Morphological variation of Juniperus oxycedrus subsp. oxycedrus (Cupressaceae) in the Mediterranean region

ARTICLE IN PRESS

Flora 202 (2007) 133–147 www.elsevier.de/flora

Morphological variation of Juniperus oxycedrus subsp. oxycedrus (Cupressaceae) in the Mediterranean region Małgorzata Klimkoa, Krystyna Boratyn´skab, Jose Maria Montserratc, Yakov Didukhd, Angel Romoc, Daniel Go´meze, Magdalena Kluza-Wielocha, Katarzyna Marcysiakf, Adam Boratyn´skib, a

Department of Botany, August Cieszkowski Agricultural University, Wojska Polskiego 71 C, 60-625 Poznan´, Poland Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Ko´rnik, Poland c Consejo Superior de Investigaciones Cientificas, Institute of Botany, Passeig del Migdia s/n., 08038 Barcelona, Spain d M. G. Kholodny Institute of Botany of National Academy of Sciences of Ukraine, 2 Tereschenkivska str., 01601 Kyiv, Ukraine e Consejo Superior de Investigaciones Cientificas, Instituto Pirenaico de Ecologia, Avda. Regimiento de Galicia s/n., 22.700 Jaca (Huesca), Spain f Department of Botany, University of Kazimierz Wielki, Ossolin´skich 12, 85-064 Bydgoszcz, Poland b

Received 10 February 2006; accepted 24 March 2006

Abstract The intra- and inter-population variation of Juniperus oxycedrus subsp. oxycedrus were examined biometrically on the basis of morphological characters of needles, seeds and cones. Cluster analysis of Euclidean distances and discriminant analysis were performed to verify the hypothesis of geographic differentiation and origin from the Westand East-Mediterranean Pleistocene refugial regions. The West-Mediterranean populations of J. oxycedrus subsp. oxycedrus differed from the eastern ones. The intra-populational differentiation of three of the West-Mediterranean populations was significantly higher than within the other West- and all the East-Mediterranean ones. Results confirm the hypothesis of probable different origins of the East- and West-Mediterranean populations of the taxon. r 2006 Elsevier GmbH. All rights reserved. Keywords: Juniperus oxycedrus; Taxonomy; Variation; Polymorphism; Biometry; Pleistocene refugia

Introduction Taxonomically Juniperus oxycedrus belongs to the section Juniperus ( ¼ Oxycedrus) of the genus Juniperus. It is a variable species, especially in the central and western parts of the distribution range. This was the reason for distinguishing four subspecies (Farjon, 2005; Greuter et al., 1984), differing in habit, dimension of Corresponding author.

E-mail address: [email protected] (A. Boratyn´ski). 0367-2530/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2006.03.006

cones and width of needles (Amaral Franco, 1963, 1968, 1986; Farjon, 2005; Lebreton et al., 1991, 1998): J. oxycedrus L., Sp. Pl. 1038 (1753) Syn.: Juniperus rufescens Link in Atti Congr. Sci. Ital. 7: 878 (1846) – subsp. oxycedrus – subsp. badia (H. Gay) Debeaux, Fl. Kabylie 411 (1894) Syn.: J. oxycedrus var. badia H. Gay – subsp. transtagana Franco in Fedde’s Repert. Sp.

ARTICLE IN PRESS 134

M. Klimko et al. / Flora 202 (2007) 133–147

Nov. Regni Veget. 68(3): 166 (1963) Syn.: Juniperus navicularis Gand., in Bull. Soc. Bot. France 57: 55 (1910) – subsp. macrocarpa (Sibth. et Sm.) Ball in J. Lin. Soc., Bot. 16: 670 (1878) Syn.: Juniperus macrocarpa Sibth. et Sm., Fl. Graec. Prodr. 2: 263 (1816). J. oxycedrus subsp. transtagana Franco (Amaral Franco, 1963, 1964; Farjon, 2005; Greuter et al., 1984) has been sometimes treated as an independent species – J. navicularis Gand. (Amaral Franco, 1986; Farjon, 2001). The typical subspecies – J. oxycedrus subsp. oxycedrus – is a small tree or only a shrub, has reddish-brown cones about 1 cm across and needles most frequently 1.0–1.5 mm width. It occurs in the Mediterranean region throughout the species range (Fig. 1), but mainly somewhat inland and in the mountains, up to altitudes of 2300 m in Europe, 2000–2100 in Asia and even 2500 m in North Africa. It grows in various types of Mediterranean forest, also sometimes forming its own communities, as for example in Greece, Anatolia and Morocco (Boratyn´ski et al., 1992; Browicz, 1982; Charco, 1999). It is a drought-resistant, but lightdemanding, pioneer species (Bondi, 1990; Charco, 2001; Farjon, 2005; Que´zel and Barbero, 1981; Que´zel and Pesson, 1980; Zohary, 1973). In spite of the description of taxa based on morphological characters, the morphological variation of J. oxycedrus has not been studied intensively. The only known biometric investigation of cone variability (Lebreton et al., 1991, 1998) was based on not very numerous individuals, sampled mainly from France, Spain, North-West Africa and Turkey, and used such characters as the diameter and weight of cones and the number of seeds per cone. The results stressed the

differences between subsp. oxycedrus and subsp. macrocarpa and a lack of significant differences between subsp. oxycedrus and subsp. badia. The results were confirmed in a parallel study of prodelphinidin contents in the needles (Lebreton et al., 1991, 1998). Sometimes are mentioned the slight differences between J. oxycedrus subsp. oxycedrus and J. oxycedrus subsp. badia leaves. The first taxon has a rounded, while the second one a keeled abaxial face of needle (Roloff and Ba¨rtels, 1996). Morphometric comparisons of J. oxycedrus subsp. oxycedrus sampled from western and eastern parts of the Mediterranean regions have been performed on several populations, on a few characters of cones and seeds by Lebreton et al. (1998). The average values of diameter of cones and number of seeds per cone calculated for 25 individuals from Turkey appeared lower than those from France (Lebreton et al., 1998). The considerable differences found lately between J. oxycedrus and the other closely related taxa, demonstrated in the composition of leaf essential oils and fingerprinting by RAPDs, suggested that most of them should be treated at the specific level (Adams, 2000), but this has not been accepted (Farjon, 2005). Differences among 27 individuals of J. oxycedrus subsp. oxycedrus based on DNA RAPDs, nrDNA sequences, terpenoid composition of the needles and the position of the widest part of the leaves were lately the basis of description of Juniperus deltoides R. P. Adams (Adams, 2004). The morphological differences were found as slight and inconspicuous, while differences between the biochemical data were significant. The analysis of related taxa, as J. oxycedrus subsp. macrocarpa (J. macrocarpa), subsp. transtagana (J. navicularis) and subsp. badia, revealed the strong separation of subsp. macrocarpa. The individuals of all other verified taxa formed two agglomerations, the western with

Fig. 1. Natural range of Juniperus oxycedrus subsp. oxycedrus (after Boratyn´ski et al., 1992; Browicz, 1982; Charco, 2001; Jalas and Suominen, 1973).

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

individuals sampled as subsp. transtagana, subsp. badia and a part of subsp. oxycedrus, and the eastern with individuals of subsp. oxycedrus (Adams et al., 2005, Fig. 3). The sufficiently great differences between western and eastern individuals of J. oxycedrus subsp. oxycedrus served as the basis for description of J. deltoides (Adams, 2004), than recognized as one of many still not described cryptic species within the J. oxycedrus complex (Adams et al., 2005, p. 786). The large east–west area of distribution of J. oxycedrus subsp. oxycedrus (Fig. 1) and the differentiation on the biochemical and genetic levels suggest an origin from various Pleistocene refugia. The eastern populations, e.g. from Anatolia, Crimea and Balkan Peninsula may have developed from the East-Mediterranean Pleistocene refugia in Anatolia and the Arabian Peninsula, while the western ones from the WestMediterranean refugial regions in the southern part of the Iberian Peninsula and North-Western Africa, as has been suggested several times for forest trees and small mammals (Bennet et al., 1991; Bilton et al., 1998; Dumolin-Lape`gue et al., 1997; Ferris et al., 1999; Hewitt, 1996, 1999; Huntley and Birks, 1983; Willis et al., 2000). The hypothesis of the present study is that the probable different origins of the species in the Eastand West-Mediterranean were responsible not only for the differences in biochemical traits, DNA RAPDs and ITS sequences, but also for generating the morphological differentiation among populations of J. oxycedrus subsp. oxycedrus.

Table 1.

2 3 4 5 6 7 8 9 10 11 12 13

Materials and methods Plant material and characters studied Thirteen populations of J. oxycedrus subsp. oxycedrus were sampled in 2001–2003 (Table 1). A total of 30 ripe cones and 30 1-year-old needles were collected from the central parts of long shoots, from sunny, mostly southoriented parts of the crowns (including the south-eastern and south-western expositions), at a height of 1.0–2.0 m, from about 20–30 individuals within eight populations (Table 1). Additionally, five populations were represented

Table 2. Analysed characters of cones, seeds and needles of Juniperus oxycedrus subsp. oxycedrus No.

Character

Accuracy (mm)

1 2 3 4 5 6 7 8 9 10 11

Cone length Cone diameter Seed number per cone Seed length Seed width Seed thickness Needle length Needle width Ratio of cone length/width (1/2) Ratio of cone diameter/seed number (2/3) Ratio of seed length/width (4/5)

0.1 0.1 0.1 0.1 0.1 0.1 0.1

Location of studied populations of Juniperus oxycedrus subsp. oxycedrus

Number of sample 1

135

Localization

Acronym

Longitude

Latitude

Altitude (m)

Number of individuals

Ukraine, Crimea, Chekan Kaya near Novyy Svet Turkey, near Konya Greece, Falakron Mts, near Pirgi Greece, Olymp slope above Litokhoron Bosnia, between Mostar and Medjugorie Croatia, near Zaostrog Spain, Mallorca island Spain, Huesca, Benabarre Spain, Sierra de Valdancha, SW of Morella Spain, Puerto de la Selva Spain, Costa del Azahar, near Torreblanca France, W of St. Pierre sure Mere near Narbonne Morocco, High Atlas, Tizi-n-Test Pass SW of Asni

CRIMEA

E351020

N441580

80

22

TURKEY GREECE_1 GREECE_2

E321200 E241040 E221280

N371500 N411150 N401010

900 1400 1000

4 20 3

BOSNIA

E171450

N431150

600

12

CROATIA MAJORCA SPAIN_1 SPAIN_2

E171160 E031000 E001280 W001140

N431120 N391300 N421070 N401300

30 200 1400 1100

15 39 17 35

SPAIN_3 SPAIN_4

E031160 E001120

N421200 N401110

20 100

46 6

FRANCE

E031050

N431090

170

56

MOROCCO

W071000

N311040

2100

30

ARTICLE IN PRESS 136

M. Klimko et al. / Flora 202 (2007) 133–147

by smaller numbers of individuals (Table 1, in italics). Altogether, 305 individuals were sampled. Eight morphological characters and three ratios were examined to determine the variation of every individual (Table 2). Cones, seeds and needles were measured and statistically analysed as in the case of J. oxycedrus subsp. macrocarpa (Klimko et al., 2004). A total of 9150 cones and 9150 needles were measured and characterized.

Statistical analysis Each individual was analysed separately, and later the local populations were compared using multivariate statistical methods (Łomnicki, 2000; Zar, 1999). The normality of frequency distribution of character values was examined to state the possibility of using statistic methods (Zar, 1999). The correlations among characters were examined to eliminate possible high redundant ones (Zar, 1999) and to check the possibility of using the analysis of a discriminant function (Sokal and Rohlf, 2003; Tabachnik and Fidell, 1996). The discriminating value of characters was verified with Tukey’s post hoc test and with the analysis of the discriminant function of characters (Łomnicki, 2000; Zar, 1999). The shortest Euclidean distances were calculated in an agglomerative grouping by the method of the closest neighbourhood according to Ward, and dendrograms were constructed to examine the relationships within and between populations (Karon´ski and Calin´ski, 1973; Sokal and Rohlf, 2003). A discriminant analysis was performed and the position of the specimens was tested along the first main discriminant variables to find differences among populations and individuals (Morrison, 1990; Sokal and Rohlf, 2003; Underwood, 1997).

Results Evaluation of characters The frequency distribution of the examined characters was normal or only slightly left- or right-biased. This enables statistical analyses. The average values of cone length and diameter (characters 1 and 2) are about 9 mm and these varied significantly among the western and eastern populations. The largest cones were found in the Spanish populations, and especially in the Puerto de la Selva (SPAIN_3) population. The number of seeds in a cone and seed dimensions (characters 3–6) are less different, but present the same tendency as cones (Table 3). The needles of the eastern populations are somewhat longer and wider (characters 7 and 8) than those of the western ones (Table 3), but the shape of needles (ratio of needle

length/width) is more similar within all the populations compared. The shape of cones (cone length/width, character 9) is rather similar among the populations, but that of seeds (length/width ratio, character 11) is slightly different in the East and Central populations, when compared to the West-Mediterranean ones (Fig. 2, Table 3). The variation coefficients for particular characters vary between 3.7% and 38.5% (Table 3). Generally, needle length and the ratio of cone diameter/seed number (characters 7 and 10) are the most variable, while seed length, the shape of the cone, the length of the cone and the shape of needle (characters 4, 9, 1 and 11, respectively) are the most stable characters. Populations from CRIMEA and MOROCCO are the most stable ones, while those from BOSNIA, CROATIA and FRANCE are the most variable (Table 3). Length and diameter of cones (characters 1 and 2) are highly significantly correlated (Table 4). These features of cones affect only seed width (character 5). The needle length and width (characters 7 and 8) are also correlated on a statistically significant basis. Likewise, the width of needles shows a positive significant correlation with the cone diameter (characters 8 and 2, respectively). The fact that there are rather low, if any, correlations among the dimensional characters of seed is surprising (compare Table 4). The correlations are statistically significant; however, not very highly. For this reason all the characters were utilized in statistical comparisons of individuals and populations. All the characters differentiated statistically significantly at the level of po0.01 at least among a few sampled populations in Tukey’s post hoc test (Fig. 3). For that reason all the characters were used in further analyses. The highest numbers of statistically significant differences were found for the cone length, seed width and needle width (characters 1, 5 and 8, respectively). Discriminant analysis indicated that the length of cones and the ratio of cone length/diameter (characters 1 and 9) did not vary between the samples in a statistically significant way. All the other traits discriminated between the samples at the level of p ¼ 0.01 or very close to it (Table 5). The width and length of leaves (characters 8 and 7, respectively) and the ratio of seed length/width (character 11) had the greatest discriminant power demonstrated with the lowest values of partial Wilk’s l (Table 5).

Intra-populational variation The individuals within the populations differed at various levels. Generally, the western populations included individuals, which differed more significantly than those from the central and eastern part of the region (Fig. 4). This suggests non-uniformity of samples

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

137

Table 3. Statistic description of analysed 11 characters of Juniperus oxycedrus subsp. oxycedrus (numbers 1–11 as in Table 2); populations represented by less than 10 individuals omitted Statistics

Sample

Character 1

2

7.52 8.72 8.11 8.59 9.91 10.38 9.66 11.88 9.82 9.59

8.03 8.98 8.66 8.91 9.42 10.22 8.95 12.56 9.44 9.78

2.97 2.41 3.07 2.89 3.06 3.04 2.31 2.77 3.31 2.77

5.73 6.08 6.10 6.02 6.74 6.59 6.21 7.28 6.43 6.79

2.83 3.61 3.25 3.14 3.98 3.98 4.25 4.64 4.01 4.12

2.50 3.11 2.81 2.57 2.87 3.08 3.27 3.54 3.09 3.20

12.19 13.71 14.72 15.19 13.94 15.90 10.72 13.15 9.97 17.70

1.66 1.48 1.71 1.57 1.62 1.54 1.05 1.79 1.21 1.87

0.94 0.98 0.94 1.00 1.06 1.05 1.08 0.95 1.04 0.98

2.70 4.10 2.83 3.12 3.10 3.55 4.50 4.67 2.93 3.60

2.04 1.74 2.01 1.94 1.72 1.69 1.50 1.58 1.62 1.66

All samples

9.05

9.01

2.79

6.29

3.70

2.91

13.84

1.55

1.02

3.39

1.76

Minimum

CRIMEA GREECE_1 BOSNIA CROATIA MALLORCA SPAIN_1 SPAIN_2 SPAIN_3 FRANCE MORROCO

6.00 6.00 5.20 6.80 7.70 8.50 7.50 8.60 7.80 6.60

5.50 7.00 5.00 5.00 7.80 7.90 6.40 8.80 6.00 7.90

2.00 1.00 2.00 1.00 2.00 2.00 1.00 1.00 2.00 1.00

3.00 5.40 5.20 5.00 5.40 5.60 4.80 6.00 4.50 5.00

2.20 2.60 2.20 2.00 2.90 3.00 3.40 4.00 2.50 2.60

1.60 2.20 1.80 1.90 2.00 2.20 2.40 3.20 1.60 1.50

7.50 8.00 9.00 8.50 9.00 11.00 8.00 8.00 6.50 7.00

1.00 1.00 1.00 1.00 1.00 1.00 0.90 1.00 1.00 1.00

0.86 0.78 0.85 0.89 0.94 0.88 0.95 0.72 0.88 0.80

2.10 2.40 2.50 1.00 2.50 1.43 1.64 2.10 1.60 2.80

1.56 1.45 1.72 1.46 1.32 1.31 0.95 1.20 1.08 1.14

Maximum

CRIMEA GREECE_1 BOSNIA CROATIA MALLORCA SPAIN_1 SPAIN_2 SPAIN_3 FRANCE MORROCO

10.20 10.00 12.00 13.00 13.70 12.70 12.20 18.50 10.80 12.50

10.10 11.10 10.80 14.00 13.80 12.30 11.80 17.60 12.40 12.10

4.00 3.00 4.00 5.00 6.00 6.00 5.00 6.00 6.00 4.00

7.00 7.30 6.50 7.40 9.20 7.70 7.50 9.00 7.30 8.00

3.60 4.00 3.40 5.70 5.60 5.40 5.80 6.00 5.20 6.60

3.50 4.00 4.00 3.90 5.40 3.70 4.30 5.40 4.00 4.80

18.50 23.00 21.00 23.00 23.00 23.00 18.00 21.00 14.50 22.00

2.00 2.10 2.00 2.00 2.50 2.00 1.50 2.00 2.00 2.00

1.07 1.11 1.10 1.13 1.33 1.22 1.37 1.15 1.21 1.16

3.40 4.55 3.60 4.00 5.00 5.80 8.80 4.20 5.50 5.50

3.00 2.34 2.73 2.75 2.55 2.24 2.06 2.25 2.88 2.57

Variation coefficient

CRIMEA GREECE_1 BOSNIA CROATIA MALLORCA SPAIN_1 SPAIN_2 SPAIN_3 FRANCE MOROCCO

6.19 3.72 9.59 19.54 8.46 6.50 8.74 9.23 8.49 6.48

7.22 6.34 11.32 23.73 10.57 9.38 8.28 10.48 11.15 6.70

4.31 17.56 9.76 15.00 16.05 15.29 18.33 18.56 16.04 12.04

6.78 4.76 11.67 10.82 9.41 7.05 8.68 8.02 9.01 6.69

9.01 12.81 29.03 16.32 10.33 11.22 11.42 13.05 12.98 9.27

8.02 10.55 38.46 17.47 11.07 9.30 9.70 11.44 18.17 7.64

12.94 12.30 13.74 30.46 9.21 12.98 13.39 17.72 18.52 19.83

13.31 11.55 15.35 10.83 11.43 8.71 12.97 14.38 11.29 9.83

6.50 6.40 6.59 14.46 7.50 15.87 5.56 8.38 7.56 5.77

7.74 25.57 13.26 23.62 17.56 15.29 23.38 18.69 20.47 16.42

7.73 11.72 10.52 7.48 13.70 8.92 9.16 9.30 11.17 9.67

8.19

9.02

12.51

7.78

12.09

12.76

15.73

11.61

7.84

15.71

8.57

Mean

CRIMEA GREECE_1 BOSNIA CROATIA MALLORCA SPAIN_1 SPAIN_2 SPAIN_3 FRANCE MORROCO

All samples

3

SPAIN_2, SPAIN_3 and FRANCE, which differentiated into two groups of individuals at a high level (25–30) when they were conglomerated using the shortest Euclidean distances according to the Ward method. The individuals within all the other samples differentiated at the level of about 15, which is the second level of differentiation within samples SPAIN_2, SPAIN-3

4

5

6

7

8

9

10

11

and FRANCE (Fig. 4). This suggests the composition of the latter three samples with two morphologically different types of individuals. The differences among them are determined mostly by the length and diameter of cones (characters 1 and 2), the length and width of needles (characters 7 and 8), the number of seeds per cone (character 3) and dimensional features of seeds

ARTICLE IN PRESS 138

M. Klimko et al. / Flora 202 (2007) 133–147

Table 4. Correlation coefficients between 8 measured characters of Juniperus oxycedrus subsp. oxycedrus from all populations sampled Characters 1

2

2 3 4 5 6 7 8

0.04 0.18** 0.01 0.61** 0.12* 0.15** 0.29** 0.10 0.06 0.39** 0.07 0.05 0.07 0.07 0.00 0.30** 0.06 0.04 0.02 0.01 0.51**

0.87** 0.04 0.20** 0.71** 0.27** 0.02 0.11

3

4

5

6

7

*Significance at level p ¼ 0.05. **Significance at level p ¼ 0.01 (character numbers follows Table 2).

Inter-populational differences

Fig. 2. Morphology of cones (1) and seeds (2) of Juniperus oxycedrus subsp. oxycedrus; bar means 1 cm (photo M. Klimko).

(characters 4 and 5). The frequency distribution of the values of these traits for all individuals analysed together is not exactly unimodal; however, it does not show bimodality, as could be expected. Also the intrapopulational analysis of discrimination of samples SPAIN_2, SPAIN_3 and FRANCE shows rather their multivariate unimodality with outstanding only of single individuals.

The only three pairs of populations without statistically significant differences in the Tukey’s T-test were found among 10 samples represented by more than 12 individuals (Table 6). More closely related to each other are Crimea to Bosnia in comparison with Bosnia to CROATIA, and MALLORCA to SPAIN_1. The other compared samples differed from each other in a statistically significant way with respect to at least one, but more frequently occuring, of several characters. The largest value of statistically significant differences to the others was demonstrated by CRIMEA from the eastern and SPAIN_2 and SPAIN_3 from the western parts of the species range (Table 6). An analysis of discriminant variables showed the differentiation of the studied populations more precisely. The first discriminant variable U1, responsible for 41% of variation, describes the largest differences between samples SPAIN_2 and SPAIN_3, surprisingly, but it does not differentiate the eastern populations from the western ones. Such differences, however, are clearly visible in the space determined with variable U2, which accounts for about 34% of the total variation. All the compared samples from the Crimea, the Balkan Peninsula and Turkey form a separate conglomeration, rather slightly differentiated by variable U1. Inversely, the group of the western populations, close in the space determined by variable U2, is strongly differentiated in the space of U1 (Fig. 5). The latter result can be interpreted as a significantly higher level of variation of the western populations. Cluster analysis using Euclidean distances segregated all the samples compared within the two groups (Fig. 6). The first of them was formed by the populations from the Crimea, Turkey and the Balkan Peninsula, while the second one contained all the others. The differences among the western populations are greater than among the eastern ones. In the eastern populations BOSNIA, CROATIA and CRIMEA were closely connected

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

139

Fig. 3. Result of Tukey T-test for 11 characters of 10 samples of Juniperus oxycedrus subsp. oxycedrus represented by more than 12 individuals; *significance at level p ¼ 0.05; **significance at level p ¼ 0.01 (sample numbers as in Table 1; character numbers as in Table 2).

ARTICLE IN PRESS 140

M. Klimko et al. / Flora 202 (2007) 133–147

Table 5. Discriminant power testing for the characters of Juniperus oxycedrus subsp. oxycedrus No.

Character

Partial Wilk’s l

p

1 2 3 4 5 6 7 8 9 10

Cone length Cone diameter Seed number per cone Seed length Seed width Seed thickness Needle length Needle width Ratio of cone length/width (1/2) Ratio of cone diameter/seed number (2/3) Ratio of seed length/width (4/5)

0.936 0.860 0.917 0.836 0.881 0.841 0.740 0.530 0.943 0.855

0.089 0.000 0.017 0.000 0.000 0.000 0.000 0.000 0.157 0.000

0.805

0.000

11

among themselves, more than what could be observed among the others. Among the western populations the most closely related were MALLORCA, SPAIN_1 and, surprisingly, MOROCCO – geographically the most distant one.

Discussion Variation of characters The values of particular characters received in our studies (Table 3) did not differ drastically from data reported in the species descriptions in basic floras and taxonomical works. The average values of length and diameter of the cone (characters 1 and 2, respectively) found in our study as predominantly being between 8 and 12 mm (Table 3) are similar to values reported by Amaral Franco (1964, 1986), Christensen (1997), Lebreton et al. (1998), Arista et al. (2001), Charco (2001) and Farjon (2005) (Table 7). Also the length and width of seed (characters 4 and 5) found as 6–7.3 mm and 2.8–4.6 mm, respectively, were similar to those reported earlier by Christensen (1997) and Arista et al. (2001) (Table 7). The average number of seeds per cone (character 3) was found in several samples higher than expected. The number of ovules in the single female cone of junipers of section Juniperus, to which J. oxycedrus belongs, is generally reported as three. Some atypical cones containing less or more than three ovules has been also found (Schulz et al., 2003). Less than three seeds in one particular cone can result from (1) the abnormal type of female cone which has developed a restricted number of ovules, and (2) lack of success of pollination and fertilization of a normal, 3-ovule cone (Juan et al., 2003). The latter is quite a frequent situation, described

on J. oxycedrus subsp. oxycedrus and J. oxycedrus subsp. macrocarpa (Cantos et al., 1998; Ortiz et al., 1998; Juan et al., 2003). More than three seeds in a particular cone result from abnormal number of ovules. Female cones with four and six ovules have been found in J. oxycedrus subsp. oxycedrus and in Juniperus communis (Arista et al., 2001; Schulz et al., 2003, Fig. 2D and E), which allow development of up to six seeds in one cone. The frequency of abnormal female cones was estimated as 25 per 128 analysed in total in the study by Schulz et al. (2003), but cones with more than three ovules was only a part of them. We have found cones with more than three seeds in all analysed samples except of GREECE_1 (Table 4). The maximal number of seeds in particular populations was 4 in three samples, 5 in two, and 6 in four samples (Table 3). This indicates the quite frequent occurrence in J. oxycedrus subsp. oxycedrus of female cones with two whorls of ovules, as described for J. communis by Schulz et al. (2003, Fig. 16). The cones containing six seeds were found only in the populations from West-Mediterranean region, namely in samples SPAIN_1, SPAIN_3, MALLORCA and FRANCE (Table 3). Cones containing more than three seeds had generally similar length and diameter (characters 1 and 2, respectively), as those normally developed, with three seeds inside. The number of seeds in one cone did not correlate with dimensional characters of cones, but it was inversely correlated at a statistically significant level with seed width (character 5) only (see Table 4). The average number of seeds per cone in four of the analysed samples was higher than three, what indicated (1) the high frequency of individuals with abnormal female cones containg four and more ovules, and (2) the sufficiently high level of success in pollination and fertilization. The latter has been described earlier as rather low (Arista et al., 2001; Ortiz et al., 1998). The leaves of J. oxycedrus subsp. oxycedrus are linearacicular needles, sometimes ‘‘boat-shaped’’ (Farjon, 2005). Needles at the central and apical part of the shoots are always the longest ones. Needles at the basis of the yearly increment of the shoot are significantly shorter, but have the same width as those from the central and apical parts. The widest part of the needle on individuals of J. oxycedrus subsp. oxycedrus in Greece was described as situated central or somewhat below the mid-length of the needle (Christensen, 1997). Only the shortest needles at the basal part of long shoots are widest at base (Farjon, 2005), but these have not been tested in the present work, according to methods by Klimko et al. (2004). The average length and width of needles (characters 7 and 8, respectively) found in our examinations are similar as reported earlier, but have a slightly narrower range (Table 7). This probably

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

30

30

CRIMEA

25

25

20

20

15

15

10

10

5

5

0

. . . . . . . . . . .. . . . . . . . . . .

0

BOSNIA

25

20

15

15

10

10

5

5 .

.

. .

.

. .

.

.

..

.

0

.

. .

. .

.

. .

. .

MALLORCA

.

. .

. .

SPAIN_1

25

25

20

20

15

15

10

10

5

5 ...... ...... ..... ..... ...... ..... ......

30

0

. .

.

.

.. .

.

.

.

.

.

.

. . . .

30 SPAIN_2

25

SPAIN_3

25

20

20

15

15

10

10

5

5 .... ... ... ... ... ... .. ... ... ... ... ..

0

..........

..........................

..........

30

30 FRANCE

MOROCCO

25

25

20

20

15

15

10

10

5

5

0

CROATIA

30

30

0

. . . . . . . . . . . . . . . . . . .

25

20

0

GREECE_1

30

30

0

141

.. ...... ...... .......

...... .......

...... ...... .......

...

0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fig. 4. Intra-populational variability of 10 most numerous samples of Juniperus oxycedrus subsp. oxycedrus analysed using the Ward agglomeration on the shortest Euclidean distances among individuals.

resulted from using the needles collected on central parts of shoots, without the longest, apical and shortest, basal ones. We also found many individuals with boat-

shaped needles within populations from the eastern part of the J. oxycedrus subsp. oxycedrus range and individuals with delta-shaped needles in the western

ARTICLE IN PRESS 142

M. Klimko et al. / Flora 202 (2007) 133–147

Table 6. Numbers of statistically significant differences (Tukey T-test for 11 characters) at level p ¼ 0.01 (straight number) and p ¼ 0.05 (cursive number) among 10 samples of Juniperus oxycedrus subsp. oxycedrus represented by more than 12 individuals each CRIMEA GREECE_1 BOSNIA CROATIA MALLORCA SPAIN_1 SPAIN_2 SPAIN_3 FRANCE GREECE_1

6 6 BOSNIA 0 0 CROATIA 1 2 MALLORCA 6 6 SPAIN_1 8 9 SPAIN_2 7 7 SPAIN_3 7 7 FRANCE 9 9 MOROCCO 8 8

3 4 1 2 4 4 2 3 6 6 5 7 5 5 2 3

0 0 4 5 4 5 8 8 7 7 6 6 3 4

4 4 4 4 8 8 8 8 6 6 5 6

0 0 7 7 8 8 3 3 1 2

6 6 7 8 2 2 1 1

9 9 3 3 6 7

10 10 5 5

4 4

3 SPAIN _ 3

2 1

SPAIN _ 2 FRANCE

MALLORCA

SPAIN _ 4

0 U2 (34%)

SPAIN 1 _ MARROCO

GREECE _ 1

-1

CROATIA TURKEY

-2 BOSNIA CRIMEA

-3 GREECE _ 2

-4 -5 -3.5

-2.5

-1.5

-0.5

0.5

1.5

2.5

3.5

U1 (41%)

Fig. 5. Result of discriminant analysis based on 11 characters for Juniperus oxycedrus subsp. oxycedrus populations.

populations. The deltoid needles were described by Adams (2004) as typical for J. deltoides, a new cryptospecies distinguished by him from J. oxycedrus subsp. oxycedrus mostly on the basis of DNA sequencing, RAPDs and leaf terpenoids, from 27 individuals dispersed in the Mediterranean region (Adams, 2004; Adams et al., 2005). We found that between these two types are many intermediates, which prevail among populations.

Intra-populational variation The level of intra-populational variation of J. oxycedrus subsp. oxycedrus was found as very different in various samples. Most variable were the samples SPAIN_2, SPAIN_3 and FRANCE from the western part of the species range, with a high variation coefficient (Table 3). The intra-populational differentiation of individuals among these three samples had

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

143

MORROCO MALLORCA SPAIN_1 FRANCE SPAIN_3 SPAIN_4 SPAIN_2 CROATIA BOSNIA CRIMEA TURKEY GREECE_1 GREECE_2 0

2

4

6

8

10

12

14

Fig. 6. Dendrogram constructed by Ward method of cluster analysis on the Euclidean distances between samples of Juniperus oxycedrus subsp. oxycedrus.

significantly higher level, than inside of any other sample (Fig. 4). The high morphological polymorphism of the samples SPAIN_2, SPAIN_3 and FRANCE can be explained as: (1) developed during the long period of their isolation, (2) a trace of ancient hybridization between J. oxycedrus subsp. oxycedrus and J. oxycedrus subsp. macrocarpa and (3) the present-day hybridization via long distance transportation of pollen of J. oxycedrus subsp. macrocarpa, as it was described within other wind-pollinated species (Potts and Reid, 1990; Petit et al., 2004).

Inter-populational variation The differentiation of the compared populations has a multivariate character and is determined mostly by 9 of the 11 characters employed (Fig. 3). The other two characters differentiate only between single samples. The statistical significance of differences between populations in such a number of characters indicates a large scale of variation of J. oxycedrus subsp. oxycedrus. It also elucidates the occurrence of the great number of taxa closely related to the typical red juniper (compare Farjon, 2001, 2005). The other juniper species compared biometrically demonstrated statistically significant differences in various numbers of similar characters. Three populations of Juniperus phoenicea were differentiated at the statistically significant level by 12 of the 14 compared characters (based on the data of Mazur et al., 2003), three populations of Juniperus excelsa by 9 of the 11 characters (Mazur et al., 2004), while also three samples of J. oxycedrus subsp. macrocarpa were differentiated by 4 of the 11 characters (Klimko et al.,

2004). The latter finding was explained as typical for J. oxycedrus subsp. macrocarpa (Klimko et al., 2004); however the effect of colonization cannot be excluded (Lewandowski et al., 1996). The great number of characters which differentiate significantly between the studied populations of J. oxycedrus subsp. oxycedrus in our study may also owe their origin to hybridization and introgression between them and other subspecies of J. oxycedrus, mainly J. oxycedrus subsp. macrocarpa. The occurrence of junipers related to J. oxycedrus sensu lato was described from Central-European Tertiary sediments (Kvacˇek, 2002; Łan´cucka-S´rodoniowa in: Kornas´ and Medwecka-Kornas´ , 2002, p. 424). The distribution of the tertiary Juniperus species in Europe was restricted in the Quaternary to the Pleistocene refugia of tertiary floras in southern Europe, SouthWest Asia, North-West Africa, Madeira and the Canary Islands (Reinig in: Kornas´ and Medwecka-Kornas´ , 2002, p. 433), together with many other plant and animal taxa (Bennet et al., 1991; Bilton et al., 1998; Dumolin-Lape`gue et al., 1997; Ferris et al., 1999; Hewitt, 1996, 1999, 2004; Willis et al., 2000). However, junipers are drought resistant and light demanding trees (Auclair, 1996; Quezel and Barbero, 1981; Quezel and Pesson, 1980; Zohary, 1973), and for this reason probably did not occur inside the refugia, but rather on peripheric sites around them. A positive reaction of Juniperus (unfortunately, the pollen has not been distinguished at species level in the paleobotanic studies) to the arid periods during the late Pleistocene/Holocene and its suppression by more humid-demanding, predominantly broad-leaved trees were reported from the Iberian Penisula (Carrio´n et al., 2001a, b, 2003, 2004).

ARTICLE IN PRESS

(5–)6–7.3(–9.0) (2.2–)2.8–4.6(–6.6) (6.5–)10–20(–23) (0.9–)1–1.9(–2.0) — — 6–25 1.1–2.0 7.7 4.3 10–20 (1.2–)1.4–2.0(–2.5) — — — — — — — — 5.3–9 2.5–8 — — — — 8–15 (25) 1.0–1.5

— — 8–20 1–1.5

(6–)8–12(–18) (1–)3(–6) 6–13 — 6–11(12) (1–)3 7.9 2.1 8.8 2.6 8–10 —

2. Diameter of cone 3. Seed number per cone 4. Seed length 5. Seed width 7. Needle length 8. Needle width

8–10 — 8–11 1.9 (1–4)

Spain (Amaral Franco, 1986) Character

Spain Morocco (Arista et al., (Charco, 2001) 2001)

France (Lebreton et al., 1998)

Turkey (Lebreton et al., 1998)

Greece (Christensen, 1997)

In general (Farjon, 2005)

Our finding

M. Klimko et al. / Flora 202 (2007) 133–147

Table 7. Comparison of separate character values of Juniperus oxycedrus subsp. oxycedrus reported (Amaral Franco, 1986; Arista et al., 2001; Christensen, 1997; Farjon, 2005; Lebreton et al., 1998) and found in our study; in bracts extreme values

144

Nevertheless, the thermal demands of J. oxycedrus were probably a reason for the isolation of its populations during the Pleistocene. This long-term spatial isolation probably resulted in the beginning of processes of taxonomic differentiation (Comes, 2004; Petit et al., 2003). The late Tertiary–early Quaternary isolation of refugial areas results, among others, in the present-day recognition of several taxa, including typical J. oxycedrus subsp. oxycedrus and/or closely related taxa. These are, in the main, Juniperus cedrus Webb et Berthel. in Madeira and Canary Islands, Juniperus brevifolia (Seub.) Ant. in the Ac`ores, J. oxycedrus subsp. transtagana in the south-west part of the Iberian Peninsula and J. oxycedrus subsp. macrocarpa in the coastal regions of the Mediterranean Sea (compare: Amaral Franco, 1963, 1964, 1986; Farjon, 2001, 2005; Greuter et al., 1984; Lebreton et al., 1991). The areas of distribution of Juniperus cedrus, J. brevifolia and J. oxycedrus subsp. transtagana are restricted to the refugial regions only, while inversely, J. oxycedrus subsp. oxycedrus belongs to the common taxa, broadly distributed both in the West- and EastMediterranean regions (Boratyn´ski et al., 1992; Browicz, 1982; Charco, 2001; Jalas and Suominen, 1973). Its area of distribution (Fig. 1) covers several regions recognized as areas with Pleistocene refugia of Tertiary floras. Material in our study comes from three such main regions: (1) the North-West African, (2) the Iberian and (3) the Balkan. Geographical isolation of those areas surely influenced the geographic variation of J. oxycedrus subsp. oxycedrus. In our study the most clearly visible is the differentiation of the sampled populations on dendrograms constructed on the shortest Euclidean distances (Fig. 6), but it is also recognizable in the distribution of the analysed samples between the two first discriminant variables (Fig. 5). It is noteworthy that samples including only a few individuals (TURKEY, GREECE_2 and SPAIN_4) are also characteristic of the region where they occur. The result stresses the differences between the West- and East-Mediterranean populations, confirming thus their origin from at least two different refugial areas. The great differences between West- and East-Mediterranean individuals of J. oxycedrus subsp. oxycedrus were lately found on nrDNA sequencing (ITS1, 5.8S, ITS 2), RAPDs and leaf terpenoids (Adams, 2004; Adams et al., 2005). These differences were even the reason for distinguishing of two separate species within J. oxycedrus subsp. oxycedrus – J. oxycedrus var. oxycedrus in Adams’ work (2004) – the typical J. oxycedrus in the West and new species J. deltoides in the East-Mediterranean region. The lack of morphological differences between those two taxa suggested to treat J. deltoides as cryptospecies (Adams et al., 2005). In our opinion it is rather an intra-subspecific quantitative variation in biochemical characters, which confirms the geographic isolation and

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

restricted gene flow between West- and East-Mediterranean populations of the J. oxycedrus subsp. oxycedrus. The differences between populations inside the WestMediterranean region are generally greater than in the East (Fig. 6), and connections of particular of them are sometimes surprising. The close relation between the population of J. oxycedrus subsp. oxycedrus from the Atlas Mountains and the other West-Mediterranean samples is an example of such a similarity. It could indicate a rather short-time isolation of the compared population and/or permanent gene flow. It is also of interest that the closest affinity of the African sample was found to be to the populations from MALLORCA and SPAIN_1, and not to the southernmost SPAIN_2. Such a similarity among the Iberian and African samples was also found in Juniperus thurifera (Jime´nez et al., 2003). The genetic analysis (RAPD of the length of the intergenic spacer in the chloroplast) related to the geographical structure of J. thurifera on the Iberian Peninsula and in Africa showed also a surprising isolation of the populations from the Spanish Central Cordilleras, but a closer relation to the South-Iberian and African samples (Jime´nez et al., 2003). This was explained by another way of migration during the late Tertiary and Quaternary (Jime´nez et al., 2003, Fig. 6). The present-day range of J. oxycedrus subsp. oxycedrus (Fig. 1) suggests also other possible paths of their migration. We can conclude that the geographical nature of the polymorphism of J. oxycedrus subsp. oxycedrus confirms the theory of its long-term isolation during the Pleistocene in separate centres in the West- and EastMediterranean. The taxon colonized subsequently the Mediterranean basin from at least two different centres.

Acknowledgements We would like to express our gratitude to Ilona Wysakowska for her help in the measurement procedures and to Samuel Pyke for editing the English version of the manuscript. The study was partly supported by the Department of Botany, the August Cieszkowski Agricultural University of Poznan´ and the Institute of Dendrology of the Polish Academy of Sciences at Ko´rnik. The collection of material was possible to a large extent, thanks to the official cooperation of the latter institution with the Institute of Botany in Barcelona (CSIC) and the M. G. Kholodny Institute of Botany in Kyiv (NANU).

References Adams, R.P., 2000. Systematics of Juniperus section Juniperus based on leaf essential oils and random amplified poly-

145

morphic DNAs (RAPDs). Biochem. Syst. Ecol. 28, 515–528. Adams, R.P., 2004. Juniperus deltoides, a new species, and nomenclatural notes on Juniperus polycarpos and J. turcomanica (Cupressaceae). Phytologia 86, 49–53. Adams, R.P., Morris, J.A., Padney, R.N., Schwarzbach, A.E., 2005. Cryptic speciation between Juniperus deltoides and Juniperus oxycedrus (Cupressaceae) in the Mediterranean. Biochem. Syst. Ecol. 33, 771–787. Amaral Franco, J.do, 1963. Taxonomic notes on Juniperus oxycedrus L. and J. macrocarpa Sm. Feddes Repert. 68, 163–167. Amaral Franco, J.do, 1964. Juniperus L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea, vol. 1. Cambridge University Press, Cambridge, pp. 38–39. Amaral Franco, J.do, 1968. Distribuic¸a˜o de zimbros e pomo´ideas na Penı´ nsula Ibe´rica. Collect. Bot. 7, 449–481. Amaral Franco, J.do, 1986. Juniperus L. In: Castroviejo, S., Laı´ nz, M., Lo´pez Gonza´lez, G., Montserrat, P., Mun˜oz Garmendia, F., Paiva, J., Villar, L. (Eds.), Flora Iberica, vol. 1. Real Jardı´ n Bota´nico, CSIC, Madrid, pp. 181–188. Arista, M., Ortiz, P.L., Talavera, S., 2001. Reproductive cycles of two allopatric subspecies of Juniperus oxycedrus (Cupressaceae). Flora 196, 114–120. Auclair, L., 1996. L’appropriation communautaire des foreˆts dans le Haut Atlas marocain. Cah. Sci. Human. 32, 177–194. Bennet, K.D., Tzedakis, P.C., Willis, K.J., 1991. Quaternary refugia of north European trees. J. Biogeogr. 18, 103–115. Bilton, D.T., Mirol, P.M., Mascheretti, S., Fredga, K., Zima, J., Searle, J.B., 1998. Mediterranean Europe as an area of endemism for small mammals rather than source for northwards postglacial colonization. Proc. R. Soc. London Ser. B 265, 1219–1226. Bondi, E., 1990. Population characteristics of Juniperus oxycedrus L. and their importance to vegetation dynamics. Giorn. Bot. Ital. 124, 330–337. Boratyn´ski, A., Browicz, K., Zielin´ski, J., 1992. Chorology of Trees and Shrubs in Greece. Polish Academy of Sciences, Institute of Dendrology, Ko´rnik. Browicz, K., 1982. Chorology of Trees and Shrubs in SouthWest Asia and Adjacent Regions, vol. 1. Polish Science Publishers, Warszawa, Poznan´. Cantos, M., Cuerva, J., Za´rate, R., Troncoso, A., 1998. Embryo rescue and development of Juniperus oxycedrus subsp. oxycedrus and macrocarpa. Seed Sci. Technol. 26, 193–198. Carrio´n, J.S., Andrade, A., Bennett, K.D., Munuera, M., 2001a. Crossing forests thresholds. Inertia and collapse in Holocene sequence from south-central Spain. Holocene 11, 635–653. Carrio´n, J.S., Munuera, M., Dupre´, M., Andrade, A., 2001b. Abrupt vegetation changes in the Segura mountains of southern Spain. J. Ecol. 89, 783–797. Carrio´n, J.S., Yll, E.I., Walker, M.J., Legaz, A.J., Chain, C., Lo´pez, A., 2003. Glacial refugia of temperate, Mediterranean and Ibero-North African flora in south-eastern Spain: new evidence from cave pollen at two Neanderthal sites. Global Ecol. Biogeogr. 12, 119–129.

ARTICLE IN PRESS 146

M. Klimko et al. / Flora 202 (2007) 133–147

Carrio´n, J.S., Yll, E.I., Willis, K.J., Sanchez, P., 2004. Holocene forest history of the eastern plateaux in the Segura Mountains (Murcia, southeastern Spain). Rev. Paleobot. Palynol. 132, 219–236. Charco, J., 1999. El Bosque Mediterra´neo en el Norte de A`frica. Agencia Espan˜ola de Cooperacio´n International, Madrid. Charco, J., 2001. Guı´ a de los A`rboles y Arbustos del Norte de A`frica. Agencia Espan˜ola de Cooperacio´n International, Madrid. Christensen, K.I., 1997. Juniperus L. In: Strid, A., Tan, K. (Eds.), Flora Hellenica, vol. 1. Koeltz Scientific Books, Ko¨nigstein, pp. 10–14. Comes, H.P., 2004. The Mediterranean region – a hotspot for plant biogeographic research. New Phytol. 164, 11–14. Dumolin-Lape`gue, S., Demesure, B., Fineschi, S., Le Corre, V., Petit, R.J., 1997. Phylogeographic structure of white oaks troughout the European continent. Genetics 146, 1475–1487. Farjon, A., 2001. World Checklist and Bibliography of Conifers, second ed. The Royal Botanic Gardens, Kew. Farjon, A., 2005. A Monograph of Cupressaceae and Sciadopitys. Royal Botanic Gardens, Kew. Ferris, C., King, R.A., Hewitt, G.M., 1999. Isolation within species and the history of glacial refugia. In: Hollingsworth, P.M., Bateman, R.M., Gornall, R.J. (Eds.), Molecular Systematics and Plant Evolution. Taylor & Francis, London and New York, pp. 20–33. Greuter, W., Burdet, H.M., Long, G., 1984. Med-Checklist, vol. 1. Botanischer Garten and Botanisches Museum, Berlin-Dahlem. Hewitt, G.M., 1996. Some genetic consequences of ice ages, and their role in divergence and speciation. Biol. J. Linn. Soc. 58, 247–276. Hewitt, G.M., 1999. Post-glacial re-colonization of European biota. Biol. J. Linn. Soc. 68, 87–112. Hewitt, G.M., 2004. Genetic consequences of climatic oscillations in the quaternary. Philos. Trans. Biol. Sci. 359, 183–195. Huntley, B., Birks, H.J.B., 1983. An Atlas of Past and Present Pollen Maps for Europe: 0–13 000 Years Ago. Cambridge University Press, Cambridge. Jalas, J., Suominen, J., 1973. Atlas Florae Europaeae. The Committee for Mapping the Flora of Europe and Societatis Biologica Fennica Vanamo, Helsinki. Jime´nez, J.F., Werner, O., Sa´nchez-Go´mez, P., Ferna´ndez, S., Guerra, J., 2003. Genetic variation and migration pathway of Juniperus thurifera L. (Cupressaceae) in the western Mediterranean region. Isr. J. Plant Sci. 51, 11–22. Juan, R., Pastor, J., Ferna´ndez, I., Diosdado, J.C., 2003. Relationships between mature cone traits and seed viability in Juniperus oxycedrus L. subsp. macrocarpa (Sm.) Ball (Cupressaceae). Acta Biol. Cracov. 45 (2), 69–78. Karon´ski, M., Calin´ski, T., 1973. Grouping in multivariate populations on the basis of Euclidean distances. Algor. Biometr. Statyst. 17, 117–129. Klimko, M., Boratyn´ska, K., Boratyn´ski, A., Marcysiak, K., 2004. Morphological variation of Juniperus oxycedrus

subsp. macrocarpa (Cupressaceae) in three Italian localities. Acta Soc. Bot. Pol. 73, 113–119. Kornas´ , J., Medwecka-Kornas´ , A., 2002. Geografia Ros´ lin (Plant Geography). Wydawnictwo Naukowe PWN, Warszawa. Kvacˇek, Z., 2002. A new juniper from the Palaeogene of Central Europe. Feddes Repert. 113, 492–502. Lebreton, Ph., Bayet, C., Muracciole, M., 1991. Le statut syste´matique du Gene´vrier oxyce`dre Juniperus oxycedrus L. (Cupressace´es): une contribution d’ordre biochimique et biome´trique. Lazaroa 12, 21–42. Lebreton, Ph., Pe´rez de Pas, P.L., Barbero, M., 1998. Etude syste´matique du sous-genre Oxycedrus (section oxycedroides) du genre Juniperus (Cupressaceae). Ecol. Mediter. 24 (21), 53–61. Lewandowski, A., Boratyn´ski, A., Mejnartowicz, L., 1996. Low level of isozyme variation in an island population of Juniperus oxycedrus subsp. macrocarpa (Sm. ex Sibth.) Ball. Acta Soc. Bot. Pol. 65, 335–338. Łomnicki, A., 2000. Wprowadzenie do Statystyki dla Przyrodniko´w (Introduction to the Statistics for Biologists). Wydawnictwo Naukowe PWN, Warszawa. Mazur, M., Boratyn´ska, K., Marcysiak, K., Go´mez, D., Tomaszewski, D., Didukh, J., Boratyn´ski, A., 2003. Morphological variability of Juniperus phoenicea (Cupressaceae) from three distant localities on Iberian Peninsula. Acta Soc. Bot. Pol. 72, 71–78. Mazur, M., Boratyn´ska, K., Marcysiak, K., Didukh, Y., Romo, A., Kosin´ski, P., Boratyn´ski, A., 2004. Low level of inter-populational differentiation in Juniperus excelsa M. Bieb. (Cupressaceae). Dendrobiology 52, 39–46. Morrison, D.F., 1990. Wielowymiarowa Analiza Statystyczna (Multivariate Statistical Analyses). PWN, Warszawa. Ortiz, P.L., Arista, M., Talavera, S., 1998. Low reproductive success in two subspecies of Juniperus oxycedrus L. Int. J. Plant Sci. 159, 843–847. Petit, R.J., Aguinagalde, I., Beaulieu, J.-L., Bittkau, C., Brewer, S., Cheddadi, R., Ennos, R., Fineschi, S., Grivet, D., Lascoux, M., Mohanty, A., Mu¨ller-Starc, G., Demdesure-Musch, B., Palme´, A., Martı´ n, J.P., Rendell, S., Vendramin, G.G., 2003. Glacial refugia: hotspots but not melting pots of genetic diversity. Science 300, 1563–1565. Petit, R.J., Bode´ne`, C., Ducuoso, A., Roussel, G., Kremer, A., 2004. Hybridization as a mechanism of invasion in oaks. New Phytol. 161, 151–164. Potts, B.M., Reid, J.B., 1990. The evolutionary significance of hybridization in Eucalyptus. Evolution 44, 2151–2152. Que´zel, P., Barbero, M., 1981. Contribution a´ l’e´tude des formations pre´-steppiques a` Gene´vriers au Maroc. Bol. Soc. Broter. Ser. 2 (53), 1137–1160+6 tables. Que´zel, P., Pesson, P., 1980. Biogeographie et e´cologie des conifers sur le pourtour mediterrane´en. In: Actualite´s d’Ecologie Forestie`re. Gautier-Villars, Paris, pp. 205–255. Roloff, A., Ba¨rtels, A., 1996. Geho¨lze – Bestimmung, Herkunft und Lebensbereiche, Eigenschaften und Verwendung. Ulmer, Stuttgart. Schulz, Ch., Jagel, A., Stu¨tzel, T., 2003. Cone morphology in Juniperus in the light of cone evolution in Cupressaceae s.l. Flora 198, 161–177.

ARTICLE IN PRESS M. Klimko et al. / Flora 202 (2007) 133–147

Sokal, R.R., Rohlf, T.J., 2003. Biometry: the Principles and Practice of Statistics in Biological Research. 8th Printing. Freeman W.H. and Comp., San Francisco. Tabachnik, B.G., Fidell, L.S., 1996. Using Multivariate Statistics. Harper Collins College Publishers, New York. Underwood, A.J., 1997. Experiments in Ecology. Cambridge University Press, Cambridge.

147

Willis, K., Rudner, E., Su¨megi, P., 2000. The full-glacial forests of Central and Southeastern Europe. Quat. Res. 53, 203–213. Zar, J.H., 1999. Biostatistical Analysis, fourth ed. PreniceHall, New Jersey. Zohary, M., 1973. Geobotanical Foundations of the Middle East, vols. 1–2. Fischer, Stuttgart.