Amaryllis Vidalis1, Aristotelis Papageorgiou2, Seraphim Hatziskakis2, Ioannis Tsiripidis3, Oliver Gailing1, Reiner Finkeldey1

1 Department of Forest Genetics and Forest Tree Breeding, Büsgen Institute, Georg-August-University Göttingen, Büsgenweg 2, 37077 Göttingen, Germany

2 Department of Forestry, Environment and Natural Resources, Democritus University of Thrace, P.O. Box 129, Pantazidou 193, 68200 Orestiada, Greece

3 School of Biology, Department of Botany, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

Patterns of genetic diversity in Fagus sylvatica L. in Rodopi Mountains of N.E. Greece

PL LL LW LMW LA LP NS AN LI MWI PI RC Elatia 0,95 7,86 4,85 4,02 25,97 19,07 8,00 36,41 163,00 51,16 12,11 1,06

Echinos 0,88 8,56 5,21 4,59 30,74 20,78 9,60 38,48 166,00 53,59 10,31 1,07

Papikio 0,97 8,74 5,33 4,68 31,56 21,15 9,20 38,48 165,00 53,32 11,03 1,07

Dadia 0,83 8,73 5,22 4,62 31,10 20,92 9,30 37,37 168,00 52,81 9,56 1,08 Table 1. Average values of morphological traits measured per population

0% 20% 40% 60% 80% 100%

Dadia

Papikio

Echinos

Elatia

ФT1

ФT2ФT3

Figure 2. Phylotypes frequencies within populations

Hs Ht Gst AFLPs 0,234 0,255 0,089

cpDNA haplotypes 0,551 0,925 0,405

Elatia Echinos Papikio Dadia Number of polymorphic zones AFLPs 26 26 28 33

Percentage of polymorphic zones AFLPs 76,47 76,47 82,35 97,06

Expected heterozygosity - AFLPs 0,212 0,213 0,243 0,269

Haplotypes numbers - cpDNA 3 4 8 9

Expected "heterozygosity" - cpDNA 0,153 0,438 0,707 0,813

0% 20% 40% 60% 80% 100%

Dadia

Papikio

Echinos

Elatia

145-115 146-110 146-115 147-110 147-115 148-110 148-115149-110 150-110 151-110 152-110 153-110 154-110 156-110

Table 2. Diversity and differentiation of populations (Nei 1987)

Table 3. Genetic diversity within populations

Figure 3. Frequences of cpDNA haplotypes

According to the most accepted taxonomic classification of beech in Europe, Fagus sylvatica can be subdivided into two subspecies (ssp. sylvatica and ssp. orientalis) [1]. The

current beech populations of Europe may have a common origin from refugial populations which survived the last glaciation's era [2]. The wider area of Greek Rodopi is being

considered as potential refugium for European beech [3]. In previous research, high variation in morphological traits was found in Greek beech populations. At molecular level,

variation has been generally found higher in the Mediterranean part of Fagus sylvatica distribution, as compared to that found in central and western European populations [4].

Specifically, in cpDNA of beech, former research has revealed no or minor polymorphism in large areas of its natural distribution [4].

Material and methods

PL Petiole length LL Lamina length LW Lamina width

LMW Distance between the lower part of the lamina and the maximum width point on the axis

LA Lamina area LP Lamina perimeter NS Number of secondary nerves

AN Angle between the primary and the central secondary nerve

Morphological traits

Molecular markers

AFLPs [5] Primer pairs

Restriction enzymes EcoRI- MseI

Preselective amplification E01/M03 (Keygene)

Selective reaction E37/M68

LI LL/LW · 100 MWI LMW/LL · 100

PI PL/LL · 100

RC

Radius of a cycle with an area equal to LA divided by the radius of a cycle having a perimeter equal to LP

DNA was extracted from buds using the DNeasy® Plant Kit (Qiagen)

Results

Morphological traits Molecular markers

Elatia Echinos Papikio Dadia

Elatia 0 6,068 4,966 1,522

Echinos 0,04 0 0,276 3,619

Papikio 0,07 0,03 0 3,028

Dadia 0,056 0,018 0,013 0

Conclusions

• The beech populations of Rodopi mountains show high levels of genetic diversity within populations in leaf morphological traits, AFLPs and cpSSRs

• The levels of genetic variation within populations were found high compared to previous surveys in European populations

• Clinal variation patterns were observed at both morphological traits and molecular markers; variation increases from the west to the east

• Differentiation among populations was, as expected, stronger at maternally inherited cpDNA, a sign of uninterrupted history of beech occurrence in the area.

• The results support the assumption that the Rodopi massif is an introgression zone, where the European beech (ssp. sylvatica) meets the eastern beech (ssp. orientalis)

• Several haplotypes found in the easternmost population were also reported for ssp. orientalis in Turkey [7]

Hs: total diversity within populations

Ht: total diversity

Gst: total differentiation

Table 4. Genetic distance (Nei 1972) between populations cpDNA SSRs above, AFLPs below diagonal

PCA, revealed three independent components

expressing 84,6% of the total variation. Based on

these components and the Eucledian distances

between the individuals, a grouping was conducted,

where all the trees belonging to the same group were

discribed by the same „phylotype“ (ФT).

ELATIA

ECHINOS PAPIKIO

DADIA

ФT1: corresponds to characteristics that fit leaf morphology of ssp. sylvatica

ФT2, ФT3: show intermediate morphological traits between the two subspecies,

but resembling more ssp. orientalis

AIMS OF THE STUDY: - Description of variation patterns in beech on the Rodopi Mountains (morphological traits and molecular markers)

- Test of the hypothesis of possible introgression between the two subspecies.

Chloroplast SSRs Primer pairs [6]

Applied ccmp4, ccmp7, ccmp10

Polymorphic ccmp7, ccmp10

Figure 1. Location of the populations

Introduction

References

[1] Denk T (2003) Phylogeny of Fagus L. (Fagaceae) based on morphological data. Plant Syst Evol 240:55–81 [2] Taberlet P, Fumagalli L, Wust-Sausy A, Cosson J (1998) Comparative phylogeography and postglacial colonization routes in Europe. Mol Ecol 7:453–464 [3] Pott R (1997) Invasion of beech and establishment of beech forests in Europe. Annali di Botanica LV:27–58 [4] Magri D, Vendramin GG, Comps B, Dupanloup I, Geburek T, Gömöry D, Latałova M, Litt T, Paule L, Roure JM, Tantau I, van der Knaap WO, Petit RJ, de Beaulieu J-L (2006) A new scenario for the Quaternary history of European beech populations: paleobotanical evidence and genetic consequences. New Phytol 171:199–221 [5] Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414 [6] Weising K, Gardner RC (1999) A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of decotyledonous angiosperms. Genome 42:9–19 [7] Gailing O, von Wuehlisch G (2004) Nuclear markers (AFLPs) and chloroplast microsatellites differ between Fagus sylvatica and Fagus orientalis. Silvae Genet 53:105–110

Acknowlegements

The authors are grateful to Olga Artes and Ludger Leinemann for their technical support and advice and Dimitris Kasimiadis and Artemis Kafkaletou-Diez for their help during sampling and the morphological trait measurements

Plant material: 4 populations of 25 individuals were randomly sampled (leaves and buds)