Botanical Journal of the Linnean Society, 2016, 180, 365–385. With 4 figures
Effect of the Aegean Sea barrier between Europe and
Asia on differentiation in Juniperus drupacea
(Cupressaceae)
1
1
KAROLINA SOBIERAJSKA1, KRYSTYNA BORATYNSKA
, ANNA JASINSKA
, MONIKA
1
2
3
DERING , TOLGA OK , BOUCHRA DOUAIHY , MAGDA BOU DAGHER-KHARRAT3,
1
*
ANGEL
ROMO4 and ADAM BORATYNSKI
1
Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 K
ornik, Poland
Department of Forest Botany, Faculty of Forestry, Kahramanmaras Sutcu Imam University, 46100
Kahramanmaras, Turkey
3
Laboratoire Caract
erisation G
enomique des Plantes, Facult
e des Sciences, Universit
e Saint-Joseph,
Campus Sciences et Technologies, Mar Roukos, Mkalles, BP: 1514 Riad el Solh, Beirut 1107 2050,
Lebanon
4
Institute of Botany, Consejo Superior de Investigaciones Cientıficas-Ajuntament de Barcelona,
IBB-CSIC-ICUB, Passeig del Migdia s/n, Parc de Montju€ıc., 08038 Barcelona, Spain
2
Received 26 June 2015; revised 2 December 2015; accepted for publication 18 December 2015
Juniperus drupacea is an eastern Mediterranean mountain tree with a disjunct geographical range. We
hypothesized that this disjunct occurrence (the Peloponnese in Europe and the Taurus and Lebanon Mountains
in Asia) should be reflected in the patterns of genetic and morphological diversity and differentiation. Nuclear
microsatellite markers (nSSR) and biometric variables of the cones and seeds were examined on material sampled
from four populations in Europe and eight in Asia. The Asian populations were characterized by a higher level of
genetic diversity than the European populations. The genetic differentiation among populations was moderate
but significant (FST = 0.101, P < 0.001). According to the clustering performed with BAPS, six genetically and
geographically groups of populations were found: I and II from the Peloponnese; III from the Taurus Mountains;
IV and V from the Anti-Taurus Mountains; and VI from the Lebanon Mountains. The level of genetic
differentiation among these six groups (4.30%, P = 0.012) probably reflects long-lasting genetic isolation during
the Pleistocene, as limited genetic admixture was found. In accordance with genetic analysis, the biometric
investigations indicated a high level of morphological divergence between the European and Asian
populations of the species, with further differentiation between the populations from the Taurus and Lebanon
Mountains. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385.
ADDITIONAL KEYWORDS: Bayesian clustering – biogeography – biometrics – East Mediterranean –
multivariate analyses – nSSR – plant diversity – plant variation – STRUCTURE clustering.
INTRODUCTION
The Mediterranean Region is one of the global biodiversity hotspots (Myers et al., 2000), one of the
important global centres of endemism and speciation
(Greuter, Burdet & Lang, 1984; Tan, Iatrou & Johnsen, 2001; Thompson, 2005) and the main Pleistocene refugial region for the European Tertiary
*Corresponding author. E-mail: [email protected]
floras (Hewitt, 1996; M
edail & Diadema, 2009). The
present geographical ranges of organisms in the eastern Mediterranean basin result from the geological
alternations and subtropical climate cooling, which
started with the regression of the Thetys (Thompson,
2005; Popov et al., 2006). The catastrophic effects of
the Messinian salinity crisis (Krijgsman et al., 1999)
and the Pliocene land-plate movements that formed
the Peloponnese and Aegean Islands (Popov et al.,
2006; Ivanov et al., 2011) also had a great influence
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
365
366
K. SOBIERAJSKA ET AL.
on contemporary geographical ranges of species
(Greuter, 1979; Thompson, 2005).
In the western Mediterranean basin, the Strait of
Gibraltar, which opened at the end of the Messinian
(c. 5.3 Mya; Krijgsman et al., 1999), is commonly recognized as a significant biogeographic barrier that
caused vicariant speciation processes in the European and African parts of the geographical ranges of
several taxa (e.g. Terrab et al., 2007, 2008;
Rodrıguez-S
anchez et al., 2008; Dzialuk et al., 2011;
Sez kiewicz et al., 2013; Boraty
nski et al., 2013; Dering et al., 2014; S
anchez-Robles et al., 2014). In the
eastern Mediterranean basin, there was no such
restricted isolation between Europe and Asia. The
Dardanelles and Bosphorus Straits, which divide
Europe from Asia, have been narrow since the late
Miocene (Ivanov et al., 2011) and are currently 6 km
and c. 3.4 km wide, respectively. Thus, during the
Pliocene and Pleistocene, neither strait formed such
an impermeable geographical barrier as the Strait of
Gibraltar. Additionally, the Bosphorus and Dardanelles dried out, at least during the coldest periods
of the Pleistocene (Hughes, Woodward & Gibbard,
2006), allowing land bridges to form between Europe
and Asia and increasing the possibility of migration
(Eastwood, 2004; Magyari et al., 2008). The Thracian
Plain in the Balkans, which is rather impenetrable
for high mountain plants (Ansell et al., 2011), was
not a barrier for temperate organisms (Bilgin, 2011;
Magyari et al., 2008). In contrast, the Aegean Sea
between the Balkan and Anatolian Peninsulas was a
considerable barrier between Europe and Asia. The
contemporary flora of the Aegean Islands contains
numerous endemic plant species (Georgiou & Delipetrou, 2010), but the flora is poorer than that of
islands located closer to the Peloponnese and
Anatolian shores (Rechinger, 1943; Strid, 1996). Consequently, the patterns of variation described for
several woody plant taxa based on various genetic
markers and morphological characteristics suggest
the possibility of migrations between Europe and
Asia via the Bosphorus and Dardanelles (e.g. Marcysiak et al., 2007; Fady & Conkle, 1993; Fady &
Conord, 2010; Bilgin, 2011; Douaihy et al., 2011,
2012). The possibility of such migration during the
Pleistocene has also been confirmed in plant palaeoremnants (Magyari et al., 2008). Historical analysis
of speciation in the genus Abies Mill. (Linares, 2011)
and recent studies on the genetic differentiation of
the eastern Mediterranean species A. cephalonica
Loudon, A. nordmanniana Spach, A. bornmuelleriana Mattf. (Liepelt et al., 2010) and Juniperus
excelsa M.Bieb. (Douaihy et al., 2011, 2012) demonstrate the possibility of migration between Europe
and Asia via the Bosphorus and Dardanelles in the
past, but also indicate the restriction of gene flow
between Europe and Asia for some organisms (Bilgin, 2011). However, these studies mostly concern
the organisms of mountain systems that are characterized by temperate or sub-Mediterranean climates
(Magyari et al., 2008). More thermophilic eastern
Mediterranean tree species, which have adapted to
supra-Mediterranean and/or oro-Mediterranean climate conditions, such as Cedrus libani A. Rich. and
Abies cilicica (Antoine & Kotschy) Carri
ere (Qu
ezel
& M
edail, 2003), exhibit genetic differentiation
between the Taurus, Anti-Taurus and Lebanon
Mountains (Bou Dagher-Kharrat et al., 2007;
Sez kiewicz et al., 2015). These species, however, do
not occur in Europe. In searching for the woody and
oro-Mediterranean (Rivas-Martınez, Pe~
nas & Dıaz,
2004) plant taxa that occur in Europe and Asia, we
identified Juniperus drupacea Labill. as a good target species with which to investigate the influence of
isolation by the Aegean Sea on species differentiation
processes between Asia and Europe.
The location of the divergence and evolution of
J. drupacea remains unknown. All species of Juniperus L. evolved in arid and semi-arid regions, where
plant remnants have extremely restricted opportunities to be fossilized and preserved until modern times
(Axelrod, 1975; Palamarev, 1989; Willis & McElwain,
2002). For this reason, fossil data concerning Juniperus spp. are rare and dispersed (Kvacek, 2002;
Stockey et al., 2005) and only include remnants
known from Miocene and Pliocene deposits in Europe
(Axelrod, 1975; Palamarev, 1989; but see the comments in Kvacek, 2002). To date, no fossils of prickly
Juniperus spp. have been reported from the geological period of divergence of J. drupacea.
The current geographical range of J. drupacea
(Fig. 1) covers the southern parts of the Peloponnese
in Europe (Boraty
nski & Browicz, 1982; Tan, Sfikas
& Vold, 1999; Maerki & Frankis, 2015), the southern
parts of Anatolia and the mountains of Syria and
Lebanon in the Levant, with a disjunction of c.
800 km between European and Asian localities
(Browicz, 1982; Qu
ezel & M
edail, 2003). The main
part of the species range in Asia is also disjunct and
is divided into several centres, the most important
being located in the Taurus, Anti-Taurus, Amanos
and Lebanon Mountains, regions that have been recognized as refugia of the Tertiary floras (M
edail &
Diadema, 2009). However, J. drupacea or its ancestor was more widely distributed during the late Tertiary and early Quaternary as a component of the
Tethyan and later Mediterranean sclerophyll flora
(Palamarev, 1989; Kvacek, 2002). The geographical
range of J. drupacea became restricted during Pliocene climate cooling and ultimately decreased to the
present extent during the Pleistocene glacial periods.
It is hypothesized that it reached the southern
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
367
moderately drought-resistant J. drupacea (Zohary,
1973; Yaltırık, 1993) might have survived the Pleistocene in mountain regions and migrated downwards
during the glacial and upwards during the interglacial periods (Hewitt, 1996, 2004; Eastwood, 2004).
The frequency of occurrence might have decreased
during more humid periods as an effect of the competition with more moisture-demanding trees and
might have accelerated during drier periods, when
such competition was weaker (Uzquiano & Arnaz,
1997; Carri
on, 2002; Eastwood, 2004; Hajar, Khater
& Cheddadi, 2008; Orland et al., 2012). The reduction of fir and cedar forests by humans during recent
millennia might also be a reason for some extension
of the occurrence of J. drupacea (Talhouk, Zurayk &
Khuri, 2001; Douaihy et al., 2011; Awad et al., 2014).
The alternative origins and/or long-lasting isolation between the European and Asian populations of
J. drupacea implies genetic and morphological differences. The discontinuous geographical range in Asia,
where J. drupacea occurs on several mountain ridges
(Browicz, 1982), also suggests its further genetic and
morphological differentiation. The aim of the study
was to test these hypotheses using nuclear
microsatellite markers (nSSRs) and the morphological characteristics of cones, seeds and needles.
Figure 1. A, Geographical distribution of Juniperus drupacea and origin of the 12 analysed populations on the
background of the mountain ranges; grey areas – present
range of the species, circles and acronyms – locations of
the tested populations (for details, see Table 1). Genetic
structure of populations: (B) proportional assignment of
individuals in sample to the genetic clusters I (green), II
(red), III (blue), IV (yellow), V (violet) and VI (light blue),
as detected in admixture analysis of three microsatellite
loci, conducted with BAPS software; (C) proportional
assignment of individuals in sample to the genetic clusters I (dominance of red), II (dominance of green) and III
(dominance of blue), as detected in admixture analysis
conducted with STRUCTURE software.
Peloponnese by migrating from the Balkan Peninsula, whereas it populated Anatolia from the more
eastern source regions (Palamarev, 1989), or that the
ancestor of the species might have evolved on the
Anatolian continental plate as early as the Eocene/
Oligocene boundary (Mao et al., 2010) and then
migrated with the Balkan subcontinent when it split
from Anatolia and joined Europe during the Miocene
(Popov et al., 2006; Ivanov et al., 2011). Finally, the
Pleistocene climate oscillations with cold glacial and
warm interglacial periods influenced the distribution
of J. drupacea. Thermophilic, light-demanding and
MATERIAL AND METHODS
STUDY SPECIES
Juniperus drupacea is a dioecious tree that reaches
20–40 m in height and 1.2 m in trunk diameter at a
height of 1.3 m (Elicin, 1977; Karaca, 1994; Christensen, 1997; Maerki & Frankis, 2015). The crown of
the young trees is conical, but becomes more irregular in older individuals. The prickly, boat-shaped
needles are arranged in alternate whorls of three
and are 10–15 mm long and 2–4 mm wide, with two
whitish bands on the adaxial side (Christensen,
1997; Farjon, 2005; Adams, 2014). The seed cones
are ovoid or globose, brown and pruinose when ripe,
and are c. 20–25 mm in diameter and are composed
of two to four whorls of alternate, imbricate scales.
The three seeds are connate and form a drupe-like
stone. Despite its specific morphological characteristics, no infraspecific taxa have been described in
J. drupacea (Farjon, 2005; Auders & Spicer, 2012).
The peculiar morphological characteristics of
J. drupacea among all other taxa of Juniperus led to
the description of section Caryocedrus Endl.; the distinct genus Arceuthos Antoine & Kotschy was proposed but later rejected (e.g. Gaussen, 1967, 1968;
Elicin, 1977; Christensen, 1997; Farjon, 2005, 2010;
Adams, 2014). The position of J. drupacea in the
genus is still unclear and disputed (Farjon, 2005;
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Appendix S1. Genetic structure of 12 populations of J. drupacea and membership of individuals for K = 6, as
detected in admixture analysis conducted with STRUCTURE software; sample codes as in Table 1.
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
Average across all
populations
Lebanon, Mchati
Lebanon, Ehmej
Lebanon, Dufram
Average
Average across Turkey
Turkey, Anti-Taurus,
Baskonus
Turkey, Anti-Taurus,
Bostanli
Average
34.05
34.1333
34.4167
37.45
TAT_2
LEB_1
LEB_2
LEB_3
37.55
37.18
TT_3
TAT_1
36.3333
TT_2
37.0333
GRT
37.05
37.0333
GRP_3
TT_1
37.1
GRP_2
Turkey, Taurus,
Akseki
Turkey, Taurus
Ermenek
Turkey, Taurus,
C
amliyayla
Average
37.3333
GRP_1
Greece, Parnon,
Agios Petros
Greece, Parnon,
Kosmas
Greece, Parnon,
Gerakas
Greece, Taygetos,
Anavriti
Average
Latitude [o]
Acronym
Locality
35.7667
35.8167
36.1
36.4167
36.6
34.6167
33
31.8
22.3833
22.7
22.7333
22.5333
Longitude [o]
1080
1340
1250
980
1400
1050
1200
1500
900
650
800
1000
Elevation
[m]
15
17
27
30
33
29
28
30
33
32
31
33
N
–
–
KOR 47192
KOR 47323
KOR 47296
KOR 45430
KOR 47334
KOR 44656
KOR 48753
KOR 48754
KOR 48756
KOR 47869
KOR 47933
KOR 47934
KOR 47901
KOR 47903
Voucher
0.500 ( 0.071)
7.47
( 0.43)
6.8
0.607 ( 0.082)
6.6
( 0.20)
6.15
( 0.54)
8.0
7.0
7.1
7.37
( 0.45)
6.88
( 0.82)
0.497 ( 0.103)
0.444
0.667
0.420
0.510 ( 0.111)
0.449 ( 0.092)
0.689
6.4
0.525
0.437
0.464
7.8
6.9
0.430 ( 0.067)
6.22
( 0.91)
7.7
0.600
0.484
0.510
0.365
0.363
Observed
heterozygosity
HO
4.7
7.1
6.4
6.7
Allelic
richness
AR
0.675
( 0.044)
0.595
( 0.057)
0.657
0.740
0.715
0.704
( 0.035)
0.667
( 0.062)
0.720
0.698
( 0.062)
0.631
0.783
0.635
0.612
( 0.042)
0.677
0.661
0.640
0.552
0.596
Gene
diversity
HE
10.3%
0.069inbr
0.0%
0.399inbr
0.068
( 0.035)
0.109
( 0.133)
0.143
0.077inbr
0.304inbr
0.174
( 0.095)
0.136
( 0.111)
0.033
5,3%
( 0.02)
6.1%
( 2.2)
10.8%
7.7%
0.1%
6.2%
( 4.5)
6.09%
( 4.14)
5.1%
2.0%
( 0.02)
5.5%
1.3%
0.239inbr
0.239
( 0.131)
0.104
9.5%
( 3.0)
4.7%
6.7%
0.065
( 0.023)
0.078
0.043
7.0%
14.0%
0.102inbr
0.047
Null alleles
Fixation
index
FIS
Table 1. The genetic characteristics and origin of populations of Juniperus drupacea analysed (N, number of individuals in population), bolded average values
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
369
370
K. SOBIERAJSKA ET AL.
et al., 2008). Clustering of groups of individuals was
run in BAPS for ten replicates for K = 1–13. The
number of populations was inferred as the combined
maximum likelihood and highest posterior probability estimates over all ten replicates. The admixture
analysis was conducted with a fixed number of clusters inferred with the cluster analysis. After testing
runs in STRUCTURE, the final analysis was run
with the number of groups K set for 1–12, with ten
independent runs for each K. The admixture model
with correlated allele frequencies and a burn-in period of 100 000 and 200 000 Markov chain Monte
Carlo (MCMC) iterations for each run according to
the recommendation of Gilbert et al. (2012) was
used. K was chosen using Evanno’s DK (Evanno,
Regnaut & Goudet, 2005) using Structure Harvester
(Earl & von Holdt, 2012). The results obtained with
STRUCTURE were also explored with CLUMPAK to
align the runs across the tested K values and to
obtain the consensus graphical representation of the
final population genetic structure.
An analysis of molecular variance (AMOVA) was
performed with ARLEQUIN 3.11 (Excoffier, Laval &
Schneider, 2005) for groups inferred with BAPS,
STRUCTURE and geographical regions (Peloponnese
vs. Anatolia vs. Lebanese Mts.). Statistical significance was obtained after 9999 random permutations.
The relationships between geographical distance
and genetic distance were tested with the Mantel
test (Mantel, 1967) with 999 random permutations in
PopTools 3.2.3 (Hood, 2010). The genetic distances of
Cavalli-Sforza & Edwards (1967) with INA correction
(including null-allele correction) were estimated with
FreeNA software. The linear regressions for genetic
diversity parameters (AR, HO and HE), with respect
to latitude, longitude and elevation, were confirmed
to trace the geographical trends in the spatial distribution of genetic variation.
BIOMETRY
Morphological characters used and measurement
procedures
The characters and biometric procedures were
adopted from studies on J. oxycedrus and related
taxa (Klimko et al., 2007; Boraty
nski et al., 2014).
Initially, 14 morphological characters and ten ratios
were examined, but the total number was reduced to
17 (Table 2) during the study by eliminating those
that lacked variability, the most redundant ones and
extreme variables with possible high error. The dry
cone and ‘drupe’ characteristics, such as cone length
(CL), cone diameter (CD), drupe length (DL) and
drupe diameter (DD), were taken manually using an
electronic calliper (31C624, Topex.PL; accuracy of
measurement 0.02 mm). The cone and the drupe
diameters were calculated as means from two measurements perpendicular to each other. The cone
scale numbers (CSN) were counted using dry cones.
The needle characters were collected from needle
images using WinFolia (REGENT,
Inc.) software.
The needles were soaked in 70% alcohol for 24 h and
were then scanned using an Epson Perfection V-700
photo scanner. The needle length (NL), maximum
needle width (NW), width at 50% of needle length
(NW5), width at 90% of needle length (NW9), needle
area (NA) and needle circumference (NC) were measured automatically and the distance from the maximal width to the needle base (DNW) was taken
manually. The width of the stomata band (STB) was
taken manually and the number of stomata on 1 mm
of needle (STN) was counted using scanning electron
microscope (SEM) images (Hitachi S-3000N, Tokyo,
Japan), taken with a fixed working distance and a
magnification of 990.
Data analysis
To assess the possibility of conducting multivariate
statistical analyses and parametric tests, the symmetry, unimodality and homoscedasticity of data were
verified (Sokal & Rohlf, 2003; Zar, 1999). The data distribution was verified using Shapiro–Wilk’s test. The
homoscedasticity of direct data variances was verified
with the Brown–Forsythe test, as implemented in
STATISTICA 9 (StatSoft, Krak
ow, Poland). Before
further analyses, data were standardized using STATISTICA procedures to avoid the influence of variation resulting from different types of characters.
Each individual and population was characterized
by an arithmetic mean, standard deviation and variation coefficient to determine the variation ranges.
Pearson’s correlation coefficient was used to identify
interactions between characters and to detect potential redundant variables. A discrimination analysis
was performed to calculate the discriminatory power
of characters among populations, to eliminate redundant variables and to reveal population groupings
(Sokal & Rohlf, 2003). Population groupings were
also verified using agglomeration (dendrograms)
according to Ward’s method on the Euclidean distances of nearest neighbourhood distances among
populations and Mahalanobis’ distances and Kmeans cluster analysis (Sokal & Rohlf, 2003). The
differentiation among populations and regions for
characters with a normal distribution was verified
using the honest significant difference (HSD) Tukey’s
T-test and for characters with a skewed distribution
using the Kruskal–Wallis’ test. STATISTICA software was used for statistical analyses.
A Mantel test (Mantel, 1967) was performed on
the matrices of geographical distances to compare
them to those of Euclidean distances (Manni,
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
Cone length [mm]
7.2
Cone diameter
[mm]
8.0
Cone scale number
9.5
Drupe length [mm]
7.0
Drupe diameter
[mm]
7.8
Needle length
[mm]
18.5
Needle maximal
width [mm]
10.3
Width at 90% of
needle length
[mm]
26.0
Distance of
maximal width
to needle base
19.0
CL
CD
20
CSN
DL
DD
NL
NW
NW9
DNW
Character
Code
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
18.8
14.7
25.6
8.7
16.0
10.4
20.9
7.1
7.93
6
12
10.5
13.4
10.8
16.8
7.4
12.0
9.0
15.3
7.1
12.3
7.8
19.6
17.7
2.6
1.7
3.7
10.2
0.4
0.1
1.0
30.0
3.0
0.3
6.5
21.1
GRP_1
18.4
12.4
23.5
8.7
15.6
10.6
21.1
7.1
8.18
6
12
7.9
13.2
10.3
22.2
6.8
11.7
8.9
13.8
6.0
12.1
6.8
20.8
18.9
2.4
1.7
3.2
9.9
0.4
0.1
0.8
36.4
3.1
0.5
6.5
20.6
GRP_2
19.1
14.3
23.5
6.0
17.8
9.9
22.1
7.5
8.32
6
12
10.7
13.6
10.1
17.7
6.1
12.3
10.1
15.5
7.1
13.8
7.0
21.5
17.4
2.3
1.3
3.8
15.7
0.3
0.1
0.8
36.7
3.1
0.1
7.1
22.4
GRP_3
18.3
9.5
23.5
7.7
16.4
8.9
23.1
10.8
8.74
6
12
8.3
13.3
8.9
17.5
7.1
11.8
8.8
14.4
6.5
12.8
5.3
18.2
15.5
2.3
1.4
3.5
12.6
0.3
0.1
0.7
36.2
3.0
0.1
6.3
23.7
GRT_1
Population (acronyms as in Table 1)
20.9
7.8
13.3
7.0
25.4
22.4
2.5
1.7
3.5
9.6
0.6
0.2
1.0
16.4
3.5
1.5
7.7
20.7
19.7
8.2
13.9
12
8.6
15.8
25.6
7.8
9.24
21.6
16.0
26.6
8.0
20.1
TT_1
20.9
16.0
26.2
6.6
19.7
13.9
23.5
6.7
9.31
6
12
10.1
15.2
11.1
18.7
7.9
13.5
10.1
18.4
6.9
12.1
7.1
17.2
14.3
2.4
1.7
3.3
8.1
0.6
0.2
0.9
19.0
3.1
0.4
7.0
16.1
TT_2
19.8
13.5
27.4
8.5
19.2
12.5
24.3
7.4
9.31
6
12
6.1
14.7
8.7
19.0
10.2
12.6
6.9
15.9
12.2
13.0
7.2
20.2
14.8
2.6
1.6
3.7
10.2
0.6
0.2
1.2
21.6
3.7
1.8
6.9
14.3
TAT_1
20.6
13.7
26.1
6.7
20.0
15.9
25.6
7.9
9.76
6
12
9.0
15.4
13.0
17.5
5.4
13.4
10.6
17.9
7.7
13.6
6.4
23.2
21.5
2.5
1.5
3.4
9.3
0.6
0.2
1.1
23.4
3.5
0.9
6.8
19.9
TT_3
21.5
15.3
29.1
8.0
20.3
12.8
28.5
11.6
10.0
6
15
10.8
15.7
10.0
19.4
7.2
13.3
8.1
16.6
9.9
14.4
8.6
23.7
16.8
2.6
1.5
4.0
13.5
0.6
0.1
0.9
22.7
4.0
1.4
7.8
18.8
TAT_2
23.5
20.2
27.1
5.5
23.1
18.4
27.1
6.2
11.88
9
15
10.1
17.8
14.5
20.4
5.9
14.4
12.3
17.0
7.2
12.5
7.8
21.9
24.2
2.4
1.7
3.5
8.1
0.6
0.2
0.9
20.0
3.9
1.6
6.2
12.8
LEB_1
22.2
18.7
25.6
4.5
22.0
17.3
25.5
7.6
10.27
6
12
11.0
16.8
15.0
19.8
5.7
14.1
11.8
17.3
7.7
13.2
7.3
23.3
25.1
2.5
1.7
3.4
8.2
0.6
0.2
0.9
21.5
4.2
1.2
8.6
20.6
LEB_2
21.8
10.0
33.4
7.2
19.9
13.5
24.4
8.8
10.35
3
15
9.4
16.6
13.0
19.8
6.2
13.9
10.9
16.4
7.1
13.2
8.2
20.1
25.1
2.5
1.8
3.6
8.2
0.6
0.3
1.1
21.5
4.2
2.2
7.5
20.6
LEB_3
0.895**
0.924*
0.945
0.925*
0.876**
0.863**
0.783**
0.838**
0.851**
Wilks’ k
Table 2. Values of analysed characters for 12 populations of Juniperus drupacea: mean (Mean), minimal (Min), maximal (Max), variation coefficient (V) and discrimination power testing among populations: **P ≤ 0.01, *P ≤ 0.05
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
371
NSH
CSH
45.8
NW9/NW
24.7
NL/NW9
Location of needle
max. width
(DNW/NL 9 100)
[%]
Needle shape
(NL/NW)
18.1
Drupe shape
(DL/DD)
6.5
Number of
stomata on
1 mm of needle
17.5
Cone shape
(CL/CD)
7.1.
Width of stomatal
band [mm]
15.6
STB
STN
Character
Code
Table 2. Continued
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
Mean
Min
Max
V
13.6
Mean
Min
Max
V
Mean
Min
Max
V
36.6
12.8
148.0
44.3
0.16
0.04
0.36
27.5
0.549
0.180
0.780
15.5
156.6
83
235
18.5
1.17
0.96
1.76
5.3
1.12
0.88
1.38
5.0
4.80
2.69
7.67
15.3
24.6
2.8
42.2
12.5
GRP_1
41.6
21.1
163.0
46.0
0.16
0.04
0.37
34.8
0.545
0.318
0.766
15.9
155.8
95
232
13.2
1.19
0.46
1.87
6.7
1.13
0.88
2.03
5.0
5.00
2.96
8.50
16.0
25.6
5.5
49.2
14.1
GRP_2
54.2
15.6
208.0
49.6
0.14
0.08
0.44
37.1
0.577
0.290
0.847
20.7
152.5
85
221
17.9
1.08
0.82
1.85
8.4
1.10
0.75
1.41
7.9
6.15
2.73
10.29
17.1
23.1
1.1
46.5
21.0
GRP_3
46.5
15.1
167.0
50.2
0.15
0.03
0.33
31.1
0.539
0.354
0.871
15.9
148.6
83
252
16.7
1.13
0.48
1.64
8.0
1.14
0.79
1.60
6.4
5.81
2.65
10.12
20.3
23.5
0.9
45.3
20.8
GRT_1
Population (acronyms as in Table 1)
22.8
12.6
127.0
35.2
0.24
0.09
0.41
13.2
1.51
7.3
5.31
2.19
11.55
23.8
27.0
12.2
45.7
11.7
1.39
8.0
1.14
0.574
0.379
0.835
11.4
143.2
87
234
12.0
1.08
TT_1
22.9
11.6
65.0
27.3
0.24
0.08
0.37
17.8
0.538
0.354
0.758
14.8
119.8
59
197
19.0
1.07
0.54
1.47
6.9
1.13
0.87
1.47
5.9
5.13
2.22
7.50
14.8
26.0
4.1
58.1
10.0
TT_2
22.6
9.9
101.0
35.1
0.25
0.08
0.46
21.4
1.30
6.5
1.17
0.99
1.44
6.1
5.08
2.57
7.96
16.4
28.7
14.5
50.0
11.4
0.542
0.121
0.813
17.2
148.1
31
224
20.2
1.03
TAT_1
24.6
10.4
86.0
41.6
0.25
0.09
0.41
21.7
0.556
0.266
0.824
19.6
151.7
93
246
23.1
1.04
0.61
1.41
7.7
1.15
0.84
1.44
6.4
5.52
2.44
9.00
20.3
26.7
10.1
52.8
16.5
TT_3
29.1
13.6
130.0
47.8
0.21
0.06
0.41
24.0
0.554
0.330
0.790
18.0
136.8
92
214
18.2
1.08
0.74
1.74
12.0
1.18
0.93
1.85
10.1
5.51
3.19
8.25
14.7
27.7
12.3
46.7
11.0
TAT_2
23.8
11.3
109.5
65.5
0.26
0.09
0.43
22.4
0.518
0.351
0.687
15.2
167.1
92
273
21.3
1.02
0.86
1.29
5.3
1.24
1.00
1.54
5.5
5.19
3.22
10.71
23.5
31.8
9.7
48.7
12.7
LEB_1
26.4
11.9
108.5
54.6
0.23
0.07
0.47
24.5
0.541
0.423
0.774
10.1
144.8
88
218
15.3
1.02
0.45
1.20
4.6
1.20
1.03
1.37
6.1
5.36
2.77
8.68
19.4
32.3
9.5
50.9
12.2
LEB_2
21.8
11.8
67.0
32.0
0.25
0.11
0.45
20.3
0.592
0.368
0.808
11.9
157.8
102
225
14.9
1.10
0.50
1.44
6.3
1.20
1.03
1.41
5.7
5.15
3.03
7.91
16.1
32.4
19.0
52.8
9.4
LEB_3
0.951
0.946
0.919*
0.934*
0.934*
0.826**
0.770**
0.766**
Wilks’ k
372
K. SOBIERAJSKA ET AL.
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
Guerard & Heyer, 2004) with the implementation of
PopTools (Hood, 2010). The geographical distances
were taken from the geographical coordinates of the
localities in Map Info 9.5 (Pitney Bowes).
To detect characters that distinguished between
groups of populations detected by the discrimination
and the percentage of the variation that was responsible for differences among them, hierarchical
analysis of variance was performed (Sokal & Rohlf,
2003); this was also applied to the K-grouping and
agglomeration analyses. The JMP Software (SAS
Institute, SAS Campus Drive, Cary, NC, USA) was
used for the calculations.
RESULTS
GENETIC
DIVERSITY AND DIFFERENTIATION
No evidence for linkage disequilibrium was detected
between pairs of SSR loci in each of the populations.
Six, 12 and 34 different alleles were found at loci
Jc032, Jc035 and Jc037, respectively. Locus Jc035
showed a statistically significant excess of homozygotic alleles, whereas the other two loci were in
Hardy–Weinberg equilibrium (HWE). Jc035 was the
locus for which the highest frequency of null alleles
was detected (Table 3). We thus suspected that the
excess of homozygosity was partly due to null alleles.
The estimates of within-population genetic diversity
are shown in Table 1. The highest (8.0) allelic richness (AR) was observed in one of the Lebanese populations (LB_1) and the lowest AR (4.7) in one of the
Greek populations (GRT). Similarly, another Turkish
population (TAT_2) exhibited the highest observed
heterozygosity (0.689), whereas the lowest value
(0.363) was detected in a Greek population (GRP_1).
The highest value (0.740) of genetic diversity (HE) was
observed in a Lebanese population (LEB_2) and the
lowest (0.552) in a Greek population (GRP_2).
373
The permutation test did not indicate any statistically significant differences in genetic diversity estimates (AR, HO and HE) among Greek, Turkish and
Lebanese populations of J. drupacea. The linear regressions among latitude, longitude, elevation and genetic
variation estimates also showed no clear relationships,
the only exception being HE values, which were positively related to the longitude (r2 = 0.406, P = 0.026).
However, there was a visible west–east trend (from
Greece to Lebanon) of increase in HE: HEGR = 0.612,
HETT = 0.698, HETAT = 0.675, and HELEB = 0.704.
In all studied populations of J. drupacea, a significant excess of homozygotes was detected (P < 0.05).
In most cases, high values of inbreeding index (FIS)
were related, with inbreeding as a causative factor.
The mean frequency of null alleles across the populations studied was 6.09%.
Global differentiation among populations was significant (FST = 0.108, P < 0.001) and the estimation
with the ENA correction that considers null alleles
was only slightly lower, with FST = 0.101. The permutation test did not reveal any significant difference between FST and RST, suggesting a lack of
impact of mutations on genetic structure. Bayesian
clustering conducted with BAPS revealed six clusters
among 12 analysed populations, with log(marg.
3730.3017 (Fig. 1). Cluster I contained
likelihood) =
three Greek populations (GRP_1, GRP_2 and
GRP_3), and the fourth Greek population was placed
in Cluster II. All Lebanese populations were grouped
in Cluster III (LEB_1, LEB_2 and LEB_3) and the
Turkish populations were distributed among the
other three clusters: populations TT_1 and TT_2 in
Cluster IV; populations TAT_ 1 and TAT_2 in Cluster V; and population TT_3 in Cluster VI. The
admixture analysis indicated a limited sharing of
gene pools among inferred clusters.
The geographical pattern inferred with STRUCTURE differed slightly from that inferred using
Table 3. Characteristics of three nSSR loci used in the study of genetic structure of Juniperus drupacea.
Locus
Range
Jc032
F:acattgcaaatatggggtaa
R:ttgagtagttgttgagttattaag
Jc035
F:ttggtttattctccccatct
R:cccccagttattctaaacatt
Jc037
F:ggcaattagtaaggcacaag
R:taaggtggatatcaccaagg
154–170
N
HO
HE
6
0.748
0.738
0.014
0.0000
120–148
12
0.728
0.894
0.186*
0.0775
137–215
34
0.943
0.912
0.035
0.0051
FIS
Null
N, total number of alleles detected; HO, observed heterozygosity; HE, expected heterozygosity; FIS, fixation index; Null,
estimation of null-allele frequency.
*P = 0.05.
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
374
K. SOBIERAJSKA ET AL.
BAPS. Firstly, three genetic clusters (Fig. 2) were
found to be an optimal structure and the grouping
was as follows: I – Greek population GRP_1-GRP_3
and one Lebanese population LEB_2; II – Turkish
populations TT_1, TT_2, TAT_1 and TAT_2; III –
Lebanese populations LEB_1 and LEB_3, Turkish
population TT_3 and Greek population GRT (Fig. 1C).
Secondly, a wide sharing of gene pools among populations representing different regions was inferred.
For example, 54.4% of the genes of the Greek population GRT were in common with the gene pool of
cluster III, which contained Lebanese populations
LEB_1 and LEB_3 and 40.8% belonged to cluster I, in
which the remaining populations from Greece were
grouped. The Lebanese population LEB_2 also almost
equally shared its genes with cluster I (50.6%) and
cluster III, into which the other Lebanese populations
were grouped. Under alternative scenarios of clustering suggested by Evanno’s DK (Fig. 2), the model in
STRUCTURE indicated substructuring with K = 6
(Supporting Information, Appendix S1, Fig. 1), which
referred to the genetic structure defined by BAPS.
Accordingly, substructuring in the Greek and Turkish
populations was revealed. The Greek population GRT
and Turkish population TT_3 were placed in separate
clusters and the division between the Taurus (TT_1
and TT_2) and Anti-Taurus populations (TAT_1
and TAT_2) resulted in two separate clusters for
these populations.
The AMOVA performed for genetic clusters
detected with BAPS indicated that 4.3% (P = 0.012)
of the total genetic diversity resides among these
groups, whereas for STRUCTURE, the amount was
35
30
Delta K
25
20
15
10
5
0
2
4
6
K
8
10
Figure 2. Number of clusters detected using method of
Evanno’s DK among 12 Juniperus drupacea populations,
conducted with STRUCTURE software (Supporting Information, Appendix S1).
found to be 10.6% (P = 0.015). The AMOVA also
detected 10.55% (P = 0.013) of the diversity among
samples from the Peloponnese, Anatolia and Lebanon (Table 4). The comparison of geographical and
genetic distances among populations using the Mantel test revealed low (r2 = 0.318) but significant relationships (P < 0.05).
MORPHOLOGICAL
VARIATION AND DIFFERENTIATION
Character variation and correlation
The mean values of most of the analysed characters
were specific for a particular population, but showed
a frequency distribution in most cases that overlapped more populations. The most variable characters were NL/NW9, NW9 and NA. The most stable
characters, with the lowest values of variation coefficient (V), were DL/DD, DL, DD, CL, CD and CL/CD
(Tables 2 and 5). The V values for particular characters between European and Asiatic populations did
not differ significantly, with the exception of the
most variable characters (NL/W9 and NW9/NW).
The European and Asian populations revealed similar levels of morphological variation.
Pearson’s correlation coefficient between character
pairs was slightly different for each sample, but the
characters of cones and drupes (CL, CD, DL and DD)
were positively correlated at a statistically significant level (P ≤ 0.01) in all samples. In addition, needle characters (NL, NW, NW5 and NC) were
significantly correlated (P ≤ 0.01). The needle characters were correlated at a much lower level with the
characters of cones and drupes. The most closely correlated characters (|r| > 0.95) were highly redundant.
Therefore, NW5, NA and NC were omitted from subsequent analyses, with NW5 being closely associated
with NW and NA and NC with NL. The ratios characterizing the shape of the cone (CL/CD), drupe (DL/
DD) and needle (NL/NW and NW9/NW) and the location of the maximum needle width (DNW/NL 9 100)
were included in the analyses and the original values
of DNW and NW9 were excluded as being highly
redundant; the former to DNW/NL 9 100 and the
latter to NW9/NW. Finally, statistical analyses were
conducted on 17 characters: nine that were measured, two counted and six recalculated (Table 2).
Most characters had a normal or close-to-normal frequency distribution and after standardization, also
have a homogeneous level of variation.
Character differentiation
Tukey’s T-test and the Kruskal–Wallis test detected
that the mean values of most characters from Europe
and Asia differed at a statistically significant level
(Table 2). No differences were found only among needle characters, such as NL, STB and NLW. These
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
375
Table 4. Hierarchical population structure in analyzed population of J. drupacea, as evaluated using AMOVA
(*P ≤ 0.05, ***P ≤ 0.001)
Source of variation
d.f.
Sum of squares
Fst Variance components
BAPS
Among groups
5
53.745
0.04305
Among populations within groups
6
31.681
0.07548
Within populations
664
645.057
0.11528
Total
675
730.484
STRUCTURE
Among groups
2
31.415
0.03862
Among populations within groups
9
52.650
0.08273
Within populations
664
646.419
0.11815
Total
675
730.484
Geographic regions (Peloponnese, Taurus and Anti-Taurus, Lebanon)
Among groups
2
33.013
0.04714
Among populations within groups
9
51.052
0.07861
Within populations
664
646.419
0.12205
Total
675
730.484
characters were variable, with V oscillating around
16–20%, but without large differences among their
mean values, even for particular populations, not only
between Europe and Asia (Table 2). Among the other
analysed characters, STN did not distinguish
European and Lebanese populations at a statistically
significant level; likewise NW, NW9 and NL/NW9
did not distinguish Anatolian and Lebanese populations. The other characters revealed statistically
significant differences among the compared regions
(Table 5).
Generally, the Asian populations contained individuals with larger cones and seeds (CL, CD, DL and
DD), a greater number of seed scales on the cones
(CSN) and slightly wider needles (NW), with a
greater distance between the base and the widest
point (DNW and DNW/NL 9 100). Asian populations
were additionally differentiated from Anatolian and
Lebanese populations by cone and seed characters.
The highest values for characters were found in
Lebanese populations (Tables 2 and 5).
Differentiation of populations
Among 17 analysed characters, nine showed high
discriminatory power among populations, which was
significant at P ≤ 0.01, and another four showed discriminatory power at P ≤ 0.05 (Table 2). The highest
discrimination powers were found for STB, STN and
CSN, with Wilk’s k values of 0.766, 0.770 and 0.783,
respectively. The first two variables that discriminated between populations accounted for 74.6% of
the total variation. On the plane between the twofirst variables compared, the populations formed
three separate groupings (Fig. 2A). The populations
from Europe formed the most distinct group, as
Percentage of variation
4.30 *
7.22***
88.47***
–
10.60 *
9.52***
88.19***
–
4.71***
7.49***
87.80***
–
determined by the first variable (U1), which was primarily dependent on CD, DL, SCN, CL and NW9/
NW. Among Asian populations, the Turkish populations from the Taurus and Anti-Taurus Mountains
could be distinguished from those from the Lebanese
Mountains. The differences between the Asian populations were determined mostly by the second variable (U2), which was dependent to the greatest
degree upon STN and NW9/NW. The Lebanese populations, however, did not form a condensed group,
but were discriminated by both variables (Fig. 3A).
The discrimination among individuals was fully
determined by the two-first variables, which
accounted for 100% of the variation. European individuals only penetrated the Asian group slightly,
whereas the Anatolian and the Lebanese individuals
formed two partly intermingled groups (Fig. 3B).
The European group differed from the Anatolian and
Lebanese groups at a statistically significant level in
14 and 13 characters, respectively. The Anatolian
group differed from the Lebanese group in nine characters (Table 5). The proper ordination of individuals
into populations was nearly 61%, on average, and
varied from 45% in population LEB_2 to 93% in
LEB_3. The ordination of European vs. Asian groups,
however, was generally much more successful (85%
on average), at 95%, 94% and 75% in European,
Turkish and Lebanese groups of populations, respectively. Such high levels of fit of the ordination of
individuals to the European/Asian groups of populations indicate the high probability of the proper
inclusion of each individual (Fig. 4B). The comparison of geographical and Euclidean distances
between populations using the Mantel test revealed
a significant dependence (r2 = 0.687, P < 0.001).
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
1248
1248
1248
625
625
1244
1244
1243
1244
363
363
1248
625
1244
1244
1243
1243
CL
CD
CSN
DL
DD
NL
NW
NW9
DNW
STB
STN
CLD
DLD
NLW
DNWL
NLW9
NW9W
18.7
16.5
8.3
13.4
11.9
12.7
2.4
0.4
3.1
0.6
153.6
1.2
1.1
5.4
24.2
44.5
0.15
Mean
9.5
8.9
6.0
8.9
8.8
5.3
1.3
0.1
0.3
0.3
83
0.5
0.8
2.7
2.9
11.1
0.1
Min
25.6
23.1
12
22.2
15.5
21.5
3.8
1.0
7.1
0.9
252
1.9
2.0
10.3
49.2
208.0
0.4
Max
7.9
9.6
9.9
6.8
6.9
17.9
13.2
35.1
21.8
16.8
16.3
7.9
6.3
20.2
17.5
50.2
32.6
V
1433
1433
1433
744
744
1481
1481
1481
1481
407
410
1433
744
1481
1481
1481
1481
N
20.8
19.8
9.5
15.3
13.3
13.3
2.5
0.6
3.6
0.6
140.3
1.1
1.2
5.3
27.3
24.4
0.2
Mean
13.5
12.5
6
8.6
6.9
6.4
1.5
0.1
0.4
0.1
31
0.5
0.8
2.2
4.1
9.9
0.1
Min
29.1
28.5
15
19.7
20.9
25.4
4.0
1.2
7.8
0.8
246
1.7
1.9
11.6
58.1
130.0
0.5
Max
B Taurus and Anti-Taurus Mts
8.2
8.6
9.5
8.3
9.6
19.0
10.9
21.2
19.5
16.4
20.4
8.5
7.4
18.5
12.6
40.4
20.4
V
515
515
515
263
263
588
588
588
588
171
171
515
263
588
588
588
588
N
22.4
21.3
10.7
17.0
14.1
13.2
2.6
0.6
4.2
0.6
156.9
1.1
1.2
5.2
32.2
23.7
0.2
Mean
C Lebanon Mts
10.1
13.5
3
12.9
10.9
7.3
1.7
0.2
1.2
0.4
88.0
0.5
1.0
2.8
9.5
11.3
0.1
Min
33.4
27.1
15
20.4
17.3
23.3
3.6
1.1
8.6
0.8
273.0
1.4
1.5
10.7
52.8
109.5
0.5
Max
6.9
10.0
11.8
6.6
7.2
20.3
10.0
20.1
16.4
13.2
17.4
7.0
5.9
18.9
10.9
50.3
21.7
V
0.0000
0.0000
0.0000
0.0000
0.0000
0.2564
0.0000
0.0000
0.0000
0.7589
0.0000
0.0000
0.0000
1.0000
0.0000
0.0000
0.0000
A/B
0.0000
0.0000
0.0000
0.0000
0.0000
0.9382
0.0000
0.0000
0.0000
0.3307
0.2428
0.0000
0.0000
0.7786
0.0000
0.0000
0.0000
A/C
0.0000
0.0000
0.0000
0.0000
0.0000
1.0000
0.1247
0.8512
0.0000
0.4587
0.0000
1.0000
0.0000
1.0000
0.0000
0.3904
1.0000
B/C
Tukey’s and Kruskal–
Wallis tests p between
pairs of groups
N, number of measurements for a particular character; due to there being fewer than ten shoots, cones and/or seeds for some number of individuals in analysed
populations, the real number of measurements was somewhat smaller than the assumed; Min., minimal value of character; Max., maximal value of character;
V, variation coefficient; P, level of statistic significance of differences between mean values of characters; A–C, geographical groups of populations as indicated
in Table 1.
N
A European populations
Character
code
Asian populations
Table 5. Average values, minima, maxima and variation coefficients of analysed characters of cones, seeds and shoots with leaves of three groups of Juniperus
drupacea populations distinguished by discrimination analysis (Fig. 2A)
376
K. SOBIERAJSKA ET AL.
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
Figure 3. Results of discrimination analysis for Juniperus drupacea according to the discrimination variables U1
and U2: (A) for populations; (B) for individuals (lines indicate 95% confidence intervals for each of three geographical groups of populations); acronyms of populations as in
Table 1.
DISCUSSION
GENETIC
DIVERSITY
The presented results are the first to describe the
level of diversity and differentiation of J. drupacea
across almost the entire natural range of the species.
The divergence time of J. drupacea was estimated to
be as early as prior to c. 30–35 Mya, at the boundary
of the Eocene and Oligocene (Mao et al., 2010), and
might be the main reason for the low transferability
of the nSSR markers. Problems of cross-amplification
have also been reported for J. excelsa Willd.
(Douaihy et al., 2011), in which successful amplification was observed for three loci out of the 31 tested,
and for J. oxycedrus (Boraty
nski et al., 2014), three
loci out of eight tested resulted in high-quality
amplification. We also tested eight pairs of primers
for nSSRs, but only three could be included in the
analysis. Although the three loci used here showed
repeatable and high-quality amplification products,
with a total of 54 alleles identified, they represent
377
only a small fraction of the whole genome and this
might introduce some bias. Furthermore, transferred
microsatellites frequently show an increased level of
null alleles, also influencing patterns of differentiation. Anticipating this potential problem, we performed statistical procedures that included the
influence of null alleles.
The level of genetic diversity of J. drupacea was
relatively high, but was comparable to that exhibited
by other taxa of Cupressaceae and of gymnosperms
in general, estimated with a different set of genetic
markers (e.g. Meloni et al., 2005; Michalczyk et al.,
2006; Provan et al., 2008; Terrab et al., 2008;
&
Douaihy et al., 2011; Dzialuk et al., 2011; Y€
ucedag
Gailing, 2013; Dering et al., 2014; Teixeira,
Rodrıguez-Echeverrıa & Nabais, 2014; Sez kiewicz
et al., 2015). The values of HE for J. oxycedrus, using
the same three markers, were similar (compare
Table 1 to Boraty
nski et al., 2014: table 1).
The high genetic diversity found in populations of
J. drupacea is typical for Mediterranean conifers
(Fady-Welterlen, 2005; Fady & Conord, 2010) and
other organisms (Conord, Gurevitch & Fady, 2012).
This phenomenon can be explained by the possible
long-lasting in situ persistence of populations of
Mediterranean conifers (Eastwood, 2004; start here
Hughes et al., 2006; Magyari et al., 2008; M
edail &
Diadema, 2009) without a strong negative influence
of climate cooling and, consequently, with only moderate genetic bottlenecks and other negative demogenetic events (Fady-Welterlen, 2005; Fady et al.,
2008; Conord et al., 2012). The well known pioneer
character of Juniperus spp., including J. drupacea
(Zohary, 1973; Yaltırık, 1993), might alleviate negative climate influences and have a positive influence
on the conservation of the local genetic pool. Additionally, long-lived, wind-pollinated, primarily
outcrossing tree species typically display great
within-population diversity. However, the high
excess of homozygosity due to inbreeding (mating
between close relatives in the case of dioecious
Juniperus spp.) detected in some populations should
be considered undesirable and worrying, because of a
direct negative relationship between the low genetic
diversity resulting from inbreeding and a long-lasting population persistence (Keller & Waller, 2002).
MORPHOLOGICAL
VARIATION
The mean values of characters that describe the size
and shape of the cones and needles detected in this
study were similar or smaller than those reported in
floras (Table 6). It is expected that our data closely
reflect actual mean values of the characters, because
they are based on large numbers of individuals. The
marginal values for particular characters were lower
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
378
K. SOBIERAJSKA ET AL.
Figure 4. Agglomeration of Juniperus drupacea populations using Euclidean distances from biometric data (A) and geographical distribution of three groups of populations detected from K-means clustering (B); acronyms of populations as
in Table 1.
than those reported in the Flora of Turkey (Coode &
Cullen, 1965), Nouvelle flore du Liban et de la Syrie
(Mouterde, 1966), Flora Hellenica (Christensen, 1997)
and in conifer monographs (Farjon, 2005; Debreczy &
R
acz, 2011; Adams, 2014). These differences might be
due to the measurement of limited numbers of individuals and the predominant presence of the largest cones
and needles in the herbaria by the authors of floras.
Herbarium collections additionally aim to conserve well
developed specimens, i.e. those with the biggest cones
and longest needles, whereas we sampled randomly.
Variation in the cones, drupes and needles of J. drupacea has not been verified biometrically until now and
no infraspecific taxa have been distinguished (Farjon,
2005, 2010; Auders & Spicer, 2012).
The level of variation in particular characters of
J. drupacea was comparable with that detected for
J. oxycedrus, J. deltoides R.P.Adams and J. macrocarpa (Klimko et al., 2004, 2007; Boraty
nski et al.,
2014). The dimensions of cones and drupes in J. drupacea were positively correlated, as were the dimen-
sions of needles, but no correlations or only weak ones
were detected between cone and needle characters.
This confirms the patterns of correlations in characters described for other Juniperus taxa (e.g. Klimko
et al., 2004, 2007; Marcysiak et al., 2007; Mazur
et al., 2003, 2010, 2015; Boraty
nski et al., 2013, 2014).
GEOGRAPHICAL
PATTERNS OF DIVERSITY AND
DIFFERENTIATION
Genetic diversity
The high genetic diversity appeared to be unevenly
distributed among populations and, depending on
the software used, three or six geographically isolated population clusters were detected (Fig. 1B, C).
The permutation test did not reveal significant differences between HE from Greek, Taurus, AntiTaurus and Lebanese Mountain populations. However,
a trend of increasing mean HE values for the Peloponnese, Taurus and Lebanon Mountains (HEGR = 0.612,
HETT + TAT = 0.687, and HELEB = 0.704, respectively)
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
1.5–4.0 (2.6)
6.4–25.4 (13.3)
6.9–20.9 (13.6)
8.6–20.4 (16.2)
12.5–28.5 (20.2)
15
8.8–15.5 (11.9)
8.9–22.2 (13.4)
9.5–25.6 (18.7)
10.1–33.4 (21.2)
3–4
2.8–3.5 ( 4)
3–5
2–4
1.3–3.8 (2.4)
Adams, 2014;
Farjon, 2005;
Coode & Cullen, 1965;
Christensen, 1997;
Mouterde, 1966;
Debreczy & R
acz, 2011
Our data for
Europe – range and
(average) values
Our data for Asia – range
and (average) values
4 mm
10–25 mm
(4 ) 10–20 ( 24)mm
11–23
11–21
12–15
15–25
5.3–21.5 (12.7)
7–15 mm
10–18 mm
20–25 mm
12–24 mm
20–25
(12 ) 13–21 ( 25)
25
15–25 ˂ 30
8.9–23.1 (16.5)
15–30 mm
Data source
NW
NL
DD
DL
CD
CL
Character
Table 6. Values of cone and needle characters in Juniperus drupacea known from literature compared to data found in the present study for Europe and Asia
(bold); character acronyms as in Table 2
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
379
was observed. The lower HE values in Europe than
in Asia confirm our hypothesis regarding the lower
genetic diversity in isolated populations of the species and is congruent with the central-marginal
hypothesis that proposes generally lower levels of
diversity in marginal populations (Hamrick, Godt &
Sherman-Broyles, 1992; Eckert, Samis & Lougheed,
2008). This is also confirmed by the lowest level of
AR, which was detected in the small westernmost
population of J. drupacea in the Taygetos of the
Peloponnese (GRT). The highest value of AR was
found in the isolated and small south-easternmost
Lebanese population (LB_1).
Our result confirm the general trend of decreasing
genetic diversity noted from the eastern to western
Mediterranean regions (Fady-Welterlen, 2005; Fady
& Conord, 2010; Conord et al., 2012). This geographical pattern of plant diversity distribution has been
explained by a higher aridity of the western Mediterranean during Pleistocene cold periods than in the
eastern part of the region (van Andel & Tzedakis,
1996; Fady & Conord, 2010). The trend of decreasing
within-population diversity from the east to west
might also be indicative of the eastern origin of
J. drupacea and its westward migration to its current distribution, as suggested for several other
Mediterranean conifers (Fady-Welterlen, 2005: fig. 6;
Fady & Conord, 2010), with a loss of genetic diversity due to repeated bottleneck events and/or founder
effects during colonization.
The highest level of diversity, found in the southeasternmost populations of J. drupacea in the Lebanon Mountains, did not confirm the tendency for a
decreased genetic diversity in marginal populations
(Hamrick et al., 1992) that has been detected in several species (e.g. Eckert et al., 2008). Generally, the
high level of diversity found in Lebanese populations
of J. drupacea can be interpreted as long-lasting
persistence in the Lebanon Mountains and possible
enhancement resulting from species migration and
gene flow from the more northerly populations
during Pleistocene cold periods. The ecological characteristics of J. drupacea, including a high requirement for light, and its moderate tolerance to
drought, have caused the fragmentation of its current geographical range into populations that occupy
spatially isolated mountain chains. However, during
Pleistocene glacial periods, the species occurred at
lower elevations in the mountains (Hewitt, 1996,
2004) and, consequently, a broader area. Additionally, the species might have replaced other more
demanding trees during Holocene dry periods and
preceding interglacial periods and attained a
broader area of distribution, as has been reported
for Juniperus spp. in the Iberian Peninsula
(Uzquiano & Arnaz, 1997; Carri
on et al., 2001;
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
380
K. SOBIERAJSKA ET AL.
Gonz
alez-Samperiz et al., 2010). Following this rule,
populations from the Anti-Taurus in Anatolia might
have been in contact with Lebanese populations via
the Amanos and Syrian Mountains, which consequently increased their genetic diversity (Bou
Dagher-Kharrat et al., 2007; Douaihy et al., 2011).
Admixture analysis conducted with STRUCTURE
indicates the wide sharing of gene pools among populations from currently isolated parts of the range
(Fig. 1C). Furthermore, gene flow from the Taurus
into the Lebanese populations explains the high
level of diversity of some populations of J. excelsa in
the Lebanese populations (Douaihy et al., 2011).
However, A. cilicica, another tree that co-occurs
with J. drupacea, has a relatively low level of
genetic diversity in Lebanese populations (Awad
et al., 2014), lower than that in the Taurus Mountains. This was explained by the sensitivity of
A. cilicica to Pleistocene and Holocene drought episodes and human impact (Sez kiewicz et al., 2015).
Differentiation
The level of genetic differentiation among populations of J. drupacea, (FST = 0.101, P < 0.001) is relatively high compared with that in forest tree
populations from the temperate zone, but is similar
to the values reported for other conifer species from
the Mediterranean (Fady-Welterlen, 2005; Petit,
Hampe & Cheddadi, 2005). We used two different
clustering algorithms and obtained different results,
which highlight the different sensitivities of the
methods. The BAPS algorithm was more powerful
than STRUCTURE in the detection of a subtle geographically induced pattern of differentiation and
allowed more clusters to be detected and, more
importantly, the obtained pattern corresponded well
with the geographical locations of the populations. In
contrast, STRUCTURE indicated less structuring as
the optimal resolution, but a higher genetic admixture. The reason why STRUCTURE detected weaker
structuring might be the low number of loci used;
however, the additional structuring with K = 6
detected with STRUCTURE supports the BAPS
results. Considering this, we will further focus
mainly on the structuring results obtained with both
BAPS and STRUCTURE.
The geographical, genetic and morphological differentiation of J. drupacea shows a significant separation of Greek populations from all others and the
sub-clustering of the populations from Anatolia and
Lebanon (Figs 1–4). The differences between the
European and Asian populations of J. drupacea
might result from: (1) the different origin of these
two sets of populations and the lack of gene flow
between them over a long period; and (2) the different evolutionary trajectories of European and Asian
populations after their separation by the Aegean
Sea. Similar arguments might explain the genetic
and phenotypic differences among the three Lebanese and all the other Asian populations and also
among populations from different parts of the Taurus Mountains (Fig. 1). However, the admixture
found in the populations in the Lebanon Mountains
might indicate an accidental gene flow between them
and populations from the different parts of the Taurus Mountains (see above). The possible recent origin
of some Lebanese populations should also be taken
into consideration, as has been suggested for Cedrus
libani (Bou Dagher-Kharrat et al., 2007; Fady et al.,
2008) and J. excelsa (Douaihy et al., 2011), trees that
co-occur with J. drupacea (Browicz, 1982).
The time of the divergence of J. drupacea, estimated to be the Eocene/Oligocene transition (prior to
c. 30–35 Mya; Mao et al., 2010), is associated with
the first Antarctic glaciations and consequently, with
the cooling of the subtropical climate (Meulenkamp
& Sissingh, 2003; Barr
on et al., 2010). These processes influenced the development of the Mediterranean biome with its sclerophyll vegetation
(Axelrod, 1975; Palamarev, 1989; Thompson, 2005).
Juniperus drupacea-pliocenica Rer., a hypothetical
ancestor of J. drupacea, evolved on the plate of the
European continent (Palamarev, 1989; Kvacek,
2002). However, palaeo-remnants of this taxon and
of Juniperus in general (plants associated with dry
and/or semi-dry environments) are extremely rare
and are only fragmentarily accessible (Palamarev,
1989; Stockey et al., 2005). Nevertheless, the colonization of terrains of the contemporary Peloponnese,
Anatolia and the north-western part of the Arabian
plateau by the ancestor of J. drupacea might have
occurred as early as the early Oligocene, when these
regions experienced a temporary connection (R€
ogl,
1999; Meulenkamp & Sissingh, 2003; Popov et al.,
2006). Subsequently, the rotation of the African and
Arabian plate that led to collision with the Anatolian
plate in the early Miocene (Burdigalian, 20.44–
15.97 Mya) created a solid land connection between
these regions (R€
ogl, 1999) and might have supported
species migrations.
The separation of the Balkan plate from the Anatolian/Asiatic plate occurred during the late Miocene.
Since the Tortonian (11.60–27.24 Mya; R€
ogl, 1999),
the Aegean Sea began to form and act as a biogeographic barrier in a similar way to the Strait of
Gibraltar in the western Mediterranean basin, thus
triggering the divergence of European and Asian
taxa. Finally, the Messinian salinity crisis, which
had a profound influence on biogeographic patterns
occurring throughout the Mediterranean region,
caused further differentiation among populations.
During the late Pliocene the climate became drier
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
BIOGEOGRAPHY OF JUNIPERUS DRUPACEA
(Thompson, 2005) and rapid climatic fluctuations
during the Pliocene probably limited the range of the
species. An abrupt shift from a fairly humid climate
to dry conditions forced elevational migration of
many plants species, probably including J. drupacea.
The mountains became a shelter for many plants,
but also induced spatial isolation among populations
located on different mountain ranges, resulting in
subsequent genetic divergence (Thompson, 2005).
The major driver of vegetation development in the
Mediterranean was, and still is, water availability. In
general, the last glacial cycle made the Mediterranean climate cooler, but mostly drier (Thompson,
2005). The climatic changes induced by the Pleistocene glacial/interglacial cycles are probably the
most relevant for the present pattern of genetic differentiation in J. drupacea, because they are the
most recent and were dynamic in nature. At the end
of the last glacial cycle, the Younger Dryas period of
dryness in the eastern Mediterranean (Dean et al.,
2015) might have been a factor that limited species
distributions. The onset of the Holocene caused a relatively short period of humidity in the eastern
Mediterranean, after which three dry phases
occurred, each ending in a drier climate (Roberts
et al., 2011). Detailed palaeo-climate reconstructions
indicate that each phase was dynamic, with several
episodes of dryness interspersed with centuries of relatively wetter conditions (Roberts et al., 2011). Plants
had to respond to such abruptly changing conditions
by subsequent contractions and expansions of the
geographical ranges. Demographic processes such as
population expansion and extinction were linked to
these shifts of the geographical ranges and had a
direct influence on the genetic variability and differentiation of the populations. For J. drupacea, climatic fluctuations probably resulted in several
episodes of elevational migration. The limitations due
to competition with more moisture-demanding trees
(Uzquiano & Arnaz, 1997; Carrion, 2002; Eastwood,
2004) during wetter periods and increased isolation,
whereas the dry periods allowed for gene exchange
among previously isolated populations located on
neighbouring mountain ranges. The final aridification
process of the eastern Mediterranean climate started
in the Holocene c. 4.2–4.0 ka BP (Dean et al., 2015)
and this climatic transition had a great impact on the
distribution of the species and the directions of demographic-genetic processes, mostly by enhancing spatial and, thus, genetic isolation. Finally, the human
impact on the forests and the reduction of A. cilicica
and C. libani during the last millennia (Talhouk
et al., 2001; Douaihy et al., 2011; Awad et al., 2014)
might have allowed the expansion of J. drupacea.
The study of genetic divergence in Taxus baccata
L. indicated that apart from the influence of neutral
381
evolutionary forces induced by the geographical isolation during each glacial cycle, genetic divergence
might have been caused by selective pressures and
adaptations associated with different local or regional environments (Mayol et al., 2015). The great
heterogeneity of Mediterranean environmental conditions that can change rapidly on a small geographical scale is frequently invoked as one of the basic
explanations for the high diversity and endemism in
the Mediterranean (Thompson, 2005). Strong regional contrasts in precipitation and temperature
regimes might have been an important selective factor for divergence among populations of J. drupacea
from the Peloponnese and high mountain areas of
the Taurus and Lebanese Mountains characterized
by more severe climatic conditions.
CONCLUSIONS
Despite the notable fragmentation of the geographical range and spatial isolation among different populations, J. drupacea has retained a high level of
genetic diversity, which is typical of several Mediterranean and most eastern Mediterranean conifers.
European populations maintained a lower level of
diversity than Asian populations.
The genetic and morphological patterns of the geographical differentiation of J. drupacea are similar
and highlight the different genetic and phenotypic
characters of European vs. Asian populations. In
Europe, the population from the Taygetos is genetically different from that from the Parnon Mountain.
Asian populations were differentiated at a higher
level and formed two groups with respect to phenotypic data and four with respect to genetic data. The
phenotypic characters did not discriminate between
Taurus and Anti-Taurus populations.
The patterns of genetic and phenotypic differentiation in J. drupacea detected in the Asian part of its
geographical range resemble patterns of differentiation described for other tree species occurring in the
Taurus, Anti-Taurus and Lebanon Mountains. This
might indicate a similar history of migrations and a
similar reaction of oro-Mediterranean conifer taxa to
palaeo-environmental influences and human impact.
ACKNOWLEDGEMENTS
The research was financially supported partly by the
Polish Ministry of Science (under grant no. NN303
153037) and partly by the Institute of Dendrology
(Poland) (under statutory activity). We would like to
express our best thanks to Małgorzata Łuczak for
her excellent laboratory support.
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385
382
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Appendix S1. Genetic structure of 12 populations of J. drupacea and membership of individuals for K = 6, as
detected in admixture analysis conducted with STRUCTURE software; sample codes as in Table 1.
© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 365–385