The biogeography and genetic relationships ofJuniperus oxycedrus and related taxa from theMediterranean and Macaronesian regions

ADAM BORATYNSKI1, WITOLD WACHOWIAK1, MONIKA DERING1*,KRYSTYNA BORATYNSKA1, KATARZYNA SEKIEWICZ1, KAROLINA SOBIERAJSKA1,ANNA K. JASINSKA1, MAŁGORZATA KLIMKO2, JOSE MARIA MONTSERRAT3,ANGÉL ROMO4, TOLGA OK5 and YAKIV DIDUKH6

1Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Kórnik, Poland2Department of Botany, August Cieszkowski Agricultural University, Wojska Polskiego 71 C, 60-625Poznan, Poland3Institut de Cultura de Barcelona, Jardín Botánic de Barcelona, C/ Font i Quer 2, 08038 Barcelona,Spain4Consejo Superior de Investigaciones Cientificas, Institute of Botany, Passeig del Migdia s/n., 08038Barcelona, Spain5KSU Faculty of Forestry, Department of Silviculture, 46060 Karamanmaras, Turkey6Institute of Botany of National Academy of Sciences of Ukraine, Tereszczeknivska 2, Kyiv, Ukraine

Received 28 November 2012; revised 10 September 2013; accepted for publication 27 December 2013

Despite Juniperus spp. being an important component of Mediterranean arid and semi-arid ecosystems, there isa lack of complex studies on their biogeographical patterns. Using 16 morphological cone and seed traits and threenuclear microsatellite markers, we investigated the morphological and genetic variability of seven Mediterraneanand Macaronesian Juniperus taxa (J. oxycedrus ssp. oxycedrus, J. oxycedrus ssp. badia, J. brevifolia, J. cedrus,J. deltoides, J. macrocarpa and J. navicularis) to identify biogeographical trends and interspecific genetic rela-tionships. The highest gene diversity was measured in J. oxycedrus ssp. oxycedrus (HE = 0.716) and the lowest inJ. brevifolia (HE = 0.441). The west Mediterranean was characterized by a higher level of genetic diversity than theeast Mediterranean. A lack of significant genetic differences between European and African populations ofJ. oxycedrus suggests that the Strait of Gibraltar was not a significant barrier to gene flow, but has promoted somemorphological differentiation. The genetic and morphological results strongly support the recognition of J. mac-rocarpa, J. navicularis and J. deltoides at the species rank, whereas J. oxycedrus ssp. badia should be included inJ. oxycedrus. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174,637–653.

ADDITIONAL KEYWORDS: Bayesian clustering – genetic diversity – microsatellites – morphologicalvariation – strait of Gibraltar – systematics.

INTRODUCTION

The natural history of the Mediterranean region hasbeen marked by great climatic and geological changesrelated to the northward movement of the Africantectonic plate and its final collision with the Iberianmicroplate (Rosenbaum, Lister & Duboz, 2002). These

changes have had far-reaching consequences for theevolution of the Mediterranean flora (Rodríguez-Sánchez et al., 2008; Linares, 2011). The Messiniansalinity crisis, during which marked climatic alterna-tions occurred and transcontinental land bridgesappeared between Europe and Africa, was an impor-tant driving factor for modern biogeographical pat-terns that can be observed in the MediterraneanBasin (Krijgsman et al., 1999). The opening of the*Corresponding author. E-mail: mdering@man.poznan.pl

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Botanical Journal of the Linnean Society, 2014, 174, 637–653. With 5 figures

© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 637–653 637

Strait of Gibraltar in the early Pliocene period isbelieved to have formed a significant geographicalbarrier that prevented, or at least limited, gene flowbetween the biota from Europe and Africa(Jaramillo-Correa et al., 2010). During the Pleisto-cene, the Mediterranean region served as a refugiumfor the northern European flora. In biogeographicaland evolutionary terms, however, the Mediterraneanflora experienced great changes during this time(Benito Garzón, Sánchez de Dios & Sáinz-Ollero,2007; Médail & Diadema, 2009).

Miocene, Pliocene and Pleistocene events had sig-nificant effects on the current distribution and popu-lation genetic structure of many Mediterraneanplants, including Juniperus L. (junipers) (Thompson,2005; Mao et al., 2010). Junipers are keystone speciesof Mediterranean arid and semi-arid ecosystems, andare essential components of the high biodiversity andendemism noted in this part of the world (Greuter,Burdet & Lang, 1984; Farjon, 2010; Adams, 2011).However, the current rate of human-induced environ-mental alterations and concomitant climate changesmay threaten the long-term persistence of somejuniper species in the near future (Castro et al., 2011;Douaihy et al., 2011; Juan et al., 2012). This situationexemplifies the general concerns about the future ofthe entire Mediterranean biome, which is assumed tobe highly vulnerable to global environmental changes(Petit, Hampe & Cheddadi, 2005).

Juniperus oxycedrus L. (prickly juniper) and relatedtaxa of Juniperus section Juniperus, characterized byreddish-brown cones and two bands of stomata on theadaxial side of the needle, are distributed across theMediterranean Basin and Macaronesia (Farjon, 2010;Mao et al., 2010). In the western Mediterranean,J. oxycedrus ssp. oxycedrus L. and J. oxycedrus ssp.badia (H.Gay) Debeaux occur across the Iberian Pen-insula and northern Africa, and J. navicularis Gand. isfound along the Portuguese Atlantic coast (Farjon,2010; Adams, 2011). Juniperus macrocarpa Sibth. &Sm. is dispersed along the Mediterranean and Atlanticcoasts of the southern Iberian Peninsula. Juniperusdeltoides R.P.Adams covers the eastern MediterraneanBasin (Boratynski, Browicz & Zielinski, 1992; Farjon,2010; Adams, 2011). The Macaronesian J. brevifolia(Seub.) Antoine occurs on the Azores, and J. cedrusWebb & Berthel is found on the Canary Islands(Greuter et al., 1984; Farjon, 2010; Adams, 2011).Recently, J. maderensis (Menezes) R.P.Adams hasbeen described as growing in Madeira (Adams,2011).

The systematic position of taxa in the J. oxycedrusgroup includes J. cedrus and J. brevifolia at thespecies rank (Farjon, 2010; Adams, 2011), but thestatus of the other taxa has been debated. Adams(2011) mentions three taxa at the species rank,

J. navicularis, J. macrocarpa and J. deltoides, withthe latter being a cryptic species within J. oxycedrus(Adams, 2004; Adams et al., 2005). In addition,Adams (2004) and Adams et al. (2005) recognized twosubspecies in J. oxycedrus: J. oxycedrus ssp. oxyce-drus and J. oxycedrus ssp. badia. However, Farjon(2010) recognized the first two species at the subspe-cies rank, J. oxycedrus ssp. transtagana Franco andJ. oxycedrus ssp. macrocarpa (Sibth. & Sm.) Ball.,respectively, and included J. deltoides in J. oxycedrus.As a result of the lack of taxonomic consensus, weadopted the classification used by Adams (2011).

Phylogenetic investigation of Juniperus sectionJuniperus based on nuclear internal transcribedspacer (ITS) and five plastid DNA regions (Adams &Schwarzbach, 2012) resulted in the grouping of dif-ferent subspecies of J. oxycedrus and allied speciesoccurring in the Mediterranean region with Macaro-nesian juniper species. Juniperus oxycedrus ssp.oxycedrus, J. oxycedrus ssp. badia and J. macrocarpawere clustered with two Macaronesian species,J. cedrus and J. maderensis. Juniperus deltoidesand J. navicularis appeared to be closely related toJ. brevifolia from the Azores. Rumeu et al. (2011) alsoindicated a close phylogenetic relationship betweenthese species using the trnL intron and trnL-trnFregion. The probable divergence time between J. del-toides and a clade containing J. brevifolia andJ. navicularis was estimated at the upper Miocene–lower Pliocene (17.03–5.07 Ma) (Rumeu et al., 2011).

Although the phylogenetic relationships betweenJ. oxycedrus agg. and related taxa have been investi-gated extensively using different markers (Adamset al., 2005; Adams, 2008; Rumeu et al., 2011; Adams& Schwarzbach, 2012), comprehensive studies of thegeographical distribution of genetic diversity in thisimportant group are still lacking. Therefore, the maingoal of this study was to assess the occurrence ofgenetic and morphological variability in J. oxycedrusand related taxa, and to define existing geographicaltrends in the spatial organization of morphologicaland genetic variability. In view of the complex naturalhistory of the Mediterranean Basin, we hypothesizedthe following biogeographical patterns: (1) significantgenetic differentiation between populations from theeastern and western Mediterranean because of theirlong isolation; (2) the causative role of the Strait ofGibraltar in the pattern of genetic differentiation inJ. oxycedrus ssp. oxycedrus and J. oxycedrus ssp.badia; and (3) significant genetic differentiationbetween populations from the Atlantic islands andcoastal Atlantic populations versus Mediterraneanpopulations. In addition, by integrating genetic andmorphometric investigations, we attempted to shedmore light on the interspecific relationships in theprickly juniper group by identifying clusters of indi-

638 A. BORATYNSKI ET AL.

© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 637–653

viduals belonging to different gene pools that mayhave taxonomic implications.

MATERIAL AND METHODSPLANT MATERIAL

We sampled 41 natural populations of J. oxycedrusssp. oxycedrus, J. oxycedrus ssp. badia, J. deltoides,J. macrocarpa, J. navicularis, J. cedrus (includingone planted population) and J. brevifolia (Fig. 1).Eleven of the populations of J. oxycedrus, J. deltoidesand J. macrocarpa had been used in previous mor-phological studies (Klimko et al., 2004, 2007). Plantmaterial (i.e. cones and fragments of branchlets) wascollected from southerly exposed parts of the crownsat a height of 1–3 m from 12–30 randomly distributedindividuals for every population (Table 1). In total,1100 female individuals were sampled. In addition,one population of J. communis L. from Turkey (17individuals), a representative of the boreal species ofsection Juniperus, was included for genetic and mor-phological comparisons (Mao et al., 2010). Voucherswere deposited in the herbarium of the Institute ofDendrology (KOR; Table 1).

DNA ISOLATION AND

MICROSATELLITE AMPLIFICATION

Total DNA was extracted from dry needles with aDNeasy Plant Mini Kit (Qiagen, Hilden, Germany).Of the eight microsatellite (simple sequence repeat,SSR) primer pairs tested (Zhang et al., 2005, 2008;Michalczyk et al., 2006), three loci that were suc-cessfully amplified from all investigated taxa(including Jc035, Jc037 and Jc032) were used forfurther investigation. Loci Jc035 and Jc037 wereamplified with a Multiplex PCR Kit (Qiagen) accord-ing to the manufacturer’s instructions. The polymer-ase chain reaction (PCR) thermal protocol startedwith a denaturation at 94 °C for 15 min, followed by34 cycles of denaturation at 94 °C for 30 s, anneal-ing at 57 °C for 90 s and extension at 72 °C for 90 s,and a final extension for 10 min at 72 °C. Amplifi-cation of Jc032 was performed in a final volume of10 μL that consisted of 20 ng of genomic DNA, 2 mM

MgCl2, 0.5 mg mL−1 bovine serum albumin (BSA),200 μM deoxynucleoside triphosphates (dNTPs),20 pmol of each primer, 0.05 U of VIVATaq Polymer-ase and 1 × reaction buffer (NOVAZYM, Poznan,Poland). After an initial denaturation at 94 °C for15 min, the reactions underwent ten touchdowncycles with denaturation at 94 °C for 30 s, annealingat 60 °C for 30 s (with a 1 °C reduction in tempera-ture for each cycle) and extension at 72 °C for 40 s.The second phase of PCR consisted of 25 cycles withdenaturation at 94 °C for 30 s, annealing at 50 °C

for 50 s and extension at 72 °C for 40 s, and a finalextension for 7 min at 72 °C.

The fluorescently labelled amplification productswere pooled and analysed in a 3130 Genetic Analyzer(Applied Biosystems), with internal size standardGeneScan – LIZ 500. Genotypes were scored withGeneMapper v.4.0 (Applied Biosystems).

GENETIC DATA ANALYSIS

Linkage disequilibrium for all pairs of SSR loci ineach population was tested in FSTAT 2.9.3 (Goudet,2001). The expected (HE) and observed (HO) heterozy-gosity (Nei, 1978), inbreeding coefficient (FIS) and nullallele frequencies (Chapuis & Estoup, 2007) wereestimated with INEST 1.0 software (Chybicki &Burczyk, 2009). As a result of differences in samplesize, the allelic richness (AR), rather than the averagenumber of alleles, was calculated in FSTAT. The sig-nificance of deviation from Hardy–Weinberg equilib-rium (HWE) was tested with GENEPOP 4.1 software(Raymond & Rousset, 1995). Global differentiationwas measured with the analysis of molecular variance(AMOVA)-derived FST value, and tested with 3000permutations in ARLEQUIN 3.11 (Excoffier, Laval &Schneider, 2005).

The correlation of geographical distances with thegenetic distances of Cavalli-Sforza & Edwards (1967)with INA correction (including null alleles correction)was assessed with a Mantel test (Mantel, 1967) forJ. deltoides, J. macrocarpa and J. oxycedrus ssp.oxycedrus, and tested with 1000 random permuta-tions in PopTools v. 3.2.3 (Hood, 2010). In addition,linear regression for genetic diversity parameters (HE

and AR) with respect to latitude and longitude wasapplied to trace the geographical trends in the spatialdistribution of genetic variation across all juniperspecies in the Mediterranean in STATISTICA v.9.

To infer the genetic relationships between thestudied populations, a dendrogram based on thegenetic distances of Cavalli-Sforza & Edwards (1967),generated in FREENA with the null allele correction(INA correction, Chapuis & Estoup, 2007), was con-structed with neighbor joining (NJ) using MEGA v.4.0software (Tamura et al., 2007).

To examine the phylogeographical and/or taxo-nomic grouping of the samples, genetic clustering ofthe populations was performed with model-basedBayesian assignment as implemented in BAPS 5.4software (Corander & Tang, 2007). In BAPS, a sto-chastic optimization algorithm is implemented toevaluate the clustering solutions using the analyti-cally calculated marginal likelihoods (Coranderet al., 2008). Clustering of groups of individualswas run in BAPS for ten replicates for K = 1–43.The number of populations was inferred as the com-

BIOGEOGRAPHY OF JUNIPERUS OXYCEDRUS 639

© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 637–653

Fig

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640 A. BORATYNSKI ET AL.

© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 637–653

Tab

le1.

Loc

atio

nan

dpa

ram

eter

sof

gen

etic

dive

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anal

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popu

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BIOGEOGRAPHY OF JUNIPERUS OXYCEDRUS 641

© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 637–653

Tab

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bined maximum likelihood and highest posteriorprobability estimates over all ten replicates. Theadmixture analysis was conducted with a fixednumber of clusters inferred with the previouscluster analysis.

To infer the partitioning of the total genetic varia-tion among geographical regions, we followed theapproach of Fady & Conord (2010). An AMOVA wasperformed for eastern vs. western Mediterranean,Iberian Peninsula vs. Africa, and Atlantic Islands andcoast vs. Mediterranean, using ARLEQUIN. AnAMOVA at the intraspecific level was performed forJ. deltoides (Europe vs. Asia), J. macrocarpa (Iberianvs. Apennine vs. the eastern Mediterranean) andJ. oxycedrus ssp. oxycedrus (Europe vs. Africa). A com-parison of genetic diversity parameters between theseregions (AR, HE, HO, FIS) was made in FSTAT 2.9.3(Goudet, 2001).

MORPHOLOGICAL ANALYSIS

Eleven morphological direct characters and five ratioswere examined in c. 30 individuals in every popula-tion: CL, cone length (mm); CD, cone diameter (mm);SN, seed number; SL, seed length (mm); SW, seedwidth (mm); STH, seed thickness (mm); NL, needlelength (mm); NWM, needle maximal width (mm);DNWM, distance of NWM from the needle base (mm);STBW, width of the right stomatal band (looking fromthe base to the apex of the needle) in the central partof the needle; STN, number of stomata on 1 mm2 ofthe central part of the needle; CSH = CL/CD; CD/SN;SL/SW; NSH1 = NL/NWM; NSH2 = DNWM/NL × 100(%) (Supporting Information Table S1). Ten cones andten seeds per individual were measured following theprocedures described by Klimko et al. (2004, 2007)and Marcysiak et al. (2007). Ten needles per indi-vidual were scanned and measured automaticallyusing WinNEEDLE software (Regent Incorp.). STBWand STN were measured for three randomly selectedneedles on scanning electron micrographs (SEM,Hitachi S3000 N).

Shapiro–Wilk’s W-test was used to verify the nor-mality of the frequency distribution of the characters.The homogeneity of variance was verified using theBrown–Forsythe test (Stanisz, 2007). Pearson’s r cor-relations between pairs of characters were examinedto eliminate the most redundant traits. Tukey’s hon-estly significant difference (HSD) post hoc test wasperformed to find statistically significant differencesbetween taxa. Agglomerated grouping with Euclideanand Mahalanobis distances according to Ward wasused to explore the relationships between populationswithin taxa and between taxa. The discriminantanalysis was used for the estimation of the discrimi-nation power of the particular traits investigated

(Tabachnik & Fidell, 1996; Sokal & Rohlf, 2003).Intra- and interspecific relationships were assessedon the scatter plot of the discrimination function onthe space between the first discrimination variables(forward stepwise analysis). K-means clustering ofpopulations was conducted to determine the numberof groups K that best explained the morphologicalvariation. The hierarchical analysis of variance(ANOVA) was used to evaluate the distribution of thevariation of each character among the studied taxa.STATISTICA v.9 (StatSoft, Kraków, Poland) and JMP(SAS Institute Inc.) were used to conduct the statis-tical procedures.

RESULTSGENETIC ANALYSIS

No evidence for linkage disequilibrium was detectedbetween pairs of SSR loci in each of the populations.Nineteen, 46 and 23 different alleles were found atloci Jc035, Jc037 and Jc032, respectively. Estimates ofgenetic diversity are shown in Table 1. The highestaverage HE was detected in J. oxycedrus ssp. oxyce-drus (0.716) and the lowest in J. brevifolia (0.441).For the latter species, the lowest average HO was alsofound (0.295), whereas the highest average HO wasreported for J. navicularis (0.539). Allelic richness(AR) was highest in the J. deltoides populations fromBosnia and Herzegovina (OD_BH_01), and the lowestin one of the populations of J. navicularis (N_PT_01).The deviations from HWE primarily resulted from adeficiency in heterozygotes, as indicated by the valuesof the inbreeding coefficient (FIS). These deviationswere statistically significant in most cases. However,the heterozygosity and FIS estimates were most prob-ably influenced by the null alleles that were detectedin most of the populations. In certain cases, thedeviations from HWE reflected probable matingbetween relatives because null alleles were notdetected and considerable values of FIS were noted. Alarge excess of homozygotes (FIS = 0.540, P < 0.001)was noted in the planted population of J. cedrus. Theoverall genetic differentiation between the studiedpopulations was high (FST = 0.16) and significant(P < 0.001).

The Mantel test detected positive and significantcorrelations between geographical distances andgenetic distances for J. deltoides, J. macrocarpa andJ. oxycedrus ssp. oxycedrus, suggesting an importantrole for spatial isolation in shaping the interpopula-tion genetic relationships (r = 0.631, P = 0.001;r = 0.519, P = 0.002; and r = 0.401, P = 0.040, respec-tively). A statistically significant increase in diversityfrom east to west in the Mediterranean Basin wasfound for HE (R2 = 0.3, P = 0.0016), but not for AR

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(R2 = 0.09, P = 0.1). There was no significant relation-ship between the genetic diversity parameters andlatitude (HE: R2 = 0.03, P = 0.37; AR: R2 = 0.12,P = 0.06).

The NJ dendrogram (Fig. 2) showed a clear sepa-ration of the three coastal populations of J. navicula-ris from all other populations, which were groupedinto three main subclusters. The first contained all ofthe J. deltoides populations and was divided into aGreek group (with one population from Lebanon), towhich populations of Macaronesian species (J. brevi-folia and J. cedrus) were linked, and an Asiatic group,which included populations from Turkey and Israel,with an additional Ukrainian population fromCrimea. In the second subclade, a population ofJ. macrocarpa was found, which was separated intoeastern Mediterranean and western Mediterraneangroups. The third subclade was less homogeneous, asit included populations of four taxa: J. oxycedrus ssp.oxycedrus, J. oxycedrus ssp. badia, two populations ofJ. navicularis and two populations of J. macrocarpa.French populations of J. oxycedrus ssp. oxycedruswere closely linked with two Moroccan populations,and a similar relationship was observed betweenSpanish and Moroccan populations of J. oxycedrusssp. badia. Populations of J. navicularis were clus-tered with Spanish populations of J. macrocarpa.

Bayesian clustering conducted using BAPSrevealed an optimum number of 15 clusters with a logmarginal likelihood of −15 989. The placement of thestudied populations is shown in Figure 2. The follow-ing clusters were detected: Cluster 3, J. deltoidespopulations from Greece and from Lebanon; Cluster10, J. deltoides populations from Asia and Crimea;Cluster 4, populations of J. brevifolia; Cluster 7,J. macrocarpa populations from Italy; Cluster 15,J. macrocarpa populations from Turkey and Greece;Cluster 11, populations of J. oxycedrus ssp. oxycedrusand J. oxycedrus ssp. badia, with a single populationof J. deltoides from Bosnia and Herzegovina and asingle population of J. macrocarpa from Spain. Indi-vidual clusters were detected for each of the fiveJ. navicularis populations (Clusters 1, 2, 5, 9 and 14),two J. cedrus populations (Clusters 8 and 13) and oneItalian and one Spanish population of J. macrocarpa(Clusters 6 and 12, respectively). The admixtureanalysis (Fig. 3) revealed extensive genetic admixturein populations of J. oxycedrus ssp. oxycedrus andJ. oxycedrus ssp. badia, and a minor admixture insome populations of J. macrocarpa. No admixturewas detected in the populations of J. navicularis.

AMOVA indicated that, although more than 84% ofthe variation was found within populations of thestudied taxa, a significant amount (9.65%) of thevariation (P < 0.001) was attributable to the differ-ences among them. Eastern and western Mediterra-

nean populations were significantly different (2.62%of the total variance, P < 0.002), and less geneticdiversity was found in the eastern (HE = 0.544) thanin the western (HE = 0.670, P < 0.001) Mediterranean.The Strait of Gibraltar was not recognized as a sig-nificant biogeographical barrier in structuring thegenetic diversity in J. oxycedrus ssp. oxycedrus orJ. oxycedrus ssp. badia. According to AMOVA con-ducted jointly for both taxa, genetic differencesbetween the two continents explained 0.1% of thetotal variance (P = 0.15). Low, but statistically signifi-cant genetic differentiation was found between theAtlantic island and coastal populations (Portuguese)versus the Mediterranean populations (2.51% of thetotal variance, P = 0.039). The latter also showed astatistically higher AR value (P < 0.001). Regionaldifferences in the values of HO and FIS were notsignificant.

At the intraspecific level, the highest, statisticallysignificant differentiation was noted among Iberian,Apennine and eastern Mediterranean (Crete andTurkey) populations of J. macrocarpa (11.5% of thetotal variance, P < 0.001), and between European andAsiatic (Turkish, Lebanese and Israeli) populations ofJ. deltoides (6.7% of the total variance, P < 0.001).

MORPHOLOGICAL ANALYSIS

The level of intraspecific morphological variabilitydiffered among the taxa, and the highest variabilitywas detected in J. oxycedrus ssp. oxycedrus, J. oxyce-drus ssp. badia and J. macrocarpa (Table S1, Fig. 4).The results of an intraspecific discrimination analysisshowed multivariate unimodality, with only singleindividuals as outliers beyond the 95% confidenceinterval (Fig. 4). Fourteen of the 16 morphologicalcharacters examined had statistically significant dis-crimination power (P < 0.001) at an individual level.The highest discriminant powers were measured forSTN, STH and NSH2 (Wilk’s λ = 0.680, 0.781 and0.820, respectively; Table S1). Traits CD, CL, NWMand STN determined the first discriminant variable U1

(c. 55% of the total variation), which separated J. mac-rocarpa individuals (Fig. 4). The individuals of theother taxa formed a more or less consistent group, witha distribution pattern along U2 (c. 22% of the totalvariation) which was primarily determined by SL/SW,SW, STH and CD/SN. The most outstanding groupaccording to the second discriminant variable (U2)consisted of the individuals of J. navicularis (Fig. 4A).

A discrimination analysis performed on the cen-troids of the populations more precisely revealed therelationships among the studied taxa. According tothe discriminant variables U1 and U2 (70% of the totalvariation), J. macrocarpa, J. navicularis and J. brevi-folia formed independent groups (Fig. 4). The discri-

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Figure 2. Neighbor joining (NJ) tree based on the genetic distances of Cavalli-Sforza and Edwards (1967) with INAcorrection and results of BAPS analysis [Juniperus deltoides (OD – ●), J. oxycedrus ssp. oxycedrus (OO – ), J. oxycedrusssp. badia (OB – ○), J. macrocarpa (OM – ▲), J. navicularis (N – ■), J. cedrus (C – ▼), J. brevifolia (B – □) andJ. communis (CO – ◊)].

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minant variable U1, responsible for > 54% of the totalvariation, was primarily determined by CL, CD, SLand NWM, whereas the variable U2 was determinedby NSH1, NL, SL/SW and NSH2.

Tukey’s test showed that the taxa differed signifi-cantly (P < 0.01) in the majority of their morphologi-cal traits (Supporting Information Table S2). Themost markedly different species was J. macrocarpa,which differed from the other taxa in 85% of thecharacteristics on average. This species was also dis-similar to J. oxycedrus and J. brevifolia (93% in bothcases). Other significantly different species wereJ. brevifolia and J. navicularis (84% and 76% onaverage, respectively). The results of ANOVA wereconsistent with Tukey’s test and indicated significantdifferentiation between the analysed juniper taxa inall of the characters tested, except for CSH (data notshown). The highest differentiation (> 70%) was notedfor CL, CD, SL, SW, STN and CSH.

Mahalanobis pairwise distances between the popu-lations were significant (P ≤ 0.001), except for the twoGreek populations of J. deltoides and the Corsicanpopulations of J. oxycedrus ssp. oxycedrus (data notshown). A cluster analysis based on Mahalanobis dis-tances divided the studied populations into twogroups (Fig. 5). The first group included all popula-tions of J. macrocarpa, and the first bifurcation of thesecond group indicated a separation of J. navicularisand J. brevifolia. A separate cluster was also obtainedfor J. deltoides. However, the populations of thisspecies originating from different geographicalregions were intermingled, and thus no clear geo-graphical structure was found. For J. oxycedrus ssp.oxycedrus, French populations were separated fromMoroccan populations that grouped with J. oxycedrusssp. badia. Euclidean distances generally confirmedthe pattern of differentiation described above (datanot shown).

Results obtained using the K-clustering methoddivided the studied populations into five clusters thatgenerally reflected the taxonomic classification, witheach of the taxa ascribed to an individual cluster(Fig. 5). This analysis primarily confirmed the closerelationships between J. oxycedrus ssp. oxycedrus andJ. oxycedrus ssp. badia, as both taxa shared Cluster 3,which additionally contained populations of J. cedrus.

DISCUSSIONGENETIC DIVERSITY AND GEOGRAPHICAL PATTERNS

OF DIFFERENTIATION

Given the long divergence time between differentJuniperus spp. (Mao et al., 2010), the relatively lowtransferability of the nuclear SSR loci is not surpris-ing. Douaihy et al. (2011) were able to successfullycross-amplify only three loci in J. excelsa Willd. of the31 tested. Nonetheless, the three loci studied hereshowed a relatively high level of polymorphism, with atotal of 88 alleles identified. However, the low numberof loci and size homoplasy (identity in state and not bydescent) that tends to occur more frequently at theinterspecific level (Estoup et al., 1995) may potentiallyaffect the level and patterns of genetic differentiationinferred in this work. In addition, transferred micros-atellites frequently show an increased level of nullalleles, which also influences the pattern of differen-tiation (Oddou-Muratorio et al., 2009).

Nevertheless, the levels of genetic diversity anddifferentiation detected in our study were similar tothose described recently when SSR markers wereused in J. excelsa (Douaihy et al., 2011), isoenzymeswere used in J. phoenicea L. (Boratynski et al., 2009)and inter simple sequence repeat (ISSR) and randomamplification of polymorphic DNA (RAPD) were usedin J. brevifolia (Silva et al., 2011). However, the

Figure 3. Bayesian admixture proportions (Q) of individuals for a K = 16 population model. Each individual is repre-sented with a single vertical line partitioned with coloured segments corresponding to inferred clusters; the length of eachsegment is proportional to the estimated membership coefficient.

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Figure 4. Result of discriminant analysis based on the 16 morphological characters of: Juniperus deltoides (●),J. oxycedrus ssp. oxycedrus (◆), J. oxycedrus ssp. badia (○), J. macrocarpa (▲), J. navicularis (■), J. cedrus (Δ),J. brevifolia (□) and J. communis (◊); A, for individuals; ellipses around groups of individuals of particular taxa show 95%confidence intervals; B, for populations.

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Figure 5. Dendrogram constructed on Mahalanobis shortest distances after the Ward method; number of the K-group of:Juniperus deltoides (OD – ●) J. oxycedrus ssp. oxycedrus (OO – ), J. oxycedrus ssp. badia (OB – ○), J. macrocarpa (OM– ▲), J. navicularis (N – ■), J. cedrus (C – ▼), J. brevifolia (B – □) and J. communis (CO – ◊).

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genetic diversity and differentiation were higher thanthose for amplified fragment length polymorphism(AFLP) for J. thurifera L. (Terrab et al., 2008) andJ. macrocarpa (Juan et al., 2012) and RAPD forJ. phoenicea (Dzialuk et al., 2011). A planted popula-tion of J. cedrus showed relatively high genetic vari-ability (HE = 0.459), but the considerable excess ofhomozygotes, despite the lack of null alleles, indicatesthat the seeds used for establishment purposes werecollected from a limited number of mother trees.

The pattern of juniper genetic diversity found in thisstudy contradicts the results presented for gymno-sperms by Fady & Conord (2010). These authors founda decreasing east–west diversity gradient in the Medi-terranean Basin. In contrast, our study found that thewestern juniper populations were characterized by ahigher level of genetic diversity than the easternpopulations. In terms of conifers, the Pleistoceneclimate changes were the key factors modelling thedistribution of genetic diversity across the Mediterra-nean Basin (Fady & Conord, 2010). The westernMediterranean experienced hyper-arid conditionsduring the last glacial period in comparison with theeastern Mediterranean, which probably led to strongdemographic declines affecting the level of geneticdiversity (van Andel & Tzedakis, 1996; Fady & Conord,2010; Linares, 2011). In our opinion, the westwardincrease in genetic diversity in junipers may betterreflect the speciation sequence in this group. Thedivergence time between J. oxycedrus and J. deltoides,which are currently on opposite fringes of the Medi-terranean Basin, has been estimated at approximately8–10 Mya (Mao et al., 2010). This result suggests aseparate colonization of the western and eastern partsof the Mediterranean, based on the distribution oftectonic plates and the formation of the Alpids at thattime (Rosenbaum et al., 2002). Consequently, duringthe late Miocene, the ancestral taxa diverged intoJ. oxycedrus and J. deltoides (Mao et al., 2010: Fig. 5),with the former inhabiting the western part of theMediterranean. The loss of genetic diversity in theJ. deltoides populations noted here could be explainedby repeated founder effects in the eastern part of therange of the species (Petit et al., 2005) and/or a differ-ent rate of microevolutionary change in the westernand eastern populations as a result of different envi-ronmental pressures. The further differentiation ofJ. deltoides into the Balkan and Asiatic groups can beexplained either by limited gene flow caused by rangefragmentation and/or a different refugial persistenceduring the last glacial cycles.

Morphological and genetic data concordantlyrevealed that the maritime juniper, J. macrocarpa,follows the same spatial pattern for genetic diversityas J. deltoides, which may indicate that it is of westernMediterranean origin. Similar to J. deltoides, J. mac-

rocarpa probably lost genetic diversity during its east-ward migration. Our findings agree with the latestresults of Juan et al. (2012), who reported a higherlevel of plastid diversity in the south-western Iberian(Andalusian) populations of J. macrocarpa than in thesouth-eastern (Valencian and Balearic) populations. Inaddition, Andalusian populations were characterizedby high levels of genetic diversity and the presence ofprivate and rare plastid haplotypes, which make thema reservoir of the old diversity.

The populations of J. navicularis exhibited highgenetic differentiation according to the Bayesianapproach. Such differentiation has probably emergedas the result of limited gene flow, as no geneticadmixture among populations was noted. The naturalrange of this species is currently limited and highlyfragmented populations prevail (Castro et al., 2011; A.Boratynski, pers. observ.). Forest management activi-ties aimed at the reduction of catastrophic fires haveled to severe reduction of the populations (Castroet al., 2011). In addition, there are problems withrecruitment in this species because of its low germi-nation rate caused by increasing drought stressduring the summer (Castro et al., 2011). All of thesefactors probably negatively affect the effective popu-lation sizes and make them more prone to geneticstochastic processes, and could promote the drift-induced differentiation observed here.

Substantial genetic admixture and the lack of sig-nificant genetic differences between European andAfrican populations of J. oxycedrus could simply beascribed to gene flow. However, the Strait of Gibraltarhas been shown previously to act as a biogeographicalbarrier for a few coniferous species (Terrab et al.,2007; Jaramillo-Correa et al., 2010). The most ances-tral divergence induced by the Strait, at the time ofthe Messinian salinity crisis, was proposed for Abiespinsapo Boiss., J. thurifera, Pinus nigra J.F.Arnoldand P. pinaster Aiton, Taxus baccata L. and, to someextent, P. halepensis Mill. In addition, J. thurifera,which frequently co-occurs with J. oxycedrus on theIberian Peninsula and populates similar habitats,showed a high level of differentiation between Euro-pean and African populations at genetic (Terrab et al.,2008) and morphological (Romo & Boratynski, 2007;Boratynski et al., 2013) levels. In contrast, somegenetic affinities between African and Europeanpopulations have been reported in J. phoenicea(Boratynski et al., 2009), although, in this species,isolation by the Strait of Gibraltar was considered tobe the major factor of morphological differentiation(Mazur et al., 2010). Indeed, morphologically, Africanpopulations of J. oxycedrus ssp. oxycedrus were dif-ferent from European populations, which maysuggest that specific climatic factors could be involvedin morphological divergence (Fig. 5). The genetic rela-

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tionships between juniper populations occurringacross the Strait of Gibraltar could also be caused bya relatively modern colonization of Africa, but consid-ering our data, this hypothesis is purely speculative.

The explanation of the genetic relationshipsbetween Corsican and Moroccan populations ofJ. oxycedrus could be long-distance dispersal (LDD),which played an important role in the evolutionaryhistory of the whole Juniperus genus (Mao et al.,2010; Rumeu et al., 2011). LDD was used to deter-mine the genetic similarities between the Corsicanand northern Iberian populations of J. thurifera(Terrab et al., 2008), or parallel haplotype compositionof the Corsican and Algerian populations of species ofQuercus L. (Petit et al., 2002). The intricate relation-ships between the western Mediterranean stands ofJ. oxycedrus, especially Pyrenean, Corsican andMoroccan ones, may also reflect the evolutionarypattern, even after geographical fragmentation of theancestral gene pool, similar to Q. suber L. populations(Magri et al., 2004). In this case, the fragmentationwas induced by past geological events occurring in thewestern Mediterranean and related to the progressiveseparation of the southern European microterranesduring the late Oligocene (25 Ma) that are presentlyfound in Calabria, Sicily, Corsica, Sardinia, Kabylies(Algeria), Balearics, Baetic range and Rif range(Morocco) (Rosenbaum et al., 2002).

INTERSPECIFIC RELATIONSHIPS

Although our study is not strictly phylogenetic, theresults of the genetic investigations provide indirectevidence for the genetic relationships among taxaincluded in the J. oxycedrus aggregate The results wewere able to obtain with the nuclear microsatellitemarkers are generally in line with recent phyloge-netic investigations (Mao et al., 2010; Rumeu et al.,2011; Adams & Schwarzbach, 2012) and contribute tothe information on the prickly juniper group charac-terized by systematic uncertainties.

Juniperus oxycedrus ssp. badia exhibited stronggenetic ties with J. oxycedrus ssp. oxycedrus. Theseresults correspond to those of Rumeu et al. (2011) andAdams & Schwarzbach (2012). The latter authorssuggested the inclusion of J. oxycedrus ssp. badia inJ. oxycedrus ssp. oxycedrus. The strong geneticadmixture detected here between these subspeciessupports this systematic synthesis. Previous taxo-nomic positions of these taxa at the subspecies rankwere justified by morphological distinctness (i.e.pyramidal crown shape, broad needles and largecones) (Amaral Franco, 1986; Adams, 2008, 2011) andby different terpene compositions and RAPD analysis(Adams, 2011). According to discriminant analysis,J. oxycedrus ssp. badia occupies a marginal position

in the range of the morphological variability forJ. oxycedrus ssp. oxycedrus, mostly as a result ofdifferent seed characteristics (seed width, length andthickness). However, both taxa clustered togetheraccording to K-means clustering and Mahalanobisshortest distances, corresponding to the geneticresults obtained.

Juniperus macrocarpa, which was close to J. oxyce-drus in terms of terpenoid content and RAPD analysis(Adams, 2000), appeared to be genetically well sepa-rated from the latter in our study. Only two Spanishpopulations showed close genetic affinity with Portu-guese stands of J. navicularis on the dendrogram.However, these two populations may represent theancestral gene pool. The refugium for this species hasrecently been proposed to be in the south-westernpart of the Iberian Peninsula (Juan et al., 2012).According to the Bayesian results, one Spanish popu-lation of J. macrocarpa was located in Cluster 11 withthe J. oxycedrus populations (Fig. 2). Rumeu et al.(2011) discovered a close relationship between theSpanish population of J. macrocarpa and the Tuni-sian population of J. oxycedrus (Rumeu et al., 2011:Fig. 2), and Mao et al. (2010) described a close posi-tion of J. macrocarpa with J. oxycedrus. In terms ofmorphological traits, however, J. macrocarpa was themost easily distinguished from the remaining taxainvestigated in this work, as it was well separated atboth the population and individual levels (Figs 4, 5).In addition, with large, multi-seeded cones, J. macro-carpa could be recognized as conserving the mostancestral pattern of cone construction, as an evolu-tionary tendency to decreasing cone size and numberof seeds has been observed in Cupressaceae (Schulz,Jagel & Stützel, 2003). In summary, our genetic andmorphological results support the taxonomic status ofJ. macrocarpa at the species rank (Amaral Franco,1986; Boratynski et al., 1992; Adams, 2008; Adams &Schwarzbach, 2012).

The considerable genetic differences noted betweenwestern Mediterranean J. oxycedrus and easternMediterranean J. deltoides in our study agree wellwith the latest results of Adams & Schwarzbach(2012) based on ITS and five plastid DNA regions, andwith Mao et al. (2010). Hence, the recognition ofJ. deltoides at the species rank is substantiated bythe current genetic data. This taxon was recognizedas a cryptic species in J. oxycedrus (Adams et al.,2005), and in our morphological analysis was shownto be more similar to J. oxycedrus ssp. oxycedrus andJ. oxycedrus ssp. badia (Fig. 4). The close genetic rela-tionships of J. deltoides with the MacaronesianJ. brevifolia and J. cedrus were quite surprising if weconsider the current allopatric distribution of thesejunipers. It is possible that these genetic affinitiesmay stem from some ancestral polymorphism shared

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by these species, as they are also related in terms ofplastid DNA (Adams & Schwarzbach, 2012).

Morphologically and genetically, J. navicularis andthe Macaronesian species were distant from all of theremaining Mediterranean junipers. Earlier studies onterpenoid content found that J. navicularis was closelyrelated to J. communis (Adams, 2000). In the NJdendrogram, three populations of this species formedthe most outstanding cluster, and two other standswere clustered with J. macrocarpa from the Atlanticcoast of the Iberian Peninsula (Fig. 2). Morphologi-cally, the population of J. navicularis formed a homo-geneous group and tended to be related to J. brevifolia,which corresponds to the results of phylogeneticstudies (Rumeu et al., 2011; Adams & Schwarzbach,2012). In general, the significant genetic and phyloge-netic differences noted in Portuguese J. navicularissupport the systematic separation of this taxon at thespecies rank.

For J. cedrus, our results are preliminary as weinvestigated only two populations, one of which wasplanted. In previous investigations, J. cedrus wasshown to be related to J. oxycedrus and J. macrocarpa(Adams et al., 2010; Rumeu et al., 2011). We did notobtain convergence between genetic and morphologi-cal results for this species. According to the geneticanalysis, J. cedrus was related to J. brevifolia,whereas morphologically it clustered with J. oxyce-drus ssp. oxycedrus, J. oxycedrus ssp. badia andJ. navicularis. These results partly correspond toprevious phylogenetic studies (Adams et al., 2010;Rumeu et al., 2011). To describe the position ofJ. cedrus more precisely in relation to other Mediter-ranean junipers, more natural populations should beincluded in investigations and other kinds of geneticinformation should be used.

ACKNOWLEDGEMENTS

This research was financially supported by the PolishMinistry of Sciences and Higher Education (GrantNo.: NN303 1534 33) and Institute of DendrologyPAS. The plant material collection was performedthanks to cooperation between the Spanish ResearchCouncil (CSIC) and the Polish Academy of Sciences.We thank Magda Bou Dagher Kharat for the collec-tion of samples. We thank the reviewers for theirhelpful comments and suggestions.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

Table S1. Statistical descriptions of measured and discriminant power testing among taxa: J. deltoides (OD),J. oxycedrus (OO), J. oxycedrus ssp. badia (OB), J. macrocarpa (OM), J. navicularis (N), J. cedrus (C), J. brevi-folia (B), J. communis (CO); V, variation coefficient; P, P value.Table S2. Morphological characters differentiated at the level of P ≤ 0.01 (bold) or P ≤ 0.05 among comparedtaxa: J. deltoides (OD), J. oxycedrus ssp. oxycedrus (OO), J. oxycedrus ssp. badia (OB), J. macrocarpa (OM),J. navicularis (N), J. cedrus (C), J. brevifolia (B), J. communis (CO); acronyms as in Table 1 (Tukey’s test for 16characters).

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