility variation in Pinus sylvestris: a test of sexual allocation theory. Am. J. Bot. 80, 1016–1020. Scarascia-Mugnozza, G., Oswald, H., Piussi, P., Radoglou, K., 2000. Forests of the Mediterranean region: gaps in knowledge and research needs. For. Ecol. Manage. 132, 97–109. Schro¨ter, D., Cramer, W., Leemans, R., Prentice, I.C., Aranjo, M.B., Arnell, N.W., Bondeau, A., Bondeau, A., Bugmann, H., Carter, T.R., Gracia, C.A., Vega-Leinert ACdl, M., Erhard, F., Ewert, M., Glendining, J.I., House, S., Kankaapa¨a¨, R.J.T., Klein, S., Lavorel, M., Lindner, M.J., Metzger, J., Meyer, T.D., Mitchell, I., Rengister, M., Rounsevell, S., Sabate´, S., Sitch, B., Smith, J., Smith, P., Smith, M.T., Sykes, K., Thonicke, W., Thuiller, G., Tuck, S., Zaehle, B., Zierl, 2005. Ecosystem service supply and vulnerability to global change in Europe. Science 310, 1333–1337. Searle, S.R., Casella, G., McCulloch, C.E., 1992. Variance Components. John Wiley & Sons Inc., NY. Shelbourne, C.J.A., Thulin, I.J., 1974. Early results from a clonal selection and testing programme with radiata pince. N.Z.J. For. Sci. 4, 387–398. St. Clair, J.B., Kleinschmidt, J., 1986. Genotype-environment interaction and stability in ten-year height growth of Norway Spruce clones (Picea abies Karst.). Silv. Gen. 35, 5–6. Sweet, G.B., 1995. Seed orchards in development. Tree Phys. 15, 527–530. Thomas, C.D., Cameron, A., Green, R.E., Bakkenes, M., Beaumont, L., Collingham, Y.C., Erasmus, B.F.N., Ferreira de Signeira, M., Grainger, A., Hannah, L., Hughes, L., Huntley, B., van Jaarsveld, A.S., Midgley, G.F., Miles, L., Ortega-Huerta, M.A., Townsend Peterson, A., Phillips, O.L., Williams, S.E., 2004. Extinction risk from climate change. Nature 427, 145–148. Vidakovic, M., 1974. Genetics of European black pine (Pinus nigra Arn.). Ann. Forestales Zagreb 6 (3), 57–86. Webber, J., 2004. Physiology of sexual reproduction in trees. In: Burley, J., Evans, J., Youngquist, J.A. (Eds.), Encyclopedia of Forest Sciences. Elsevier Publ., pp. 1639–1644. Weir, R., Zobel, B., 1975. Advanced generation seed orchards, Seed Orchards, For. Comm. Bulletin No. 54. London, pp. 118–127. Woessner, R.A., Franklin, E.C., 1973. Continued reliance on wind-pollinated southern pine seed orchards—is it reasonable? In: Dinus, R.J., Thielges, B.A., Wells, O.O. (Eds.), Proceedings of the 12th Southern Tree Improvement Conference, Louisiana State University, Baton Rouge, pp. 64–73.