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Environmental and Experimental Botany 59 (2007) 354–360

Genome size stability among five subspecies of Pinus nigra Arnold s.l.

Faruk Bogunic a,b,∗, Edina Muratovic b, Dalibor Ballian a,Sonja Siljak-Yakovlev b,c, Spencer Brown d

a Faculty of Forestry, Zagrebacka 20, University of Sarajevo, Sarajevo 71 000, Bosnia and Herzegovinab Laboratory for Research and Protection of Endemic Resources, Faculty of Sciences, Department of Biology, Zmaja od Bosne 35,

University of Sarajevo, 71 000 Sarajevo, Bosnia and Herzegovinac Ecologie, Systematique, Evolution, Universite de Paris-Sud, Batiment 360, UPRESA 8079, 91405 Orsay, France

d Institut des Sciences du Vegetal, CNRS UPR 2355, 91198 Gif-sur-Yvette, France

Received 7 October 2005; received in revised form 28 December 2005; accepted 22 April 2006

bstract

Despite the fact that genome size should be constant at species level, many reports of intraspecific variations exist. Thus, we carried out an analysiso determine the possible existence of nuclear DNA content variation in European black pine (Pinus nigra s.l.), a good model for such a study givents karyological uniformity, morphological polymorphism, broad geographical distribution, ecological plasticity and taxonomic heterogeneity. Theanel comprised 20 populations across the natural range of P. nigra from Europe, Northwest Africa and Asia Minor including five subspecies:ubsp. nigra, salzmanni, dalmatica, pallasiana and mauretanica. Mean 1C DNA content of the species was 23.62 pg (±0.209) assessed by flowytometry. This converts to 23.1 G base pairs. The coefficients of variation within and between populations did not exceed 2.6%. Although wead already reported the existence of significant differences for three Black pine populations in our previous work on five Pinus spp. [Bogunic, F.,uratovic, E., Brown, S.C., Siljak-Yakovlev, S., 2003. Genome size of five Pinus from Balkan region. Plant Cell Rep. 22, 59–63], intraspecific

ariation was not confirmed in the present study dealing with many more populations. Subspecific divisions of Black pine were characterised with

ollowing mean values: subsp. pallasiana—23.80 pg, dalmatica—23.79 pg, nigra—23.65 pg, salzmanni—23.55 pg, and mauretanica—23.24 pg.

positive relationship between genome size and longitude was observed (r = 0.44, p < 0.05). We conclude that the diversification of populationsf P. nigra has occurred without significant genome size changes throughout its wide geographical range from ecologically contrasting habitats. Alinal mode of genome size variation is present, in line with hypothesis of P. nigra spreading from south-western Asia towards European habitats.

2006 Elsevier B.V. All rights reserved.

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eywords: Genome size; Pinus; Clinal variation; Dry seeds; Haploid megagam

. Introduction

Genome size has been extensively investigated in the genusinus L. during the last decade (Ohri and Khoshoo, 1986;akamyia et al., 1993; Valkonen et al., 1994; O’Brien et al.,

996; Wakamyia et al., 1996; Murray, 1998; Hall et al., 2000;oyner et al., 2001; Bogunic et al., 2003; Grotkopp et al., 2004).nterspecific relationships based on genome size and biologi-al traits have been found for almost 75% pine species of theenus and the results are congruent with molecular phylogenetic

ata (Grotkopp et al., 2004). Despite the ancient life history ofhe genus, evolutionary processes have exclusively proceededhrough homoploid mechanisms (Wang, 1992). Therefore, kary-

∗ Corresponding author. Tel.: +387 33 614 003; fax: +387 33 611 349.E-mail address: faruk bogunic@yahoo.com (F. Bogunic).

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098-8472/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2006.04.006

yte; Nuclear DNA content

logical uniformity (2n = 24), characteristic of all pine species,akes this genus a good model for intraspecific genome size

nalysis.However, the nature of putative intraspecific variation

emains to be elucidated (Dolezel et al., 1998; Dolezel andartos, 2005). Despite the fact that genome size should be con-

tant at species level, many reports of intraspecific variationxist in recent literature for various plant species (Blondon etl., 1994; Reeves et al., 1998; Moscone et al., 2003; Murray,005). On one hand, the variation at intraspecific level mightesult from taxonomically unrecognised “entities” being anal-sed (Greilhuber and Speta, 1985; Murray, 2005). On the otherand, it may arise from technical errors during measurements

Greilhuber, 1988, 1998). Key issues in this respect are the inap-ropriate use of internal standards, use of different tissues andnsufficient caution of investigators (Greilhuber, 2005). Withhese reservations, intraspecific genome size variation has also

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F. Bogunic et al. / Environmental and

een reported for some Pinus species (Valkonen et al., 1994;all et al., 2000; Bogunic et al., 2003).Pinus nigra Arnold (European black pine) is a tertiary relict-

al species belonging to the group of Mediterranean pinesVidakovic, 1991; Barbero et al., 1998). It is an important pinepecies in forest management of Mediterranean areas and par-icularly in the Balkan Peninsula. European black pine belongso section Pinus, subgenus Pinus. This species exhibits highorphological, physiological and ecological variability that has

ed some authors to consider this pine as an aggregate ofmall microgeographical species (Villar, 1947; Svoboda, 1953;ukarek, 1958). More recently, some authors consider this pines a single species subdivided into a number of subspecies andarieties (Vidakovic, 1974; Barbero et al., 1998; Greuter et al.,984; Gaussen et al., 1993). In contrast, Christensen (1997) rec-gnizes only two well-defined allopatric subspecies (subsp. salz-anni (Dunal) Franco and subsp. nigra) with regional varieties.here is still no general consensus on taxonomy of Europeanlack pine.

All these classifications were mostly based on morphol-gy, anatomy and phytogeography. The most recent classifica-ion of the genus Pinus, that includes molecular phylogeneticata as well, treats this species subdivided into five allopatricubspecies: salzmanni, nigra, dalmatica (Visiani) Franco, pal-asiana (Lambert) Holmboe and laricio (Poiret) Maire dis-ributed across the Mediterranean basin (Price et al., 1998).orthwestern African populations constitute a distinct sub-

pecies: P. nigra subsp. mauretanica (Maire and Peyerimh)

eywood (Greuter et al., 1984; Barbero et al., 1998). Breeding

xperiments have shown that all geographic subdivisions of P.igra were mutually crossable and gene flow was very efficientVidakovic, 1974, 1991).

aats

able 1eographic origin of the Pinus nigra populations studied: D—subsp. dalmatica;

alzmanni

opulation Locality Latitude

1 Velo Grablje, Hvar, Croatia 43◦07′N2 Vidova Gora, Brac, Croatiaa 43◦18′N3 Podgorje, Peljesac, Croatia 42◦52′N1 Wien, Austriaa 48◦08′N2 Konjic, Bosnia and Herzegovina (BiH) 43◦32′N3 Olovo, BiH 44◦15′N4 Zepce, BiH 43◦22′N5 Donji Ugar, BiH 44◦28′N6 Borja, BiH 43◦32′N7 Sjemec, BiH 43◦49′N8 Visegrad, BiH 43◦47′N9 Bosansko Grahovo, BiH 44◦12′N10 Belgaj, Albania 41◦35′N11 Gif-sur-Yvette, Franceb 48◦50′N1 Tausanli-Balikoy, Turkey 39◦29′N2 B. Gamurlu, Turkey 36◦51′N3 Jezirkopru-Golkoy, Turkey 41◦10′N

Tikjda, Kabilie, Algeria 36◦01′N1 Sur de Cuenca, Spain 42◦12′N2 La Pobla de Benifassa, Castello, Spain 40◦48′Na Locus classicus of the subspecies.b Cultivated plantation.

rimental Botany 59 (2007) 354–360 355

In this study we carried out a survey to assess: (a) the exis-ence of possible intraspecific genome size variation amongifferent populations of P. nigra; (b) whether genome size prop-rly reflects proposed subspecific categories; (c) relationshipsetween genome size and geographic factors and some seedraits. The panel comprised 16 European, 1 African and 3 Asia

inor populations across the natural range of P. nigra includ-ng five subspecies: subsp. nigra, salzmanni, dalmatica, pal-asiana and mauretanica from ecologically contrasting habitats.he working hypothesis was that genome size differs among

ntraspecific taxa. European black pine presents a good model foruch a study given its constant karyotype structure, morpholog-cal polymorphism, broad geographical distribution, ecologicallasticity and taxonomic complexity.

. Materials and methods

.1. Plant material

The analysed plant material was collected in natural habitatsf P. nigra except one population originating from a cultivatedlantation (Table 1 and Fig. 1). In each population 10 individualrees were sampled during October and November 2004.

For supplied material from Turkey, Spain, Austria and Alge-ia we adopted collector’s determination of P. nigra populations.

e followed the nomenclature of subspecific taxa accordingo classification systems of Gaussen et al. (1993), and Price et

l. (1998). In this study we consider the Algerian populations P. nigra subsp. mauretanica (Greuter et al., 1984). Unfor-unately we had no material of the subspecies laricio in thetudy.

N—subsp. nigra; P—subsp. pallasiana; M—subsp. mauretanica; S—subsp.

Longitude Geological substrate Altitude (m)

16◦42′E Limestone 44016◦36′E Limestone 59017◦30′E Limestone 36016◦06′E – 50018◦08′E Dolomite 94018◦25′E Peridotite 45018◦00′E Peridotite 31017◦14′E Peridotite 35018◦09′E Peridotite 90019◦15′E Peridotite 94717◦24′E Dolomite 35016◦34′E Dolomitised limestone 130020◦11′E Peridotite 140002◦10′E – 6529◦07′E – 150036◦23′E – 65035◦03′E – 90001◦28′E Limestone 150001◦11′E – 90000◦21′W Limestone 800

356 F. Bogunic et al. / Environmental and Experimental Botany 59 (2007) 354–360

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.2. Methods

We generally followed the procedure of genome size deter-ination described by Marie and Brown (1993) with slightodifications in concentration of RNAse and ethidium bromide

Bogunic et al., 2003). Previously we obtained genome size dataor leaves of adult pine trees (2C DNA data) (Bogunic et al.,003), while in the present work we used the haploid megaga-etophyte as source of 1C genome size data except for two

opulations (S2 and M).Nuclear genome size was determined using an ELITE

SP flow cytometer with an argon laser emitting 40 mW at88 nm, taking fluorescence 610 ± 15 nm. Galbraith’s bufferGalbraith et al., 1983), containing 0.1% Triton and 1% (w/v)olyvinylpyrrolidone 10 000 was used for chopping. Nucleiere stained with intercalating dye ethidium bromide (Sigma,t. Louis, MO). Megagametophytes were removed from dryeed and chopped simultaneously with 2 cm2 leaf of Triticumestivum cv. Chinese Spring (internal standard, 2C = 30.9 pg,arie and Brown, 1993) in cold buffer, filtered through a 48 �m

ylon mesh and RNAse was added to 5 units/ml. Finally, ethid-um bromide was added to a final concentration of 70 �g/ml.he suspension of intact isolated nuclei was kept on ice forh before analysis of at least 5000 nuclei. Absolute 1C value

pg DNA) was calculated from the ratio between internal stan-ard’s fluorescence (2C wheat) and pine sample fluorescence.ach population comprised from 5 to 10 individuals that wereeasured separately often with repetition. The population from

lovo (N3) is the most representative population of P. nigra

oncerning yield and growth characteristics. Thus, wheneverarious panels were analysed in different sessions, we used thisccession as an external standard to check for day-to-day sta-

abth

d populations of Pinus nigra.

ility. All analyses were on the same machine with the sameperator.

.3. Statistical and morphological analysis

Data was analysed by SPSS for Windows version 12.1, usingone-way ANOVA test. For two populations from Spain (S2)

nd Algeria (M) we tested differences using t-test because dataame from leaves. First we tested mean values of genome sizeata without S2 and M accessions. In next step we preparedNOVA including S2 and M populations with their deducedalues.

To test whether genome size is correlated with latitude, longi-ude, and seed traits, Spearman’s correlation test was employedvoiding possible non-normality of data. From each popula-ion 1500 seeds (150 per tree) were analysed by measuring theollowing characters: seed length, width, height and mass. Rela-ionships between genome size and mentioned seed traits haveeen analysed at population level. The measurements were car-ied out using an electronic calliper. Seed mass was obtainedy weighing 1500 seeds per population. These were performedor only 15 populations because material from some populationsas insufficient.

. Results and discussion

In this study nuclear genome size was obtained for 20 pop-lations of P. nigra across the natural range of the species. T.

estivum cv. Chinese Spring was a suitable internal standardecause it possesses a relatively high nuclear DNA (2C) con-ent just above that of P. nigra (1C DNA). The fluorescenceistograms were quite stable and peaks had a high resolution

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c.v. of 3–4%) providing accurate analysis of pine megagameto-hytes. The use of fresh seed material sometimes has advantageselative to leaf material, particularly in conifers, that usually con-ain staining inhibitors in leaf cytosol (Sliwinska et al., 2005).n the other hand, dry seed was superior to imbibed seed, nooubt because hydrolytic enzymatic activity was not reactivated.

Mean nuclear 1C DNA content of all populations was 23.62g, namely 23.1 × 109 base pairs (using 1 pg DNA = 0.978 Mbp,rom Dolezel et al., 2003), with populations ranging from 23.31o 23.95 pg (Table 2). There were no significant differencesmploying ANOVA test among the mean values of individ-al populations (F17 = 2.481, p < 0.003) excluding the values of2 and M accessions. However, when we pooled the deducedalues of S2 and M accessions with 1C data of other popula-ions, significant differences were omitted again (F19 = 2.362,< 0.004). In both cases the within group variation was higher

han between groups. The largest interpopulational differenceas 0.64 pg equivalent to 2.6% of the C value (N6 versus N7)

Table 2). Other populations have a quite similar genome size.o significant differences were observed between geographi-

ally most distant populations: Spain versus Albania, Austria

ersus Turkey, Croatia versus Turkey.

When individual populations were pooled according to theiraxonomic affiliation (Table 2, respectively D, M, N, P, S), theubspecies had the following 1C DNA contents: dalmatica—

asBD

able 2uclear 1C DNA content (C-value) for 20 populations of P. nigra

opulation na Mean 1C DNA content (pg)

1 5 23.792 7 23.363 10 23.641 5 23.542 5 23.903 10 23.364 5 23.735 5 23.696 7 23.317 5 23.958 5 23.419 5 23.8610 5 23.5811 5 23.561 5 23.712 5 23.933 5 23.79b 5 23.24

1 7 23.552b 5 23.53

um 116

ean 23.62

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in–max 23.31–23.95

.v. (%) 0.88

.D.: standard deviation; c.v.: coefficient of variation.a Number of analyses.b The 1C value has been deduced from the experimental result 2C.

rimental Botany 59 (2007) 354–360 357

3.79 pg; subsp. mauretanica—23.24 pg; subsp. nigra—23.63g, subsp. pallasiana—23.80 pg, subsp. salzmanni—23.55 pg.he variation between groups was slightly higher than withinroup. We conclude that there was no consistent pattern of sig-ificant genome size variation among subspecific taxa regardlessf geographic distance.

The 2C values obtained from leaf material for accessions Mnd S2 were almost the double of 1C values (1.96–1.99) of allopulations studied. Valkonen et al. (1994) reported that ratios ofC/1C of Pinus sylvestris L. ranged from 1.87 to 1.99 analysingaploid and diploid tissues by flow cytometry. They assumed thatuclear suspensions from needles contained chemicals whichnhibited propidium iodide staining, reducing staining. How-ver, even under saturating conditions and with various stains,here is a slight non-linearity in euploid series (2C, 4C, etc.),hether addressing differentiated or proliferating plant cells,

specially in laser-based configurations.In our previous paper we reported a significant difference

etween genome size in two subspecies of P. nigra but we hadnalysed only three populations, using leaf material (Bogunic etl., 2003). Present estimations are ∼5% higher. Conifer leaves

re rich with tannins, causing stoichiometric errors during mea-urements (Greilhuber, 1998; Price et al., 2000). Marie andrown (1993) consider variation below 3% as acceptable, whileolezel and Bartos (2005) state that in particularly ‘difficult’

S.D. (pg) Min–max (pg) c.v. (%)

0.429 23.47–23.92 1.800.249 23.07–23.80 1.060.273 23.31–24.26 1.150.345 23.19–24.12 1.460.421 23.22–24.23 1.760.430 23.13–23.85 1.850.383 23.07–24.03 1.620.218 23.38–23.85 0.920.270 22.93–23.61 1.150.265 23.66–24.31 1.110.170 23.32–23.78 0.720.404 23.26–24.30 1.690.149 23.37–23.79 0.630.185 23.38–23.74 0.780.395 23.48–24.13 1.660.314 23.59–24.34 1.310.229 23.51–24.01 0.960.237 22.93–23.45 1.020.198 23.32–23.76 0.840.620 22.83–24.26 2.63

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58 F. Bogunic et al. / Environmental and

roups 5% variation should be acceptable. Unfortunately in thistudy we obtained only one population that we had previouslysed (N2).

Comparing present results (1C = 23.62 pg) to those alreadyublished for P. nigra, we are faced with a significantly differ-nt genome size value (26.93 pg/1C in Grotkopp et al., 2004).hese researchers used Hordeum vulgare cv. Sultan as internaltandard, taking the rather traditional value 2C = 11.12 pg DNA,hereas this value is inconsistent with many other standards.e have reassessed the same H. vulgare cv. Sultan seedstock

gainst pea, petunia and tomato, obtaining 2C = 9.81 pg, namely1.8% less (Garnatje et al., 2004). Therefore, we believe resultsf Grotkopp et al. to be overestimated due to this internal stan-ard value.

From biogeographic standpoint, limited genome size vari-tion is very interesting. Populations of a widespread speciest different latitudes, particularly spatially isolated populations,ay have undergone contrasting demographic and genetic pro-

esses through time if there was no gene flow between themRobledo-Arnuncio et al., 2004). The last glaciation periodccurred about 10 000 years ago and many populations of forestrees were restricted to small refugia with mild climate aroundhe Mediterranean area and Black sea (Huntley and Birks, 1983;rewer et al., 2002). Iberian, Apennine and Balkan peninsu-

as were main refugial centres for many species during the lastlaciation wherefrom they colonised present-day habitats (Petitt al., 2003). In that light, island populations of P. nigra fromvar (D1), Brac (D2) and Peljesac (D3) subsp. dalmatica aref a particular interest because there was no exchange of pollenor seed material with continental populations, suitable condi-ions for affecting speciation processes and genetic drift over aong period. In this way genetic processes could drive changesn genome size among populations where inbreeding may occurecause of the small size of island populations, yet such genomeize changes are not found. On the other hand, continental pop-lations (P. nigra subsp. nigra), where out crossing is extensive,ight exchange genetic material through male gametophyte andaintain genome size within limits of insignificant variation.It is well known that gymnosperm genomes contain a large

ortion of repetitive DNA of low sequence complexity suchs micro- and mini-satellite repeats that could evolve indepen-ently in different taxonomic groups (Schmidt et al., 2000).ence, rapid expansion of some microsatellite sequences in pineenomes that are highly repetitive could be one factor affect-ng interpopulational differences, and particularly in spatiallysolated populations diverging independently while adapting toontrasting environments (Karhu et al., 2000; Oline et al., 2000).orphological differences between subsp. dalmatica and nigra

from locus classicus) are clearly evident, as too ecological con-itions (Liber et al., 1999; Pavletic and Liber, 1999).

A similar situation occurs with populations of P. nigra subsp.alzmanii from the Iberian Peninsula. The Pyrenees and Massifentral acted as natural barriers for recolonization processes

nd some authors suggest that Spanish pines have not con-ributed to recolonization of the European continent followinghe last glaciation period (Prus-Glowacki et al., 2003). If thats so, the exchange of genetic material among populations of

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rimental Botany 59 (2007) 354–360

. nigra subsp. salzmanni was restricted within Iberian popula-ions, peripheral populations on the margin of the south-westernange of the species in Europe. Algerian populations originatedost likely from Iberian populations of P. nigra. During the last

laciation, the sea-level in the Gibraltar Strait was much reducednd the coastlines were closer to each other giving the oppor-unity for pine populations to colonise African habitats (Petit etl., 2003). Under such a scenario, after the last glacial period,frican populations had a chance for independent speciation.ome authors consider Morocco’s and Algerian populations (P.igra subsp. mauretanica) as distinct taxa under various namesSchwarz, 1938; Gaussen, 1960; Barbero et al., 1998). Molecularhylogeographic investigations will probably elucidate the prob-em of present distribution in relation to geohistorical events. Inhat sense Asian populations are the remnants of Tertiary floraf Southwest Asia presenting different lineages than Balkan andberian stands of Black pine (Vidakovic, 1991). Although therere no consistent differences in genome size values betweenubspecies, the variation is clinal through the species range.

Conflicting reports concerning different correlations withenome size exist in recent literature. Some researchers haveeported the existence of relationships between ecogeographic,nvironmental and biological factors and genome size for manylant groups at species and population levels (Bennett, 1972;rice et al., 1981; Caceres et al., 1998; Reeves et al., 1998;night and Ackerley, 2002; Jakob et al., 2004). Certain rela-

ionships have been inferred for pines too. A strong positiveelation between seed mass and genome size was found for 85ine species (Grotkopp et al., 2004). The authors also claim thatany life-history patterns (dispersal mode of seeds, number of

eeds, growth rate, generation time) are indirectly associatedith genome size in pines. Ohri and Khoshoo (1986) suggested

he existence of a positive relationship between genome sizend latitude, and in that sense tropical pines should have smallerenomes then temperate species. Hall et al. (2000) more recentlytudied 17 New World pines and found no relationships betweenatitude and genome size. Also, Joyner et al. (2001) affirmedo positive relationships for 13 Asian pines. In our study, aeak positive correlation was observed between genome size

nd longitude (r = 0.454, N = 20, p < 0.05) while no relation-hip was found between genome size and latitude (r = −0.019,= 20, p < 0.05). The correlation with longitude translates into a

ecrease in genome size from Southwest Asia toward Europeanabitats, in line with the probable spread of P. nigra (Vidakovic,991).

For seed traits and altitude (Table 3), no relations wereound with genome size (DNA-altitude = −0.246; DNA-eed length = −0.044; DNA-seed width = 0.288, DNA-seedeight = −0.433; DNA-seed mass = −0.197). Thus, a system ofnferred interspecific relationships based on genome size andcological and biological traits for the genus Pinus (Grotkoppt al., 2004) could not be transposed to the intraspecific level of. nigra.

In conclusion we stress that genome size is fairly stable andontinuous. The distribution of 1C DNA amount in the P. nigraomplex does not permit intraspecific distinction among fivetudied subspecies, despite low experimental variation (coeffi-

F. Bogunic et al. / Environmental and Expe

Table 3Mean values with standard deviations of seed traits for populations of P. nigra

Population Length (mm) Width (mm) Height (mm) Mass (g)

D1 5.58 ± 0.34 3.28 ± 0.30 2.09 ± 0.19 0.0075 ± 0.004D2 5.81 ± 0.53 3.43 ± 0.25 2.45 ± 0.189 0.018 ± 0.006N1 6.58 ± 0.65 3.71 ± 0.00 2.41 ± 0.24 0.021 ± 0.007N2 6.37 ± 0.40 3.55 ± 0.24 2.40 ± 0.20 0.018 ± 0.008N3 6.48 ± 0.52 3.57 ± 0.34 2.45 ± 0.21 0.021 ± 0.071N4 5.86 ± 0.20 3.28 ± 0.02 2.26 ± 0.02 0.014 ± 0.056N5 5.83 ± 0.47 3.32 ± 0.34 2.17 ± 0.22 0.015 ± 0.006N6 6.09 ± 0.42 3.66 ± 0.32 2.50 ± 0.51 0.020 ± 0.006N7 6.41 ± 0.54 3.60 ± 0.45 2.28 ± 0.22 0.020 ± 0.007N9 5.68 ± 0.58 3.24 ± 0.41 2.27 ± 0.22 0.013 ± 0.006N10 6.76 ± 0.61 3.77 ± 0.44 2.57 ± 0.25 0.025 ± 0.023P1 6.27 ± 0.49 3.55 ± 0.32 2.44 ± 0.39 0.021 ± 0.007P2 6.77 ± 0.61 3.82 ± 0.33 2.57 ± 0.24 0.029 ± 0.007P3 6.05 ± 0.59 3.45 ± 0.30 2.28 ± 0.28 0.017 ± 0.006S1 6.36 ± 0.61 3.24 ± 0.34 2.07 ± 0.35 0.018 ± 0.041

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Ggenome size in pines (Pinus) and its life-history correlates: supertree analy-

ean 6.19 ± 0.38 3.49 ± 0.19 2.34 ± 0.15 0.018 ± 0.03

ient of variation of all data was 0.88%). Nor does molecular-ytogenetic data support sharp subspecific distinction (Bogunict al., unpublished data). Therefore, genome size among popula-ions of European black pine has not been significantly affectedy recent speciation events (10 000 years). The speciation isather a very slow process of divergence of populations (Juddt al., 2002). Concerning ancient life history of gymnosperms,enome size changes in conifer species might be expected onbroad evolutionary time scale (Auckland et al., 2001). Fluc-

uation in DNA content across the species range may be anndicator of speciation in progress (Murray, 2005). Therefore,ur positive correlation between genome size and longitudesould be indicative of slow genome size changes among popu-ations while adapting to contrasting environments at differentongitudes. In absence of polyploidy and changes in chromo-ome number (Ohri and Khoshoo, 1986), minor but significantariations in nuclear genome size could be due either to fluc-uations within highly repetitive DNA such as retrotransposonsBennetzen et al., 2005) or to structural rearrangements such asmall amplifications and deletions at the individual chromoso-al level (Williams et al., 2002).

cknowledgements

The authors are indebted to Prof. Z. Kaya, Prof. J. Valles,rs. B. Heinze, T. Garnatje, V. Markev, J. Gracan and R. Ben-iloud for collecting material and providing geographic data

rom Albania, Algeria, Austria, Croatia, Spain and Turkey. Also,he authors are indebted to Mersad Omanovic for measuring theeed characters. This study was supported by grant of Cantonal

inistry of Education and Science Sarajevo Nb. 11-14-22678.

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