INTERNATIONAL CONFERENCE“Hot Spots of Ancient and Present

genetic Diversity”

17 - 20 June 2009Park Hotel Moskva, Sofia, Bulgaria

UNIVERSITY OF FORESTRYSOFIA

PATTERNS OF CPDNA DIVERSITY WITHIN A BEECH REFUGIAL AREA

Theodoros Mouratidis1, Serafeim Hatziskakis1, Nicolas George Eliades2, Ioannis Tsiripidis3, Reiner Finkeldey2 and Aristotelis C. Papageorgiou1

1. Forest Genetics Laboratory, Democritus University of Thrace, Pantazidou 193, 68200 Orestiada, Greece, email: apapage@fmenr.duth.gr

2. Abteilung für Forstgenetik und Forstpflanzenzüchtung, Büsgen-Institut, Georg-August-Universität Göttingen, Büsgenweg 2, 37077 Göttingen, Germany

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

Abstract

This study aims at the description of beech postglacial movement within a mountain. The selected mountain Paggeo is isolated, while beech forms an altitudinal continuum for more than 1000m. Previous genetic studies have indicated the possible presence of a glacial refugium in the broader region. Seven sub-populations were sampled, in order to describe the structure of this refugial population. Three cpDNA microsatellite primer pairs were used. A high variety of haplotypes and a significant differentiation among sub-populations was detected. The results indicate that besides the refugial lineage on the north side of the mountain, two additional beech lineages have arrived from other refugia and occupied specific locations.

Introduction

The European beech is an important tree for forestry in Europe and one of the most represented tree genera of the continent. According to the most recent taxonomic classification, one beech species exists in western Eurasia (Fagus sylvatica L.) with two subspecies: F. sylvatica ssp. sylvatica and F. sylvatica ssp. orientalis (Lipsky) Greuter & Burdet (Denk 2003, Denk et al. 2002, Greuter et al. 1984). The two subspecies are reported to be interfertile (Gömöry et al. 1999, Denk et al. 2002, Gailing & von Wuehlisch 2004, Magri et al. 2006, etc.). Except these two subspecies another taxon has been described, which represents intermediate forms between the former two subspecies and it is known with the name moesiaca (Lipsky) Greuter & Burdet or taurica Popl. This taxon is considered as a hybrid between subspecies sylvatica and orientalis in conduct areas of these two subspecies or an evolutionary link between them (Bergmeier & Dimopoulos 2001). It is considered now as subspecies, but with questionable taxonomic validity (Greuter et al. 1984) and it is usually classified within subspecies sylvatica (Bergmeier & Dimopoulos 2001). However, forms with intermediate characteristics have been found to have an extensive distribution all over Greece (Moulopoulos 1965, Bergmeier & Dimopoulos 2001). Moreover, the subspecies of Fagus sylvatica occurring in Greece and southeast Europe it is believed that they present different ecological optimums (Moulopoulos 1965, Dafis 1969, Bergmeier & Dimopoulos 2001, Tsiripidis et al. 2007a & b). This fact results in an altitudinal zonation of beech subspecies with the subspecies orientalis occupying the lower altitudes and the subspecies sylvatica the higher ones, while the forms classified by some authors to subspecies moesiaca are usually found at moderate altitudes. This is mainly observed at morphological traits (Moulopoulos 1965, Tsiripidis and Athanasiadis 2003, Papageorgiou et al. 2008), but it is indicated

by genetic studies as well (Papageorgiou et al. 2008, Hatziskakis et al. 2009). This altitudinal distribution of the subspecies could be the result of their differentiated adaptation in environmental conditions, or the result of the response of morphological characteristics, used for systematic classification, to the changing conditions in different altitudes.

Pollen and paleobotanical data (Huntley and Birks, 1983; Magri et al. 2006; Magri 2008) as well as genetic data (Demesure et al., 1996; Comps et al., 2001; Magri et al., 2006, Hatziskakis et al. 2009) indicate the presence of beech refugia in the Balkan Peninsula during the last glaciation. Several authors (e.g. Huntley and Birks 1983, Taberlet et al. 1998), propose that the current distribution of beech in Central Europe derives from the post-glacial recolonization of populations originating mainly from the Balkan refugia. On the contrary, other authors (e.g. Magri et al. 2006, Magri 2008) argue that the main refugial areas, from where the recolonization to Central and Western Europe occurred, were located close to the Alps in Slovenia and South-western France and that the populations deriving from the Balkan Peninsula did not migrate to Northern Europe.

A large-scale study of beech cpDNA variation in Greece revealed high levels of diversity and a complex spatial structure at cpDNA markers, in sharp contrast to the lack of variation in most of the northern distribution range of beech in Europe (Hatziskakis et al. 2009). The authors propose three main causes for these results: (i) The existence of several separated refugia in the region, (ii) the recolonization of mountains both from near-by and more distant refugia, and (iii) the formation of a stable hybrid zone between Fagus sylvatica spp. sylvatica and Fagus sylvatica spp. orientalis in the eastern part of Greece. They suggest that remigration took possibly place at different times and at different altitudinal levels and that populations originating from different glacial refugia may have different adaptive potentials and therefore occupied different environments.

The mountain massif of Paggeo in North-Eastern Greece is chosen as the area of this study. This mountain is geomorphologically isolated from other mountains. A beech “island type” population grows on Paggeo, covering a large altitudinal range from 600m up to 1800m elevation. Beech covers areas with different ecological and geographical characteristics. The large altitudinal range, the diverse topography as well the different geological substrates occurring on this mountain result in a high diversity of beech forest plant communities. This mountain is located in a broader area, where introgression between the subspecies sylvatica and orientalis has been recorded. Hatziskakis et al (2009) have found different haplotypes within this population; one of them was “private” for Paggeo and originated probably from a local refugium.

The aim of this study is to investigate the existence of cpDNA haplotype diversity within and especially among subpopulations of beech on the Paggeo mountain, covering different altitudes and environmental conditions. Comparing our results with existing information about beech lineages in Northern Greece, we will attempt to reconstruct the postglacial evolutionary history of beech in the area and to describe the degree of influence of each lineage on the current cpDNA haplotypic profile.

Materials and methods

Leaf samples were collected from 7 Fagus subpopulations growing on Mt. Paggeo, representing different geographical locations and covering all existing ecological (vegetation) types of beech forests (Fig. 1 & Table 1). From each population, 18-20 individuals with a minimum distance of 100m among trees were randomly sampled.

DNA was extracted from fresh leaves using the DNeasy Plant Kit (Qiagen) following manufacturer’s instructions. DNA amount was tested on 0.8% agarose gel after being stained with ethidium bromide. The amplification of chloroplast microsatellites was conducted with three primers: ccmp4, ccmp7 and ccmp10 (Weising and Gardner 1999). The specific primers have been used in previous studies of European and Greek beech populations (e.g. Gailing & von Wuehlisch, 2004, Magri et al. 2006, Hatziskakis et al. 2009). The amplification procedure followed Gailing & von Wuehlisch (2004). The homology of the amplification products with respective cpDNA regions (Weising and Gardner, 1999) has been confirmed by sequencing (Gailing & von Wuehlisch, 2004). Fragments were separated on the ABI 3100 Genetic Analyser (Applied Biosystems) using the internal size standard Gene Scan Standard 500 ROX (Applied Biosystems). Fragment sizes were determined with the software packages Genescan 3.7 and Genotyper 3.7 from Applied Biosystems.

Haplotypes were identified based on the variation of the polymorphic chloroplast microsatellites. Genetic diversity for the entire set of populations (ht) and as a mean of the variation within single populations (hs) was estimated using the program PERMUT&CpSSR (Pons & Petit 1996).

Results

Two out of three microsatellites showed polymorphism (ccmp4, ccmp7). A total of 8 haplotypes were identified by combining the fragment lengths of these microsatellites (Table 2). The geographic distribution of the cpDNA haplotypes is shown in Fig. 1. The most frequent haplotype was Hap 3, that exists in all subpopulations except E and is most frequent in subpopulations B, C and F. Hap 3 corresponds to the dominant haplotype in Paggeo and is very rare in the rest of Greece (Hatziskakis et al. 2009). Hap 6 is the most frequent haplotype of subpopulation D (0.80) and is found in large frequencies in subpopulations E and G. This haplotype is considered to have an eastern origin, as it is well represented in Eastern Rodopi (Papageorgiou et al. 2008, Hatziskakis et al. 2009) and in Turkey (Gailing & von Wuehlisch 2004). Hap 4, a haplotype found in Western Rodopi and in Central Greece mainly (Papageorgiou et al. 2008, Hatziskakis et al. 2009) is here found in subpopulation A (0.60) and at lower frequencies in all other subpopulations except D.

Hatziskakis et al. (2009) have described four post-glacial beech lineages in Greece, using the same microsatellites used in this study. Here, 6 haplotypes described by Hatziskakis et al. (2009) are also found, while 2 haplotypes are described for the first time. Haplotypes 1 and 4 were reported to represent a lineage from a possible refugium in Rodopi. Haplotype 3 was found frequent in Mt. Paggeo and was considered to represent a small local refugium. Haplotypes 5, 6 and 8 represent the lineage migrating from the east, found mainly in East Rodopi and Turkey (Gailing &

von Wuehlisch 2004, Papageorgiou et al. 2008, Hatziskakis et al. 2009). Haplotypes 2 and 7 are described here for the first time and appear each in one tree only. They are private for subpopulations F and E respectively.

Total haplotypic diversity in Mt. Paggeo was found high (ht=0.782) and was higher within (hs=0.505) than among subpopulations (Gst= 0.354). This indicates the mixture of different beech lineages in the subpopulations studied.

Discussion

Chloroplast DNA exhibits high levels of intra- and especially inter-population diversity in beech forests of Mt. Paggeo. Unlike to previous large-scale studies on Fagaceae in southern Europe (e.g. Vettori et al. 2004, Hatziskakis et al. 2009), we found that most of the total variation resides within populations (hs=0.505). Similar trend was observed by Papageorgiou et al. (2008) in Rodopi. Studies on cpDNA variation at the local scale, using larger samples, reveal levels of haplotype diversity within populations that is unusual in other regions of Europe.

In a previous broad-scale study on cpDNA variation of beech in Greece, Hatziskakis et al. (2009) found only two haplotypes in Mt. Paggeo, haplotype 3 (80%) and haplotype 4 (20%). Our study found 6 more haplotypes, 2 of which are rather frequent in most subpopulations. Hatziskakis et al. (2009) suggest four post-glacial beech lineages in Greece and additionally indicate the existence of possible smaller refugial lineages that did not expand much after the last glaciations. Two of these distant lineages and a local refugial lineage are represented in Mt. Paggeo, based on our results.

Haplotype 3 is the most frequent haplotype in Mt. Paggeo and is dominant in subpopulations B, C and F. This haplotype was found only sporadically in other beech populations in Greece (Hatziskakis et al. 2009). We consider that this haplotype represents a local refugial beech lineage that did not expand much after the end of the glaciation era. Subpopulations B, C and F contain haplotype 3 in frequencies larger than 0,75. These observations confirm the existence of a small local refugium on the northern side of Mt. Paggeo with a limited postglacial expansion on the slopes lying directly above it. Haplotype 3 is also found in small frequencies in almost all other subpopulations of the mountain, indicating that the migration of this lineage was gradually reduced either by topographical obstacles, such as the steep slopes, or the prior existence of other beech trees originating from other distant refugia lineages.

Haplotypes characterizing two beech lineages, described by Hatziskakis et al. (2009), are found in Mt. Paggeo. These lineages derive from distant refugia and have probably arrived at different time and on different sides of the mountain. A lineage originating from the Rodopi massif is characterized by haplotypes 1 and 4. These haplotypes are very frequent in the eastern most subpopulation A, which grows in a low altitude. Haplotypes denoting the Rodopi lineage are also found in lower frequencies in all populations except D. It seems that the Rodopi lineage arrived from the north-east side of the mountain and occupied the low altitudes on the eastern slopes. It is mixed with other lineages in other parts of the mountain, but always remains in low frequencies, probably due the prior existence of other beech populations. The Rodopi lineage has managed to expand over large distances in

Greece and occupies many areas in central and northern Greece (Hatziskakis et al. 2009). Rodopi is located on the north-east side of Mt. Paggeo and its postglacial lineage has probably influenced the genetic structure of this mountain. Unlike Mt. Rodopi, where typical haplotypes of this origin were found in high altitudes (Papageorgiou et al. 2008), this lineage occupies sites with lower altitudes in Mt. Paggeo.

The second lineage found in Mt. Paggeo is represented by haplotypes 5, 6 and 8. These haplotypes characterize a beech lineage migrating from the east, found mainly in East Rodopi and Turkey (Gailing & von Wuehlisch 2004, Papageorgiou et al. 2008, Hatziskakis et al. 2009). It is supposed to build introgression zones with the Rodopi lineage in North Greece. It is found in high frequencies all over north-east Greece, but also in populations of central Greece (Papageorgiou et al. 2008, Hatziskakis et al. 2009. In Mt. Paggeo, haplotypes of the eastern lineage dominate subpopulations D, E and G, located on the western part of the mountain. In these subpopulations, other haplotypes exist in small frequencies. Populations E and G are very variable, while D shows low levels of within population diversity. We assume that this lineage arrived from the north side of the mountain. It occupied the north and north-west slopes and moved upwards and formed the beech forests of the higher altitudes. The low diversity within subpopulations of high altitudes, may be explained by continuous founder effects occurring on the direction of migration. Furthermore, subpopulations on lower altitudes were easier accessible by other lineages and therefore became more diverse. Once again, these results are in contradiction with the results of cpDNA distribution in Mt. Rodopi (Papageorgiou et al. 2008), where the eastern lineage is found on lower altitudes. This indicates that postglacial movement is not related with specific differentiated adaptability of lineages or distance, but rather on topography and temporal differences in expansion.

The cpDNA profile of beech on Mt. Paggeo indicates a complex postglacial origin. This confirms the hypothesis formulated by Hatziskakis et al. (2009) that beech expansion in Greece is the result of simultaneous migration from smaller local refugia that did not expand much, or from more distant refugia. Different lineages are mixed in rather small areas and this results in high levels of cpDNA haplotype diversity.

Small local refugia have been assumed to play a major role in the current distribution of broadleaved species (Tzedakis et al. 2002, Magri et al 2006, Bhagwat and Willis 2008, Hu et al. 2008). This fine scale genetic study of the chloroplast genome confirmed this theory and described such a refugium located on the base of Mt. Paggeo. The postglacial migration of seed out of this refugium was restricted on the north-east slopes of the mountain, while other lineages were more efficient in colonizing large parts of the mountain.

Acknowledgements

We wish to thank Giorgos Korakis, Dimitris Kasimiadis and Amaryllis Vidalis for their support and advice during this study. This research was partially financed by the IKYDA project.

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Figure 1. Map of the study area. Contour interval is 200 m, and the lowest contour represents an altitude of 400 m. Grey shadowed areas indicate the distribution of Fagus sylvatica and black patches the sampling localities (see Table 1). The inserted map presents the geographical locations of Mt Pangeo and the other mountains of NE Greece (grey colored areas represent altitudes from 500 to 1000 m and black colored ones above 1000 m).

Table 1. Location and description of the subpopulations studied

Sub-population Latitude Longitude

Altitude (m)

Aspect (º) Sample

A 40º 55’ 08,25” 24º 11’ 47,74” 946 118 20B 40º 54’ 48,06” 24º 08’ 51,25” 1428 137 20C 40º 55’ 12,23” 24º 10’ 25,86” 1207 76 20D 40º 54’ 41,01” 24º 06’ 37,52” 1717 71 20E 40º 57’ 35,66” 24º 06’ 15,84” 708 49 20F 40º 55’ 33,86” 24º 07’ 29,68” 720 165 20G 40º 55’ 51,64” 24º 03’ 44,11” 1098 286 18

Table 2. Description and frequencies of the 8 haplotypes identified by polymorphic microsatellite fragments

Haplotype ccmp7 ccmp4 ObservationsFrequencies

A B C D E F G

1 146 bp 115 bp 5 0,20 0,00 0,00 0,00 0,00 0,05 0,002 147 bp 114 bp 1 0,00 0,00 0,00 0,00 0,00 0,05 0,003 147 bp 115 bp 55 0,20 0,75 0,85 0,10 0,00 0,75 0,114 148 bp 115 bp 22 0,60 0,20 0,10 0,00 0,05 0,05 0,115 149 bp 115 bp 10 0,00 0,05 0,00 0,00 0,35 0,00 0,116 150 bp 115 bp 36 0,00 0,00 0,05 0,80 0,40 0,10 0,507 151 bp 114 bp 1 0,00 0,00 0,00 0,00 0,05 0,00 0,008 151 bp 115 bp 8 0,00 0,00 0,00 0,10 0,15 0,00 0,17

bp: base pairs