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Eur J Forest Res (2011) 130:657–670 DOI 10.1007/s10342-010-0457-1

ORIGINAL PAPER

Absence of geographical structure of morphological variation in Juniperus oxycedrus L. subsp. oxycedrus in the Balkan Peninsula Robert Brus • Dalibor Ballian • Peter Zhelev • Marija Pandzˇa • Martin Bobinac • Jane Acevski Yannis Raftoyannis • Kristjan Jarni



Received: 26 March 2010 / Revised: 20 October 2010 / Accepted: 25 November 2010 / Published online: 21 December 2010 Ó Springer-Verlag 2010

Abstract We examined leaf and mature seed cone variation of Juniperus oxycedrus L. subsp. oxycedrus in 12 natural populations across the species range in the Balkan Peninsula. We measured 10 morphological traits from a minimum of 100 leaves in each of 190 individuals, and two morphological traits from 30–50 seed cones in each of 94 females. High phenotypic variation was found, but no geographical structure or cline across populations was detected for any of the studied traits. Mean values of comparable leaf and cone morphological traits did not differ considerably from values reported elsewhere. Gender dimorphism in leaf morphology was detected, but it was not distributed uniformly throughout the studied area. An ANOVA model with both nested and crossed effects revealed that the largest proportion of the total variation was, as expected, contained within populations, partly as among-tree variation (18–47%, depending on the trait) and partly as within-tree variation (33–77%), which

was remarkably high. Gender dimorphism explained only 0–3% of the total variation. Differences among populations (2–23%) were significant for all studied traits except one; however, PCA showed no clear geographical differentiation of the studied populations. This lack of phylogeographical structure may be the consequence of repeatedly occurring colonisation-retreat scenarios and suggests the existence of several small refugial populations scattered over a large part of the Balkan Peninsula in the Pleistocene. Further research including palaeobotanical and molecular genetic studies will be needed to better understand the forces that shaped current variation patterns of J. oxycedrus L. subsp. oxycedrus in the Balkan Peninsula. Keywords Phenotypic variation  Plant variation  Plant morphology  Biometry  Sexual dimorphism  Geographical differentiation  Pleistocene refugia

Communicated by C. Ammer. R. Brus (&)  K. Jarni Biotechnical Faculty, Department of Forestry and Renewable Forest Resources, University of Ljubljana, Vecˇna pot 83, 1000 Ljubljana, Slovenia e-mail: [email protected] D. Ballian Faculty of Forestry, University of Sarajevo, Zagrebacˇka 20, 71000 Sarajevo, Bosnia and Herzegovina P. Zhelev Department of Dendrology, University of Forestry, 10 Kliment Ochridsky Bulvd., 1756 Sofia, Bulgaria

M. Bobinac Faculty of Forestry, University of Belgrade, Kneza Visˇeslava 1, 11030 Belgrade, Serbia J. Acevski Faculty of Forestry, University of Skopje ‘Cyril and Methodius’, Bul. Aleksandar Makedonski bb, 1000, Skopje, Republic of Macedonia Y. Raftoyannis Department of Forestry and Environmental Management, Technological and Educational Institute of Lamia, 36100 Karpenisi, Greece

M. Pandzˇa Primary School ‘Murterski sˇkoji’, Put sˇkole 8, 22243 Murter, Croatia

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Introduction Juniperus oxycedrus L. subsp. oxycedrus is considered a typical subspecies of Prickly Juniper (J. oxycedrus L.) and is distributed from Morocco, Algeria and Tunisia in north Africa into Portugal, Spain, France, Italy, the Balkan Peninsula, Turkey and eastward to southern Caucasus and northern Iran (Franco J do 2002; Farjon 2005; Roloff and Ba¨rtels 2008). Throughout its distribution range, it is a common element in Mediterranean sclerophyllous shrubland, such as maquis and garrigue, and in dry woodland with termophyllous tree species, as well as in montane and mesic forest. It occurs on dry, stony slopes in thin soil on all kinds of parent rock but is rare on sand dunes; in Europe, it can grow up to 2,300 m a.s.l. (Christensen 1997; Franco J do 2002; Farjon 2005). It is an evergreen, dioecious erect shrub or small tree up to 8(12) m tall, with spreading or ascending branches, leaves (6)8–15(25) mm long and 1–1.5(2) mm wide and orange- to reddish brown seed cones that are (6)8–10(13) mm thick and normally contain three seeds (Franco J do 2002; Schulz et al. 2003; Farjon 2005; Roloff and Ba¨rtels 2008). Beside J. oxycedrus L. subsp. oxycedrus, another three subspecies are usually recognised within the species: J. oxycedrus subsp. macrocarpa (Sibth. & Sm.) Ball, J. oxycedrus subsp. badia (H. Gay) Debeaux and J. oxycedrus subsp. transtagana Franco (Franco J do 1963; Farjon 2001; Franco J do 2002; Farjon 2005). In addition, Adams et al. (2005) argue the existence of two cryptic, genetically distinct but morphologically very similar species within J. oxycedrus L. subsp. oxycedrus. These species are believed to be largely allopatric; J. oxycedrus subsp. oxycedrus is only distributed in the areas west of Italy, while the recently recognised new species, J. deltoides R. P. Adams, based on published data on DNA RAPDs, nrDNA sequence, morphology and terpenoids (Adams 2004), is thought to be only present from Italy eastward through Turkey into the Caucasus Mts. and Iran. According to this view, all J. oxycedrus L. subsp. oxycedrus populations in the Balkan Peninsula would in fact be treated as J. deltoides R. P. Adams. Much of the recent research within J. oxycedrus L. sensu lato has so far been focused on J. oxycedrus L. subsp. macrocarpa (Lewandowski et al. 1996; Cantos et al. 1998; Garcı´a-Berlanga and Serrano 2003; Klimko et al. 2004; Mun˜oz-Reinoso 2004; Redondo and Saavadera 2004; Sezik et al. 2005; Juan et al. 2006; Massei et al. 2006), which is a rare and endangered species in several parts of its range. Within J. oxycedrus L. subsp. oxycedrus, beside taxonomical studies (Adams 2003, 2004; Adams et al. 2005), research has dealt with new forms (Yaltirik et al. 2007; Avci and Zielin´ski 2008), reproduction (Cantos et al. 1998; Ortiz et al. 1998; Arista et al. 2001), demographic dynamics (Biondi 1990;

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Baldoni et al. 2004), phytosociology (Cano et al. 2007), somatic embryogenesis (Gomez and Segura 1996) or chemical composition of the plant (Milos and Radonic 2000; Koukos et al. 2002; Valentini et al. 2003; Loizzo et al. 2007). Only a few studies addressed the morphologic variation of J. oxycedrus L. subsp. oxycedrus. Lebreton et al. (1991) found the seed cones to be discriminant between subsp. oxycedrus and subsp. macrocarpa, while the mean number of seeds per cone was not. More recently, Klimko et al. (2007) performed a biometric examination, based on morphological characters of the leaves, cones and seeds, of 13 J. oxycedrus L. subsp. oxycedrus populations from East- and West-Mediterranean regions and reported significant differences between both groups of populations. In the Balkan Peninsula in particular, J. oxycedrus L. subsp. oxycedrus has been poorly studied. Most of the earlier-mentioned research on this taxon was performed on the material from either West- or East-Mediterranean (including Greek) populations with the exception of the study of Klimko et al. (2007), which included one population from Bosnia and Herzegovina and another from Croatia. When defining a new J. deltoides species, Adams (2004) only cites one representative specimen from the Balkan Peninsula outside Greece; this is a herbarium specimen collected by W. B. Turill in 1922 near Porecˇ, Istria, Croatia. Alexandrov et al. (1993) described a new locality of J. oxycedrus in Bulgaria, Milos and Radonic (2000) analysed its volatile compounds in Croatia, and Koukos et al. (2002) examined the chemical composition of the berry oil in Greece. In flowering plants, sexual dimorphism is often observed not only in sexual organs but also in traits that are not directly related to reproduction. Species can be sexually dimorphic in resource acquisition and allocation, in interactions with other community members, and in size, colour or longevity of vegetative structures (Dawson and Geber 1999; Delph 1999). Differences in leaf size between genders have been reported for several species with females often having larger and/or longer leaves than males (Wallace and Rundel 1979; Bond and Midgley 1988; Mitchell 1998; Delph et al. 2002; Iszkuło et al. 2009). For J. communis, sexual dimorphism was identified in lifetime performance (Ward 2007), in water use in drier sites (Hill et al. 1996), and males were less grazed than females (McGowan et al. 2004). Males were taller than females in J. virginiana (Vasiliauskas and Aarssen 1992), while females of J. thurifera appeared to be taller than males but had lower radial growth (Gauquelin et al. 2002). Considering these differences, sexual dimorphism might be expected in Juniperus oxycedrus L. as well. So far, no sex-related leaf dimorphism in any Juniperus species has been studied or reported. Until now, phenotypic variation of J. oxycedrus L. subsp. oxycedrus in the Balkan Peninsula has only been studied

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briefly in several studies; however, there has been no integral research that systematically analysed phenotypic variation across different factors (i.e. population, individual and gender) and geographical differentiation of this species in the entire region. Also, a hypothesis on the existence of J. deltoides R. P. Adams in the eastern Mediterranean has never been tested on a large set of samples, and sex-related leaf dimorphism in any Juniperus species and J. oxycedrus subsp. oxycedrus in particular has not been studied so far. To answer these questions, we performed a detailed analysis of leaf and seed cone morphology of Juniperus oxycedrus L. subsp. oxycedrus in the entire Balkan Peninsula.

Methods Study area In the NW part of the Balkan Peninsula, J. oxycedrus L. subsp. oxycedrus is mainly distributed along the Adriatic coast and in the adjacent areas. In Slovenia, it is a very rare species only found in isolated locations on steep slopes in low altitudes in the Mediterranean region (Brus 2008). Further to the south, it is one of the most common species of various termophyllous plant communities of the Croatian regions Istria, Kvarner and Dalmatia, and in the coastal part of Montenegro and Albania; however, it can also be found more inland either continuously in submediterranean forests or in isolated locations in more the continental part of Croatia, Bosnia and Herzegovina, and Montenegro (Vidakovic´ and Franjic´ 2004; Sˇilic´ 2005). In Greece, it is widespread on the mainland and some of the larger islands except the Ionian Islands and Cyclades and is generally found on hillslopes at 200–1,300 m, sometimes ascending to sub-alpine levels (Christensen 1997). In the central part of the Balkan Peninsula with a mildly continental climate, it is relatively common in Macedonia and in isolated locations in central, western and southwestern Serbia (Omanovic´ 1938; Jovanovic´ et al. 1997; Jovanovic´ 2000; Dinic´ et al. 2006). It is frequent and abundant on the dry sunny slopes of the mountains in Southern Bulgaria, usually at lower altitudes (Alexandrov et al. 1993). Sampling Twelve populations of J. oxycedrus L. subsp. oxycedrus were sampled in 2007 across the entire species’ range in the Balkan Peninsula (Table 1; Fig. 1). In each population, 14–17 adult individuals (at least 7 males and 7 females) were selected. The sample size was based on the number of individuals that assures the determination of statistically significant differences among populations and between genders within those and is comparable with the number of

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individuals included in similar studies (e.g., Klimko et al. 2007). The sex of each individual was determined by the presence of either seed cones or male strobili or their residue on the branches at the time of sampling. The distance between selected individuals was at least 25 m. From each individual, a normally developed branch, approx. 35 cm long, was taken from the outer, sunny portion of the south, south-east or south-west facing part of the crown at a height of 1–2 m above the ground. In the laboratory, oneyear-old leaves were collected from yearly increments of at least 10 long shoots of the same branch, which were chosen randomly. From the central part of each shoot, approximately 10 leaves were collected randomly. Altogether, a minimum of 100 leaves were sampled from each individual. From each sampled female tree, 30–50 ripe seed cones were collected from the same part of the crown. For the morphological analysis, the length and diameter (average of two measurements at an angle of 90°) of fresh cones were measured to the nearest 0.1 mm. The fresh leaves were scanned and measured with the software ‘‘WinFOLIA Pro 2005’’ of Regent Instruments Inc. The scanning accuracy was set to 0.03 mm. For each leaf, 10 morphological traits were measured or calculated (Table 2; Fig. 2). In total, 190 individuals, 19,908 leaves and 4,421 cones were analysed. Statistical analysis Standard formulae of descriptive and multivariate statistics (Sokal and Rohlf 1989; McGarigal et al. 2000) were used to obtain an objective picture of the morphological parameters. Descriptive statistics, such as arithmetic means, standard deviations and coefficients of variation were used to describe the main features of morphological variation. We found no noticeable deviation from normality for any character studied. Analysis of variance was used to determine the differences among populations, between different genders and among trees in a single population. For this purpose, a nested analysis of variance was designed where trees were nested within populations and gender. The nested design was carried out with the model: Y ¼ P þ G þ P  G þ TðPGÞ þ e The model tests the main effects of population (P) and gender (G), the interaction between them (P 9 G), and the nested effects of trees within a population and gender T(PG) on all the measured traits of leaves and cones. The model had two fixed (crossed) factors (P and G) and one random (nested) effect (T). The contribution of a hierarchical level to total variance was presented as a share of total variance. Principal component analysis (PCA) was used to determine the differences among the populations. The aim of the analysis was to combine the original variables into independent

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Table 1 Main characteristics of the studied populations Pop. No.

1

Acronym

SLO

Pop. name

Kosˇtabona, near Koper

Country

Slovenia

Sample size M

F

Tot.

8

8

16

Coordinates

45° 290 0300 N

Altitude (m)

250

13° 430 4200 E 2

CRO 1

Jablanac

Croatia

7

7

14

44° 430 0500 N 0

220

00

14° 55 07 E 3

CRO 2

Tisno

Croatia

10

7

17

43° 480 2100 N

10

15° 390 5600 E 4

BIH 1

Kravica, near Ljubusˇki, Herzegovina

Bosnia and Herzegovina

8

8

16

43° 120 2300 N 17° 340 0300 E

190

5

BIH 2

Hutovo, near Ljubinje, Herzegovina

Bosnia and Herzegovina

8

8

16

42° 570 2400 N

325

17° 480 3700 E 6

MNE

Vladimir, near Ulcinj

Montenegro

8

8

16

41° 570 4900 N

135

19° 160 4400 E 7

SRB

Gocˇ, near Kraljevo

Serbia

8

8

16

43° 330 2500 N 0

730

00

20° 40 13 E 8

MAC 1

Jabolci, near Skopje

Macedonia

8

8

16

41° 540 2300 N

620

21° 190 5000 E 9

MAC 2

Trpejca, Lake Ohrid, near Mt. Galicˇica

Macedonia

8

8

16

40° 560 5900 N

1,000

20° 460 5100 E 10

GRE

Karpenisi

Greece

8

8

16

38° 550 3200 N 0

1,280

00

21° 47 39 E 11 12

BUL 1 BUL 2

Goleshevo Yasenovo, near Kazanlak, Stara Planina

Bulgaria Bulgaria

7 8

8 8

15

41° 260 3400 N

700

16

23° 350 0100 E 42° 410 3000 N

550

25° 140 3400 E

synthetic variables that explained the greatest part of the total variation observed among the populations. In the method, a varimax rotation was used. The units that were discriminated were the populations. All computations were performed with SPSS Statistics 17.0 and Statistica for Windows software.

Results Most of the studied leaf and cone morphological traits varied greatly across the geographical range represented in the study (Table 3). In general, the differences among populations were of similar magnitude. However, population GRE differed distinctly from all other populations in several traits, such as L, A, W80, W/L and coef. The distance from the lamina’s base to the point of maximal width of the leaf (dW) was by far the most variable trait, with coefficients of variation ranging from 36.00 to 46.88% for particular populations and 42.64% on average for all samples together. The geographically intermediate and large population BIH 2 was the most variable for the largest number of studied traits (L, A,

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W/L, coef), while the least variable populations were MNE and SRB, the latter being situated at one geographical extreme and isolated. The least variable traits were cone height (H), leaf width (W) and cone diameter (D). Coefficients of variation among populations for particular morphological traits were generally of a similar order of magnitude. However, two populations, SLO and CRO 1, showed particularly large deviation from this trend in seed cone diameter (D), with very high coefficients of variation. In general, neither studied morphological traits nor coefficients of variation showed any clear patterns of change across the geographical gradient (Table 3). Gender dimorphism was established for all analysed leaf morphological traits. A comparison of the leaves of male and female sub-populations revealed significant differences in particular traits in most populations (Table 4). The differences were generally very small. In some morphological traits, however, the difference between genders was larger, and as shown in Table 5, statistically significant within the whole sample. In the population CRO 2, dW (position of the widest point of the leaf) of females was 0.224, while

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Fig. 1 Geographical distribution of the studied populations. The dark shaded area represents the distribution range of Juniperus oxycedrus L. subsp. oxycedrus (after Jalas and Suominen 1973) Table 2 Measured or calculated morphological traits

Abbr.

Morphological trait

L

Lamina length

W

Lamina maximal width

A

Leaf area

W10

Width of lamina on 10% of lamina’s length from lamina’s base up

W25 W80

Width of lamina on 25% of lamina’s length from lamina’s base up Width of lamina on 80% of lamina’s length from lamina’s base up

W90

Width of lamina on 90% of lamina’s length from lamina’s base up

W/L

Lamina width/length ratio

coef

4pi*A/P2 (P = leaf perimeter)

dW

Distance from the lamina’s base to the point of maximal width

H

Height of seed cone

D

Diameter of seed cone

dW of males was only 0.167. Likewise, in most other populations the widest point of the leaf was positioned closer to the leaf base in males than in females, but in one population (MAC 2) the situation was reversed. In other morphological traits, even more populations expressed deviation from the general pattern: in leaf length (L) and leaf area (A) these were BIH 1 and BUL 2, while in other

traits the number was even higher. However, in none of the traits were the male–female differences distributed in the same way uniformly throughout all populations. Namely, in some populations a higher value of a particular trait was found in the male sub-population, while in other populations it was found in the female sub-population. For example, in 9 populations the leaves of females were

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The PCA revealed no clear geographical differentiation of the studied populations (Fig. 3). The first principal component, which accounts for 36.0% of the total variance, had the strongest correlation with the traits that refer to the leaf width, mainly in the lower part of the leaf (morphological traits W, W10, W25, see Table 7). Component 1 slightly separated populations SLO, BIH 2 and BUL 2, which are characterised by the narrowest leaves, especially compared to other populations with broader leaves, such as CRO 1 and CRO 2 (Fig. 3). Component 2 accounted for another 33.4% of the total variance and correlated mostly with leaf length, shape and area. This component clearly separated population GRE with the shortest and stubbiest leaves from the other populations. The traits that describe the cone characteristics, especially cone diameter (D), were poorly distinguishable.

Discussion and conclusions Variation Fig. 2 Measured leaf and cone morphological traits (for description see Table 2)

significantly longer than those of males, whereas in 2 populations (BIH 1, BUL 2) the male leaves were longer. Nested analysis of variance performed on all twelve morphological variables revealed that the tested hierarchical levels (populations, genders and trees nested within populations and gender) contributed to a different extent of total variation (Tables 5 and 6). Among populations, there were significant differences in all analysed traits except for dW, where only a small proportion of the total variation (2.14%) was explained. The contribution of among-population variation to the total variation was largest for coef (23.49%), D (20.81%) and W/L (19.55%) (Table 6). The differences between genders were significant in five leaf morphological traits (Table 5), with largest proportion of the total variation in W90 (2.54%), W80 (1.63%) and dW (1.40%) (Table 6). While there was no interactive effect of gender 9 population on the mean of any trait studied, there were highly significant differences in all traits among trees within the combination of population and gender. The differences among nested trees within populations accounted for 18.24–47.18% of the total variation. The trait that most strongly differentiated the trees (L—lamina length) also showed considerable variation among populations, yet the within-tree variation was lowest for this particular trait (31.70%). For all other studied morphological traits, the largest source of variation (40.38–77.31%) was attributed to variation within trees.

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The results demonstrate the high phenotypic variability of J. oxycedrus L. subsp. oxycedrus. In accordance with genetic theory, one would expect marginal populations to show less variability and central populations to show more variability (Barrett and Husband 1989). In our research, this holds for the central population BIH and marginal population SRB. On the other hand, marginal populations SLO and CRO 1, which are the two most north-western ones in our study and represent one of the northern limits of the species distribution, surprisingly showed considerably larger variation in the cone trait D, which expressed very low variation in all other studied populations. Higher levels of variability in marginal populations compared with central ones have been reported earlier for other species, such as in isozyme studies on Picea abies (Muona et al. 1990), Picea glauca (Tremblay and Simon 1989) and Acer platanoides (Rusanen et al. 2003). Rusanen et al. (2003) suggest that this can be a result of a gene flow from central to marginal populations and too short of a time span for genetic drift to be effective. These factors have probably also played an important role in J. oxycedrus L. subsp. oxycedrus in spite of the fact that in this part of its range the species occupies a very narrow belt along the Adriatic coast creating a situation, which is normally not favourable for effective gene flow. Another unfavourable condition for effective pollination is the fact that the species is dioecious and wind pollinated (Jordano 1991), and deviations from the more general pattern in cone diameter found in these populations can be also determined by low quantities of pollen during pollination or low-density stands in

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Table 3 Descriptive statistics of leaf and cone morphological traits at the population level Population

M. trait

L (mm)

W (mm)

A (mm2)

W10 (mm)

W25 (mm)

W80 (mm)

W90 (mm)

SLO

Average

14.09

1.49

15.37

1.37

1.38

0.89

0.42

SD CRO 1

W/L 0.11

coef 0.19

dW (%) 21.17

H (mm)

D (mm)

8.01

9.15

2.23

0.16

3.35

0.16

0.16

0.14

0.13

0.02

0.04

9.46

0.84

2.23

CV (%)

15.79

10.76

21.81

11.88

11.67

15.41

30.29

18.90

19.74

44.68

10.47

24.34

Average

9.37

13.30

1.66

17.02

1.53

1.54

0.92

0.42

0.13

0.23

20.29

8.48

SD

1.75

0.21

3.16

0.21

0.20

0.14

0.11

0.02

0.04

9.15

0.84

2.35

CV

13.19

12.59

18.56

14.04

13.20

15.35

27.54

17.42

17.22

45.08

9.90

25.08

CRO 2

Average SD

13.83 1.79

1.63 0.17

17.01 3.07

1.50 0.18

1.51 0.17

0.91 0.13

0.44 0.11

0.12 0.02

0.21 0.04

19.05 8.93

8.04 0.67

8.63 0.69

CV (%)

12.96

10.69

18.03

11.87

11.55

14.46

25.31

16.74

16.33

46.88

8.29

7.98

BIH 1

Average

12.90

1.58

15.21

1.45

1.47

0.93

0.47

0.12

0.22

20.45

8.06

8.42

1.56

0.18

2.65

0.18

0.18

0.13

0.12

0.02

0.04

8.48

0.73

0.60

CV (%)

12.10

11.57

17.43

12.50

12.35

14.06

25.00

16.56

18.59

41.47

9.02

7.12

Average

13.46

1.50

15.42

1.35

1.38

0.92

0.48

0.12

0.21

23.47

8.32

8.71 0.81

SD BIH 2

SD

2.60

0.18

3.92

0.19

0.18

0.13

0.12

0.02

0.05

9.47

0.78

CV (%)

19.33

12.01

25.44

14.06

13.18

14.11

25.33

21.01

21.80

40.36

9.34

9.32

Average

13.90

1.60

17.23

1.46

1.49

0.95

0.48

0.12

0.21

20.71

8.13

8.59

1.55

0.18

2.68

0.19

0.18

0.12

0.11

0.02

0.03

8.46

0.68

0.86

CV (%)

11.12

11.40

15.58

12.71

12.09

13.05

23.49

15.35

14.21

40.84

8.38

10.03

Average

13.35

1.63

16.97

1.48

1.52

0.94

0.44

0.12

0.23

22.22

8.90

9.57

1.59

0.17

2.90

0.17

0.18

0.14

0.12

0.02

0.03

8.00

1.00

1.00

CV (%)

11.88

10.74

17.06

11.81

11.54

14.75

28.56

14.55

14.75

36.00

11.26

10.46

MAC 1

Average SD

13.79 1.99

1.56 0.17

16.50 3.15

1.43 0.17

1.45 0.17

0.93 0.15

0.46 0.13

0.12 0.02

0.21 0.04

20.84 9.18

8.85 0.71

10.25 0.80

CV (%)

14.43

10.99

19.11

12.16

11.80

16.06

28.91

17.58

18.31

44.06

8.02

7.85

MAC 2

Average

14.01

1.59

17.44

1.43

1.47

0.96

0.49

0.12

0.22

23.69

8.58

8.94

MNE

SD SRB

SD

SD GRE

2.00

0.19

3.29

0.19

0.20

0.16

0.13

0.02

0.04

9.73

0.85

0.95

CV (%)

14.25

11.95

18.85

13.28

13.48

16.63

27.47

19.97

19.02

41.08

9.96

10.60

Average

10.84

1.56

13.30

1.41

1.45

0.88

0.44

0.15

0.28

22.01

8.05

8.85

1.64

0.19

2.74

0.20

0.19

0.16

0.13

0.03

0.05

9.24

0.67

0.93

CV (%)

15.17

12.31

20.58

14.01

13.18

17.87

29.42

17.94

18.77

41.97

8.32

10.49

Average

13.84

1.61

16.79

1.47

1.49

0.93

0.45

0.12

0.21

20.49

8.56

8.63

1.96

0.21

3.17

0.21

0.21

0.15

0.13

0.02

0.04

8.97

0.60

0.63

CV (%)

14.15

12.89

18.87

14.12

13.73

16.18

28.51

18.99

19.86

43.78

7.05

7.35

Average

13.82

1.48

16.44

1.33

1.36

0.93

0.48

0.11

0.21

23.42

7.94

8.79

2.29

0.18

3.83

0.18

0.18

0.13

0.12

0.02

0.04

9.46

0.74

0.55

CV (%)

16.57

12.51

23.27

13.88

13.39

14.08

25.36

19.01

18.79

40.38

9.35

6.24

Average

13.43

1.57

16.23

1.43

1.46

0.92

0.45

0.12

0.22

21.49

8.32

8.98

SD CV (%)

2.11 15.72

0.19 12.22

3.38 20.82

0.20 13.64

0.19 13.15

0.14 15.36

0.12 27.54

0.02 19.56

0.05 20.63

9.16 42.64

0.83 10.02

1.13 12.61

SD BUL 1

SD BUL 2

SD All samples

average arithmetic mean, SD standard deviation, CV coefficient of variation

which the distance between the individuals is large. Cone weight variability can also be due to the conditions in which the cones developed (Juan et al. 2003), and we assume the high variability in marginal populations can also be a result of more variable site conditions in these populations.

Cone and leaf morphology In general, the average values of the most illustrative leaf and cone morphological traits (L = 13.43, W = 1.57, D = 8.98, H = 8.32 mm) did not differ considerably from values reported in the species descriptions or morphometric

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Eur J Forest Res (2011) 130:657–670

Table 4 Arithmetic mean values of leaf morphological traits of female (F) and male (M) sub-populations W25 (mm)

W80 (mm)

W90 (mm)

W/L

coef

dW (%)

SLO

F

14.241

1.502

15.835

1.370

1.387

0.922

0.445

0.109

0.190

0.211

M

13.939

1.480

14.893

1.360

1.373

0.856

0.395

0.108

0.184

0.213

CRO 1

F

13.545

1.626

17.144

1.479

1.509

0.931

0.425

0.122

0.223

0.228

M

13.062

1.697

16.897

1.572

1.574

0.908

0.407

0.132

0.240

0.179

CRO 2

F

14.064

1.626

17.790

1.477

1.519

0.941

0.462

0.117

0.218

0.224

M

13.661

1.629

16.468

1.513

1.505

0.894

0.416

0.121

0.213

0.167

F

12.288

1.558

14.738

1.417

1.448

0.917

0.458

0.128

0.239

0.217

M

13.517

1.609

15.684

1.480

1.494

0.941

0.474

0.121

0.209

0.192

F

13.962

1.499

16.434

1.338

1.377

0.956

0.513

0.109

0.205

0.245

BIH 2

W (mm)

W10 (mm)

Gend.

BIH 1

L (mm)

A (mm2)

Popul.

M

12.949

1.506

14.402

1.356

1.392

0.890

0.441

0.122

0.216

0.224

MNE

F M

13.855 13.944

1.620 1.589

17.379 17.080

1.472 1.452

1.507 1.472

0.978 0.915

0.509 0.446

0.118 0.115

0.217 0.212

0.215 0.200

SRB

F

13.693

1.667

17.837

1.510

1.557

0.960

0.454

0.123

0.228

0.226

M

13.005

1.587

16.104

1.446

1.479

0.913

0.421

0.123

0.229

0.219

MAC 1

F

14.113

1.563

16.976

1.423

1.450

0.939

0.462

0.112

0.206

0.213

M

13.455

1.562

16.007

1.435

1.450

0.930

0.449

0.119

0.215

0.203

F

14.518

1.565

17.940

1.414

1.445

0.978

0.520

0.110

0.208

0.228

M

13.511

1.608

16.955

1.452

1.489

0.947

0.458

0.121

0.223

0.246

GRE

F

11.095

1.555

13.767

1.384

1.445

0.927

0.464

0.143

0.277

0.244

M

10.584

1.559

12.820

1.429

1.453

0.832

0.407

0.150

0.284

0.196

BUL 1

F

14.088

1.552

16.870

1.412

1.438

0.952

0.470

0.112

0.207

0.213

M

13.547

1.670

16.691

1.535

1.558

0.904

0.425

0.125

0.219

0.195

F

13.674

1.437

15.964

1.284

1.320

0.918

0.475

0.108

0.211

0.246

M

13.962

1.518

16.908

1.371

1.399

0.949

0.485

0.110

0.208

0.223

MAC 2

BUL 2

Bold print designates statistically significant difference between female and male sub-population (t-test, P \ 0.05)

Table 5 F-ratios for the analysed traits from the nested analyses of variance Source of variation

L

W

A

W10

W25

W80

W90

W/L

coef

dW

H

D

Population

5.09***

5.13***

4.59***

5.36***

5.13***

2.07*

2.81**

7.21***

9.03***

1.54n.s.

2.95**

5.05***

Gender

1.48n.s.

3.71n.s.

1.87n.s.

8.14**

3.78n.s.

7.54**

13.43***

5.46*

0.34n.s.

11.04**

/

/

Population 9 Gender

0.64n.s.

0.71n.s.

0.58n.s.

0.78n.s.

0.68n.s.

0.69n.s.

0.74n.s.

0.77n.s.

0.77n.s.

0.65n.s.

/

/

Tree

171.91***

44.60***

102.15***

39.98***

43.65***

59.20***

50.10***

98.61***

97.48***

27.26***

52.42***

33.84***

n.s. not significant at P [ 0.05; * 0.01 \ P \ 0.05; **0.001 \ P\0.01; *** P \ 0.001

studies (Lebreton et al. 1998; Franco J do 2002; Farjon 2005; Klimko et al. 2007; Roloff and Ba¨rtels 2008). The cones in all studied populations were slightly elliptic, i.e., flattened since the D value always exceeds H; on average, the calculated ellipticity (D/H) was 1.07. This is very similar to values reported from the western Mediterranean by Lebreton et al. (1998). Also, Klimko et al. (2007) found a similarly flattened cone shape in all populations in the eastern Mediterranean region from Croatia to Crimea, while the cones from the western Mediterranean were more often oblong, i.e., cone height exceeded cone width. In addition to traditionally scored morphological traits (L, W), several traits (W10, W25, W80, W90, W/L, dW)

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were measured in our study in order to analyse the leaf shape. In the literature, detailed descriptions of Juniperus oxycedrus L. subsp. oxycedrus leaf shape are scarce. Farjon (2005) does not mention the leaf shape of this particular subspecies, Franco J do (2002) only describes it as acuminate-subulate and more or less spinous at the apex, while Christensen (1997) describes the widest point in the middle of the leaf or even above for J. oxycedrus L. subsp. oxycedrus from Greece. In our results, the widest point of the leaf was positioned substantially lower, at 21.49% of the leaf length on average and at 22.01% in the GRE population (Table 3). However, it should be mentioned that traits describing leaf shape, and dW in particular, were the

Eur J Forest Res (2011) 130:657–670

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Table 6 Partitioning of total variance by hierarchical level for studied leaf and cone morphological traits

Table 7 Correlation between morphological traits and the first three components

Population

Gender

Population 9 Gender

Tree

Within tree L

0.070

L

18.35

0.49

2.29

47.18

31.70

W

0.980

0.120

0.009

W

9.66

0.63

1.33

24.62

63.76

A

0.528

-0.690

0.368

A W10

13.82 9.27

0.51 1.28

1.73 1.35

39.39 22.65

44.54 65.44

W10

0.984

0.044

-0.114

W25

0.975

0.149

-0.017

W25

9.53

0.64

1.26

24.29

64.28

W80

0.287

-0.424

0.831

W80

4.91

1.63

1.63

31.12

60.71

W90

5.85

2.54

1.55

27.25

62.82

W90 W/L

-0.281 0.289

-0.145 0.944

0.883 -0.154

W/L

19.55

1.35

2.08

35.48

41.55

coef

0.189

0.969

0.011

coef

23.49

0.08

1.99

34.07

40.38

dW

-0.655

0.149

0.615

2.14

dW

Component 1 Component 2 Component 3 -0.979

0.163

1.40

0.91

18.24

77.31

H

0.346

-0.058

0.204

H

16.39

/

/

41.41

42.20

D

-0.020

-0.076

-0.167

D

20.81

/

/

30.71

48.48

Eigenvalue

4.32

4.01

% of explained variance 36.0

33.4

1.67 13.9

present in all studied populations with no clear geographical pattern. The leaf base shape of our samples can be seen from the comparison of W and W10 values (Table 3). The largest difference, but still relatively small, is found in population BUL 2 (W = 1.48, W10 = 1.33, W/W10 = 1.113), and on average we found W = 1.57, W10 = 1.43 and W/W10 = 1.098. The detected differences are small enough to conclude that average leaf shape in the studied area is closer to the ‘delta-shaped’ type, characterised by a relatively wide leaf base. However, variation of the leaf shape within trees is very high and within a single branch all shape types and their intermediates can be found. Gender dimorphism

Fig. 3 Distribution of the first two components according to the PCA of leaf and cone morphological traits

most variable traits with the largest coefficients of variation (Table 3). Moreover, Christensen (1997) does not specify the position of the sampled leaves within the shoot and the established differences could also be due to the different sampling method. Adams (2004) and Adams et al. (2005), when defining a new species J. deltoides, presumably distributed in the Balkan Peninsula, argue that it can be morphologically distinguished by ‘having a leaf base almost as wide as the blade’, whereas J. oxycedrus L. subsp. oxycedrus has a ‘narrowing of the leaf base’. Klimko et al. (2007) found these so-called boat-shaped and delta-shaped leaves as well as their intermediates to be

For most leaf morphological traits, the difference between male and female sub-populations was very small, but significant within single populations. This might be partly explained as an effect of a very large sample. Females and males in this study did not differ uniformly in their leaf morphological traits in all studied populations, and no geographical pattern was detected. This rather unusual distribution of gender dimorphism is difficult to interpret. Established gender dimorphism is generally a characteristic within single populations; however, it does not confirm the existence of uniform gender dimorphism in leaf morphology of J. oxycedrus L. subsp. oxycedrus in general. In earlier-mentioned examples, gender dimorphism was found in J. communis studied in one population with several subsites (Hill et al. 1996), in 4 populations (McGowan et al. 2004) and in 2 populations (Ward 2007). It was also found in several of the 20 studied populations of J. virginiana

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(Vasiliauskas and Aarssen 1992) and in most of the 9 studied populations of J. thurifera (Gauquelin et al. 2002). However, different results were reported by Marion and Houle (1996), who found no growth differences between genders in J. communis var. depressa either under mesic or stressful conditions, by Verdu´ et al. (2004), who found no gender effect in J. communis because females and males did not differ in their growth or physiological traits, and in a part by Ortiz et al. (1998), who only found segregation of the genders by niche in one population out of six in J. oxycedrus. The expected differential reproductive cost was not confirmed in J. communis subsp. alpina (Ortiz et al. 2002) and long-term results did not show major differences in growth rates and survival between males and females of J. virginiana (Quinn and Meiners 2004). The different results found within the particular samples in the average values of characters of male and female sub-populations can also result partly from the different age of compared individuals and/or populations, and possibly from the number of individuals representing particular populations. More numerous samples as well as consideration of site conditions would probably give more uniform results. Variance components across hierarchical levels We found a remarkably high within-tree variation. Random errors, caused by imprecise measurements, are one possible source of this variation. However, since the measurements were carried out with a high precision, we consider the contribution of this factor to be of minor importance. Even if the variation displayed by within-tree variation was not explicitly studied, we conclude that its major part is generated by variation among leaves within the tree, which is a largest source of variation for all studied traits except leaf length (Table 6). High within-tree variation is a typical finding in morphometric studies (Baranski 1975; Blue and Jensen 1988) and can be the result of several factors including phenotypic plasticity and/or developmental instability (Gonza´les-Rodrı´guez and Oyama 2005), leaf position on a shoot and differences of shoot position within the crown (Sokal et al. 1986; Blue and Jensen 1988; Niinemets et al. 2004). For instance, Bruschi et al. (2003) found a considerable sun-shade dichotomy in leaf morphology and anatomy in Quercus petraea. This factor accounted for the highest percentage of the within-tree variation in their research. In our study, large branches were uniformly sampled with respect to position within the crown and to light conditions, but shoots within the branch were chosen randomly. Our results do not allow us to further subdivide the within-tree variation into consistent differences depending on position and error. However, while taking measurements, we observed large differences

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(mostly in leaf shape) among leaves in spite of the fact that only one-year-old leaves from the central parts of the shoots were collected. According to Klimko et al. (2007), the leaves of J. oxycedrus L. subsp. oxycedrus in central and apical parts of the shoots are always the longest ones. If the leaves from the basal part of the shoots, which are wider at the base and shorter than other leaves, were analysed too, the variation would be even larger. Taken together, this may suggest the role of high plasticity as well as developmental effects that may be expressed at any time in the life span of the organism and are here probably expressed through large leaf shape and size variation regarding their position on the shoot. The relatively high level of among-tree variation within the populations can be partly due to the effect of phenotypic adaptation to diverse micro environmental conditions experienced by each tree, but also the result of genetic differences among individual trees. J. oxycedrus genetic variation has been poorly studied; in one population of J. oxycedrus L. subsp. macrocarpa in Korfu island, a very low level of genetic variation was found (Lewandowski et al. 1996). For the related species J. communis, the variability within populations was found to be strikingly high (Ward 2007), and most of the genetic variation was found within, rather than among, populations (Van der Merwe et al. 2000; Oostermeijer and de Knegt 2004). This is considered as typical for this wind-pollinated, dioecious species. In our study, each population was sampled in a relatively small area, microenvironmental conditions within a particular population were not very diverse, and morphological traits were likely not subjected to highly differentiated environmental pressure. Considering this, we can conclude that a high level of among-tree phenotypic variation is to a large extent also the result of genetically based variation among individuals. Geographical differentiation We found significant among-population differences in almost all analysed traits. A proportion of the total variation explained by these differences is relatively small, but this is a common situation not only within particular Juniperus species (Van der Merwe et al. 2000; Mazur et al. 2004; Oostermeijer and de Knegt 2004; Ward 2007; Sultangaziev et al. 2010), but also in other outcrossed, windpollinated species with large geographical ranges (Hamrick et al. 1992; Fady-Welterlen 2005). However, as revealed in a study of two subspecies of J. phoenicea in Spain, amongpopulation differentiation based on morphological traits can sometimes be very high (Mazur et al. 2003, 2010). Our study revealed no clear geographical differentiation of the studied populations (Fig. 3). A similar absence of geographical structure has been found in a genetic study of

Eur J Forest Res (2011) 130:657–670

J. oxycedrus subsp. macrocarpa from the three geographically distant regions in Spain (Juan et al. 2008), where most of the fragmented populations were arranged together and mixed without any geographical pattern. Morphological variation of J. seravschanica Kom. in Kyrgyzstan was not related to geographical, altitudinal or any of the environmental variables tested (Sultangaziev et al. 2010), and weak differentiation was also found in J. communis in Britain (Van der Merwe et al. 2000) and Netherlands (Oostermeijer and de Knegt 2004). Explanations of these results include a restricted geographical scale of the studied populations as well as only recent fragmentation and too short a time to develop a geographically conditioned morphological structure. These factors were probably important in shaping J. oxycedrus L. subsp. oxycedrus variation too. The specific evolutionary history of each species plays an important role in determining the level and distribution of its genetic diversity (Hamrick et al. 1992). Klimko et al. (2007), based on the revealed geographical nature of the morphological polymorphism in J. oxycedrus L. subsp. oxycedrus, hypothesised the existence of at least two Pleistocene refugial areas, one placed in the Westand another in the East-Mediterranean region. In the Balkan Peninsula, high concentrations of Juniperus pollen were recorded as early as 12,000 BP (Huntley and Birks 1983). In palynological studies, the pollen of different Juniperus species cannot be distinguished (Moore et al. 1991), but the Balkan samples most probably belonged to the most widespread J. oxycedrus (Huntley and Birks 1983) and possibly to more locally spread J. foetidissima and J. excelsa (Marcysiak et al. 2007) or J. phoenicea (Huntley and Birks 1983). This assumption is based on these species’ drought resistance and thermophily as well as their common occurrence with ecologically similar Mediterranean species, such as Phillyrea sp. and Quercus ilex in the mid-Holocene (Jahns and van den Bogaard 1998). Although recent studies clarify the locations of multiple refugia both in the southern and in the northern parts of the Mediterranean (see Me´dail and Diadema 2009), these data suggest the existence of extended glacial refugia in this region and early and fast colonisation at the opening of the late glacial. As a consequence of further forest expansion following climatic amelioration, the fragmentation of the range occurred at 10,000 BP. In the mid-Holocene, Juniperus expanded and retreated several times in this area, but always very locally (Huntley and Birks 1983). For example, such expansion was proved on the island Mljet (Jahns and van den Bogaard 1998) and in the Dalmatian mainland (Brande 1973, 1989; Gru¨ger 1996), where in the period ca. 6,000–4,400 cal BC the deciduous oak woodland was replaced by a vegetation

667

type dominated by Phillyrea and Juniperus as response to climatic change, characterised by drier summers and milder winters. The present absence of geographical structure may well be a result of early fragmentation and repeatedly occurring colonisation-retreat scenarios. All Juniperus species are prolific pollen producers, but their pollen is poorly dispersed (Huntley and Birks 1983), and normal wind pollination rates in larger populations may have been further disturbed because of the increased vegetation cover, often with a canopy of tall oak trees, and in many cases narrow distribution area along the coast and/or mountain ridges. In addition, the low density of several populations may have reduced pollination distances and created local substructures. However, other evolutionary events, for example, colonisation of deforested or fire areas by the species, could also have shaped the patterns of the current distribution of diversity. The absence of clear geographical differentiation, absence of any clinal distribution of morphological traits, as well as fast appearance in the late glacial may speak in favour of the hypotheses of the existence of a larger number of small refugial populations or groups distributed over a large part of the Balkan Peninsula rather than the existence of a unique original population somewhere in the south from where J. oxycedrus L. subsp. oxycedrus would have spread in the late glacial. In conclusion, this study has shown the high phenotypic variability of J. oxycedrus L. subsp. oxycedrus in the Balkan Peninsula and found certain gender dimorphism, but failed to confirm its uniform distribution pattern throughout the studied area. A remarkably high amount of variation was found within the trees. Significant amongpopulation differentiation of the studied populations was also revealed; however, there was no clear geographical differentiation and a relatively small proportion of the total variation was explained by the differences among populations. The results may support hypotheses of the existence of several small refugial populations scattered over a large part of the Balkan Peninsula coast that may have been exposed during the Pleistocene as result of a decreasing sea level. However, additional palaeobotanical evidence as well as extended genetic molecular research will be needed to better understand the forces that shaped the current variation pattern of J. oxycedrus L. subsp. oxycedrus in the Balkan Peninsula. Acknowledgments This research was supported by the research programme P4-0059. We thank Rade Cvjetic´anin and Zvone Sadar for their help in providing the samples, Ursˇka Galien and Danijel Borkovicˇ for technical assistance, Tjasˇa Bavcon for drawing the Fig. 2 and Blazˇ Repe for drawing the Fig. 1. We thank Thomas Nagel for improving the English.

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