A study of genetic differentiation and hybridization among oak species with divergent ecological and evolutionary profiles Thesis submitted in partial fulfilment of the requirements of the degree Doctor rer. nat. of the Faculty of Forest and Environmental Sciences, Albert Ludwigs Universität Freiburg im Breisgau, Germany by Charalambos Neophytou Freiburg im Breisgau, Germany 2010
Dean of Faculty: Prof. Dr. Jürgen Bauhus Supervisor: Prof. Dr. Siegfried Fink Second Reviewer: Assoc. Prof. Dr. Filippos A. Aravanopoulos Date of thesis’ defence: December 8, 2010
Table of contents 1. Introduction ...... 1
2. Objectives and hypotheses...... 3 2.1. Interspecific and geographic genetic differentiation patterns within and between Quercus petraea (Matt.) Liebl. and Q. robur L. based on data from natural stands growing under drastically diverse environmental conditions and possessing different evolutionary histories...... 3 2.2. Patterns of genetic differentiation and hybridization between Q. alnifolia Poech and Q. coccifera L. in the insular environment of Cyprus...... 3 2.2.1. Interspecific and intraspecific genetic differentiation between Q. alnifolia and Q. coccifera : a large scale and multipopulation approach...... 4 2.2.2. Hybridization dynamics between Q. alnifolia and Q. coccifera : genetic analysis of adult trees and progenies in a mixed stand ...... 4 2.3. Evolutionary conservation of the used microsatellite loci and phylogenetic relationships among the study species ...... 5
3. Literature review...... 6 3.1. Phylogeny of the genus Quercus in Europe and the Mediterranean Basin...... 6 3.2. Differentiation, hybridization and evolution in oaks...... 7 3.2.1. The use of morphological traits ...... 8 3.2.2. The use of molecular markers...... 9 3.3. Q. petraea and Q. robur in Europe: Species distribution and ecological requirements...... 10 3.3.1. Quercus petraea (Matt.) Liebl ...... 10 3.3.2. Quercus robur L...... 11 3.3.3. Differentiation and hybridization between Q. petraea and Q. robur ...... 11 3.4. Quercus alnifolia , Q. coccifera and Q. infectoria ssp. veneris in Cyprus: Species distribution and ecological requirements...... 14 3.4.1. Quercus alnifolia Poech...... 14 3.4.2. Quercus coccifera L...... 15 3.4.3. Quercus infectoria ssp. veneris A. Kern ...... 15 3.4.4. Differentiation and hybridization between Q. alnifolia and Q. coccifera ...... 16
4. Accomplished research...... 18 4.1. Detecting interspecific and geographic differentiation in two interfertile oak species ( Quercus petraea (Matt.) Liebl. and Q. robur L.) using small sets of microsatellite markers (Manuscript I)...... 18 4.1.1. Methodology...... 18 4.1.2. Results and discussion...... 18 4.1.3. Conclusion...... 20 4.2. Interfertile oaks in an island environment. I. Contrasting patterns of nuclear and chloroplast DNA differentiation between Quercus alnifolia and Q. coccifera in Cyprus (Manuscript II)...... 20 4.2.1. Methodology...... 20 4.2.2. Results and discussion...... 21 4.2.3. Conclusion...... 21
4.3. Interfertile oaks in an island environment. II. Limited hybridization between Quercus alnifolia Poech and Q. coccifera L. in a mixed stand (Manuscript III)...... 22 4.3.1. Methodology...... 22 4.3.2. Results and discussion...... 22 4.3.3. Conclusion...... 23 4.4. Conservation of nuclear SSR loci reveals high affinity of Quercus infectoria ssp. veneris A. Kern (Fagaceae) to section Robur (Manuscript IV)...... 23 4.4.1. Methodology...... 23 4.4.2. Results and discussion...... 24 4.4.3. Conclusion...... 24 4.5. Phylogenetic relationships among the study species based on chloroplast DNA haplotypes...... 25 4.5.1. Methodology...... 25 4.5.2. Results and discussion...... 25 4.5.3. Conclusions...... 26
5. General discussion...... 28
6. Conclusions and outlook...... 34
7. Summary ...... 37
8. Zusammenfassung...... 39
9. Περίληψη...... 42
10. References ...... 45
11. Acknowledgments...... 56
12. Publications ...... 58
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1. Introduction Oaks ( Quercus L.) constitute one of the most species rich genera of the northern hemisphere (Frodin 2004). They occupy a wide distribution range covering large parts of Europe, North America, the Mediterranean Basin and Asia spreading southwest to Columbia in South America and southeast to Indonesia. Consisting of 300 500 species (Camus 1934, Manos et al. 1999), they are a dominant element in a great variety of ecosystems ranging from Mediterranean sclerophyllous communities to temperate deciduous forests and tropical mountainous plant communities (Axelrod 1983). Numerous species, subspecies and varieties present a multiplicity of climatic, edaphic and photoperiodic adaptations. Besides their high taxonomic complexity, oaks are characterized by especially high levels of hybridization. Numerous records of intermediate forms arise from studies of both fossilized and living species (Palmer 1948, Stebbins 1950, Rushton 1993). In Europe, the genus Quercus is widely present and forms large forests in a wide ecological spectrum. Two important taxonomic groups, the sections Quercus and Cerris are represented with more than 30 species (Denk and Grimm 2010). The widespread deciduous species Q. petraea and Q. robur (section Quercus) predominate in several landscapes across the continent and play an important economical role as a valuable source of wood (Aas 2008a, Aas 2008b). A series of other deciduous species occupy diverse habitats with their centres of diversity mainly being the three main peninsulas of the Mediterranean part of Europe (the Iberian, the Italian and the Balkan Peninsulas; Roloff et al. 2008). Evergreen sclerophyllous oaks are a significant element of Mediterranean sclerophyllous communities and are adapted to the particular environmental conditions there (e.g. prolonged summer drought and fire; Mooney and Dunn 1970). Besides species with large distribution ranges and broad ecological amplitudes, several other oak taxa are geographically restricted and show a high specificity of habitat like the endemic Q. alnifolia , which is confined to the igneous rock formations of the Troodos Mountains in Cyprus (Knopf 2008). In the light of a rapid climate change, a significant worldwide shift of climatic zones is expected over the next years. The reaction of tree species consists in three main alternative strategies: adaptation, migration or extinction (Savolainen et al. 2007, Aitken et al. 2008). Species with wide ecological amplitudes are expected to shift their distribution ranges northwards, but also to occupy additional habitats within their current growing areas, due to retreat of other more demanding species. Species with more restricted habitats may be more vulnerable to extinction. On the other hand, a shift of the ecological zones northwards may offer new niches for these species (Lindner et al. 2010). Given the rapidity of global warming, fast migratory responses are needed for the species to cope with climatic changes. A facilitated gene flow of preadapted alleles (assisted migration) or even a species transfer from warmer climates may be required in the effort to cope with environmental changes (Aitken et al. 2008). The significant role of interbreeding in the evolution of genus Quercus has been increasingly recognized since the middle of the last century (Stebbins 1950, Muller 1952, Burger 1975, Van Valen 1976). Studies of hybridization in oaks contributed to a reconsideration of the biological species concept, which requires reproductive isolation among species (Mayr 1942). Empirical and scientific observations documented in a large body of literature support the notion that interbreeding amongst Quercus species is not an 2 1. Introduction
occasional phenomenon, but an active evolutionary mechanism proceeding over generations and leading to substantial exchanges of genetic variation between different taxonomical units (Muller 1952, Stebbins 1950, Burger 1975, Van Valen 1976, Petit et al. 2004, Curtu et al. 2009). The role of interspecific gene flow as a means of adaptive variation exchange is a fascinating and challenging topic of recent and current research (Petit et al. 2004, Scotti Saintagne et al. 2004a, Muir and Schlötterer 2005, Lexer et al. 2006, Lepais et al. 2009). Over the last decades, new tools of DNA analysis have given the opportunity to assess the genetic imprints of hybridization and have allowed tests of several evolutionary hypotheses in oaks. In the present study, species differentiation and interspecific gene flow are studied by focusing on oak species with divergent ecological and evolutionary profiles. Firstly, taxonomic and geographic genetic variation among very distinct environments are investigated in the interfertile Q. petraea and Q. robur , two continental oak species with a large distribution range and an analogous ecological amplitude. Secondly, hybridization and differentiation are examined in the paradigm of Q. alnifolia and Q. coccifera , two Mediterranean oak species growing in the restrictive insular environment of Cyprus and being the only potentially interfertile oaks there. Thirdly, evolutionary conservation of loci and phylogenetic relationships are resolved among the former oak species that represent two different and important sections of Quercus taxonomy. For this purpose, Quercus infectoria ssp. veneris , the third oak species of Cyprus, is additionally used forming a potential outgroup.
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2. Objectives and hypotheses 2.1. Interspecific and geographic genetic differentiation patterns within and between Quercus petraea (Matt.) Liebl. and Q. robur L. based on data from natural stands growing under drastically diverse environmental conditions and possessing different evolutionary histories The first objective of this study is to analyse interspecific and geographic patterns of genetic variation in a wide continental environment, in particular within and between Q. petraea and Q. robur . Quercus petraea and Q. robur possess a broad geographic distribution and ecological amplitude reflecting a series of adaptations to contrasting environments. The present study especially focuses on the less studied Balkan refugial provenances through an analysis of interspecific and geographic patterns of genetic variation within and between Q. petraea and Q. robur by using a multilocus approach. Insofar evidence suggests that only a restricted proportion of the genome accounts for interspecific differentiation between the two species, which has been attributed to directional selection affecting a limited number of genes and hitchhiked regions. On the other hand, gene flow has been suggested to play a homogenizing role in the rest of the genome (Bodénès et al. 1997, Petit et al. 2004, Scotti Saintagne et al. 2004a). For studying both factors of differentiation – the taxonomic and the geographic – interspecific population pairs from different environments along a large scale ecological gradient are included. It is hypothesized that loci accounting for species differentiation have a consistent geographic pattern due to directional selection acting for maintenance of species identity. On the contrary, variation at the remaining loci is governed by the homogenizing effect of interspecific gene flow and by local selection. Genomic loci which vary strongly among regions, but show low interspecific differentiation at the local scale may indicate adaptations to the regional conditions, which are shared between the species due to ‘adaptive introgression’. Moreover, selectively neutral loci are expected to be less differentiated both at the interspecific and interregional level. Genetic variation among regions and between species was tested against the ‘null’ hypothesis of absence of genetic differentiation either at the taxonomic or the geographical level. 2.2. Patterns of genetic differentiation and hybridization between Q. alnifolia Poech and Q. coccifera L. in the insular environment of Cyprus The second objective of the study is the analysis of genetic differentiation, hybridization and introgression in a restricted insular environment, in particular between Q. alnifolia and Q. coccifera . Being phylogenetically related and forming both sympatric and pure populations in Cyprus, Q. alnifolia and Q. coccifera present an ideal paradigm for the study of differentiation in the presence of hybridization and potential introgression in a restricted insular environment. Potential hybridization between these species and the third oak species of Cyprus, Q. infectoria ssp. veneris , has never been reported and may not be expected as the latter species shows an affinity to another section (Meikle 1977, Schirone and Spada 2001). 4 2. Objectives and hypotheses
Two approaches are followed for this objective. First, genetic differentiation within and between species is assessed in a large scale, using both pure and mixed populations. Second, interspecific gene flow in a fine scale is studied within a mixed stand by using adult trees and their offspring.
2.2.1. Interspecific and intraspecific genetic differentiation between Q. alnifolia and Q. coccifera : a large scale and multipopulation approach A fundamental evolutionary question regarding sympatric and potentially interbreeding species is whether the species are genetically distinct and whether interspecific differentiation varies among mixed and pure populations. In order to address this question, genetic variation is investigated in pure and sympatric populations covering a large part of the species’ distribution area in Cyprus. In the presence of hybridization it can be expected that genetic variation between species is lower in sympatry and a higher proportion of introgressed individuals occurs there. Furthermore, hybridization in contact zones is expected to result in an increased intraspecific differentiation between pure and sympatric populations. In the present study, genetic variation among regions and between species is tested against the ‘null’ hypothesis of absence of genetic differentiation either at the taxonomic or the geographical level. For testing these hypotheses, markers from both nuclear and chloroplast genomes are used. On the one hand, due to a fast decay of linkage disequilibrium, nuclear genomes reflect current or recent hybridization events (Asmussen et al. 1987). Moreover, due to an anemochoric pollen dispersal mode in oaks, formation of spatial genetic structures at nuclear DNA loci is prevented (Kremer et al. 2002). On the other hand, chloroplast genomes generally evolve slowly and lack recombination, thus better reflecting historic events of introgression (Wolfe et al. 1987). Being maternally inherited, they possess a barochoric dispersal mode in oaks, which limits the extent of gene flow and leads to the formation of spatial genetic structures (Petit et al. 1993, Dumolin Lapègue et al. 1997, Olalde et al. 2002).
2.2.2. Hybridization dynamics between Q. alnifolia and Q. coccifera : genetic analysis of adult trees and progenies in a mixed stand In order to elucidate the extent of active hybridization between Q. alnifolia and Q. coccifera in sympatry, focus is put on a mixed stand. Firstly, an estimation of genetic introgression among adults is aimed, based on phenotypic traits (leaf morphology), nuclear and chloroplast DNA markers. Secondly, the degree and possible directionality of interspecific gene flow are examined by analyzing progenies (acorns) from selected adult maternal trees representing both parental species and intermediates. In particular, by removing the maternal contribution to each offspring, a genetic characterization of male gametic contributions (pollen clouds) to each maternal tree is sought. In the lack of interspecific gene flow and assuming panmixia within species, it is hypothesized that there is no variation among pollen clouds comparing maternal trees of the same species. Alternatively, interspecific gene flow is expected to decrease genetic differentiation between pollen clouds belonging to interspecific pairs of maternal trees. Furthermore, interbreeding is expected to increase genetic variation among maternal trees at the within species level. Finally, based on multilocus genotypes, an individual characterization of
2. Objectives and hypotheses 5
male (pollen) gametic contribution to each offspring is aimed. By assigning pollen donors to species, interspecific gene flow can be quantified within progenies and within species. A possible asymmetry between species may reveal hybridization directionality. 2.3. Evolutionary conservation of the used microsatellite loci and phylogenetic relationships among the study species The third objective of the study is the establishment of phylogenetic relationships among the study species. For this purpose, the third oak species growing on Cyprus is additionally considered. A phylogenetic distinctness of this species in comparison to the sclerophyllous Q. alnifolia and Q. coccifera has been indicated by earlier studies, mostly based on morphological descriptions (Camus 1934, Meikle 1977). Additionally, a classification of this species to section Quercus along with Q. petraea and Q. robur has been suggested (Schirone and Spada 2001). However, no molecular evidence has been provided. Firstly, a comparison of amplification patterns, variability and sequence data of nuclear loci among species is aimed in this study. In general, locus conservation and amplification of homologous sequences have been shown to correlate with phylogenetic affinity in oaks (Steinkellner et al. 1997). Secondly, using chloroplast DNA markers, a study of phylogenetic relationships among all the study species is sought. Due to linkage equilibrium and slow evolution, chloroplast DNA has been widely used to infer phylogenetic relationships in angiosperms (Wolfe et al. 1987, Demesure et al. 1996, Dumolin Lapègue et al. 1997, Heuertz et al. 2006). An assignment of Q. alnifolia and Q. coccifera on one hand and Q. infectoria ssp. veneris on the other, to different sections would strongly preclude any interspecific gene flow, since effective reproductive barriers result in a lack of natural hybrids arising from intersectional crosses (Cottam et al. 1982, Rushton 1993, Manos et al. 1999). In addition, a comparison of the latter species to Q. petraea and Q. robur would provide evidence about its membership to section Quercus.
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3. Literature review 3.1. Phylogeny of the genus Quercus in Europe and the Mediterranean Basin According to the most recent classifications based on morphological and molecular data, the genus Quercus is subdivided into four main sections: Cerris with a Eurasian distribution, Lobatae (red oaks) and Protobalanus with a New World distribution and Quercus sensu stricto (white oaks) with a widespread distribution in North America and Europe (Manos et al. 1999). In contrast to previous studies based on morphology (Camus 1934, Nixon 1993), molecular data support monophyly and an early separation of the strictly Eurasian section Cerris from the other sections, which is in agreement with an initial vicariance event between Eocene and Oligocene (Manos and Stanford 2001). Regarding the other three sections, a New World origin and a more recent speciation is supported (Manos et al. 1999, Manos and Stanford 2001). Among them, only Quercus is represented in both North America and Eurasia, suggesting a link between the Old and the New World prior to breakup of the geographic corridors between the two geographic regions (Manos et al. 1999). Migration through this land bridge was possible only for deciduous oaks which were adapted to prolonged winter darkness in high latitudes (Tiffney and Manchester 2001). Two main phylogenetic clades, section Cerris and section Quercus are present in the western Eurasian region, including the whole Mediterranean Basin. The section Cerris is currently represented by 15 species (Denk and Grimm 2010). Phylogenetic affinities among the members of the section Cerris are confirmed by various cladistic studies based on molecular markers. A main finding of molecular studies from the last decade is the division of the section into two well separated entities; the ‘Ilex’ group, including all evergreen Mediterranean species except Q. suber , and the ‘Cerris’ group, including the remaining oaks of section Cerris (Manos et al. 1999, Denk and Grimm 2010). Oaks of group ‘Ilex’ ( Quercus ilex , Q. coccifera , Q. aucheri and Q. alnifolia ) are predominantly distributed around the Mediterranean Basin, being elements of sclerophyllous broadleaved communities. Oaks of group ‘Cerris’ are also spread mainly in Mediterranean countries ( Quercus macrolepis , Q. trojana and Q. libani ), whereas Quercus cerris is the only species of the section that enters into areas of Central Europe with sub continental climate. Yet, it is the only deciduous species of this phylogenetic group. The section Quercus (white oaks) is represented by ca. 18 deciduous or semi deciduous species in western Eurasia with their diversity bulk lying in the southern part of the region. Among them, several species like Q. faginea , Q. infectoria and Q. vulcanica are regionally distributed across the northern Mediterranean countries. Quercus pubescens and Q. frainetto can tolerate continental conditions and reach the interior of the European continent where they grow on the driest and warmest sites. Only two species of this section occupy large areas of Central Europe, Q. petraea and Q. robur , which are less adapted to summer dry Mediterranean climate. In general, phylogenetic subdivision within the section is difficult, since a great part of molecular differentiation is shared among species. Both rDNA and cpDNA diversity is widely shared within the section not only among European species, but also among their American counterparts indicating a recent speciation (Manos and Stanford 2001, Denk and Grimm 2010). 3. Literature review 7
Previous subdivision within the white oaks (formerly called ‘subgenus Quercus’) into sections like Robur, Prinus etc. (Krussmann 1978, Nixon 1993) is not supported by more recent molecular evidence (Manos et al. 1999, Manos and Stanford 2001, Denk and Grimm 2010). According to the same earlier taxonomic classifications, section Cerris was also placed within the ‘subgenus Quercus’. Given that this hierarchy is not supported by genetics, the terms ‘section Quercus’ and ‘section Cerris’ (following Denk and Grimm (2010)) are used in the present manuscript. 3.2. Differentiation, hybridization and evolution in oaks. Oaks are well known for their ability to interbreed in nature. Intermediate forms are well documented both in fossilized and living species suggesting that hybridization has been widespread for several millions of years (Palmer 1948, Stebbins 1950, Rushton 1993). Despite hybridization, species tend to keep their morphological, ecological and genetic identities although in most cases hybrid forms are as fertile as their parents (Burger 1975, Mir et al. 2009, Lepais et al. 2009). The example of Quercus has triggered several debates among taxonomists and has led to a challenge of classical species concepts, which require reproductive isolation as a prerequisite for speciation (Mayr 1942). Increasing evidence supports that hybridization in oaks is not an occasional phenomenon, but an important evolutionary mechanism that allows exchange of adaptive variation between different taxonomical units (Muller 1952, Van Valen 1976, Petit et al. 2004). Hybridization rates among species groups depend on several prezygotic and postzygotic reproductive barriers. Reproductive barriers are generally stronger among phylogenetically distant species, due to several physiological incompatibilities (e.g. pollen pistil interactions; Boavida et al. 2001). No natural hybrids between oak species belonging to different sections have been reported (Rushton 1993), although some artificial crossings were successful (e.g. Q. ilex x Q. robur ; Schnitzler et al. 2004). Flowering phenology sets an important prezygotic barrier to hybridization or may result in a directionality of interspecific gene flow (Muller 1952, Van Valen 1976, Belahbib et al. 2001, Varela et al. 2008). Ecological barriers among interfertile oak species may also limit hybridization. Intermediate forms are mostly observed in restricted zones where the habitats of two or more species overlap (Muller 1952, Rushton 1993). Out of these intermediate zones natural selection favours the parental species. Directional selection can lead to the re emergence of the parental species after a few generations of backcrossings (Petit et al. 2004). However, given that this selection appears to affect a limited portion of species’ genomes, species are able to exchange substantial amounts of genetic information through these hybridization zones (Bodénès et al. 1997, Lexer et al. 2006, Scotti Saintagne et al. 2004a). Based on such observations, Van Valen (1976) defined species as an ‘adaptive zone minimally different from that of any other lineage in its range and which evolves separately from all lineages outside its range’. Van Valen (1976) additionally supports that incomplete reproductive isolation permits better evolutionary adaptation by allowing the exchange of mutually beneficial genes. Various methods have been used to provide evidence about hybridization including controlled crosses, pollen viability studies, karyotype analyses, analysis of macro and
8 3. Literature review
micro morphological traits and of molecular markers (Rushton 1993). The majority of natural hybridization studies are based on morphometric traits and molecular markers. An overview of both methods is given in the next chapters.
3.2.1. The use of morphological traits Morphological characters had been the main tool for taxonomic classification within the genus Quercus from the introduction of the modern taxonomy by Linnaeus in the 18 th century until the recent preamble of molecular genetics. Various discriminant characters have been proposed for characterization of different taxonomic units. Several dichotomous keys based on flower, fruit and leaf morphology have been developed in order to describe species and sections within the genus (De Candolle 1864, Camus 1934, Schwarz 1936). With the introduction of scanning electronic microscopy (SEM), new insights into discriminative characters between species have been achieved. Foliar thrichomes and stomata shape, size and density have been used for species discrimination (Bussotti and Grossoni 1997, Aas 1998). In order to elucidate species differentiation and hybridization at the population and individual level, several quantitative methods based on morphological traits have been proposed. One of the earliest approaches was the construction of hybrid indices involving a limited number of such traits. For presentation of the data, a bivariate scatter diagram, based on two of the assessed morphometric characters, was used (Rushton 1993). This provided a quantitative estimation of natural hybridization. However, the interpretation of the results of this method may be problematic, since it is based on an arbitrary selection of discriminant characters and it relies upon the appropriateness of the used characters (Rushton 1993). The potential of analyses based on morphological traits has been increased with the introduction and development of multivariate analyses. Several different approaches have been used in oaks including principal component analysis (PCA), discriminant function analysis, cluster analysis and canonical variate analysis (Rushton 1993, Jensen 2003). The results of these methods are presented either numerically or graphically in terms of linear combinations of a set of original morphometric variables (Rohlf and Marcus 1993). The simultaneous observation of several morphometric variables allows a much more objective approach and a much more sensible biological explanation of the results (Rushton 1993). Leaf morphology has been the most important discriminator for these analyses. The use of multivariate analyses in oaks has been wide and has provided new insights about differentiation and hybridization. For instance, hypotheses about the extent and directionality of genetic introgression have been raised based on these methods (Jensen et al. 1993, Curtu et al. 2007). Although the use of morphometric traits has been valuable for taxonomists, a straightforward explanation of the results in a genetic context has been problematic. High phenotypic variation within taxonomic units makes the species boundaries fuzzy and may lead to wrong detection of hybridization. Ecological and microclimatic adaptation, as well as phenotypic plasticity, are common in oaks and increase intraspecific variation significantly (Valladares et al. 2002, Bruschi et al. 2003). Recent studies combining morphometric and genetic analyses using interfertile oaks in sympatry could not always confirm that morphological intermediacy was due to introgressive hybridization. Morphological intermediates did not necessarily match genetically admixed
3. Literature review 9
forms and, on the contrary, genetically admixed individuals possessed pure phenotypes (Curtu et al. 2007, Viscosi et al. 2009). Moreover, the establishment of phylogenetic relationships by using only morphological traits has been challenged and is not supported by more recent results from analyses of molecular markers. Putative morphological homoplasies may lead to false conclusions. For instance, phenotypically similar lobed leaves might have developed independently in different sections of oaks (Jensen 2003). Similarly, sclerophylly might have been maintained as an atavistic character in the phylogenetically remote oaks of the Mediterranean Basin, while the deciduous character in the sections Quercus and Cerris should have developed independently (Schirone and Spada 2001). The establishment of molecular methods and especially the development of numerous hypervariable DNA markers have given new opportunities for dealing with these problems.
3.2.2. The use of molecular markers The introduction of electrophoresis based analyses of proteins has been a crossroads in the history of population genetics. After their first appearance in this research area during the sixties (Lewontin and Hubby 1966), isoenzymes and alloenzymes had been gradually established as the marker of choice in plant population genetics (e.g. Gottlieb 1977). Later, and especially after the discovery of polymerase chain reaction (PCR; Mullis and Faloona 1987), numerous nuclear and organellar DNA markers have been made available. In contrast to phenotypic traits, molecular markers are completely penetrant and offer the opportunity of a random sampling of the genome, thus allowing an unbiased estimation of population genetic diversity (Hubby and Lewontin 1966). Their use for the study of population genetics in oaks has been widespread. Among others, marker assisted studies have been carried out to describe phylogeny, genetic diversity, differentiation, hybridization, spatial genetic structures, gene flow and parentage relationships. The first isoenzyme studies in oaks appeared in the late eighties and mostly dealt with diversity within and between populations and taxonomic units. A common finding of these early studies was a low interspecific differentiation between related species, partly attributed to recent speciation, and a high variation within populations (Manos and Fairbrothers 1987, Müller Starck et al. 1993). In their study with Q. rubra and Q. ilicifolia (section Lobatae), Jensen et al. (1993) concluded that isoenzymes resolved the two species worse than morphological traits (multivariate analysis of leaf morphometric traits). Isoenzymes have been used to investigate phylogenetic relationships within the genus Quercus . For instance, in an analysis of 18 oak species from eastern North America, Guttman and Weigt (1989) could differentiate well between the section Quercus and the section Lobatae, while intraspecific variation within sections was low. In addition, isoenzyme analyses provided the first molecular evidence about mating systems and hybridization directionality in natural stands of Q. petraea and Q. robur in Europe (Bacilieri et al. 1993, 1996). However, due to the low polymorphism levels and the limited number of analyzed loci, use of isoenzymes decreased significantly since PCR based methods allowed a direct analysis of DNA loci. The development of DNA markers has significantly increased the opportunities of population genetic analysis in oaks. Their higher variability allowed a better resolution in studies of genetic variation. One of the most significant observations made based on
10 3. Literature review
DNA is the contrasting differentiation pattern of nuclear vs. chloroplast genomes. While species mostly represent distinct entities in terms of molecular variance, chloroplast DNA haplotypes are shared between related species. In an early study, Whittemore and Schaal (1991) examined genetic differentiation among five morphologically distinct red oaks (section Lobatae) of eastern North America based on rDNA ITS (ribosomal DNA Internal Transcribed Spacers), a nuclear DNA marker, and cpDNA RFLPs (Restriction Fragment Length Polymorphisms). They concluded that nuclear genomes ‘may be exchanged less freely than chloroplast ones’. Similar patterns have been described among several groups of related oak species (Kremer and Petit 1993). A more detailed review based on case studies in Q. robur and Q. petraea is given in the following chapter. 3.3. Q. petraea and Q. robur in Europe: Species distribution and ecological requirements. Quercus petraea and Q. robur are the main representatives of the genus Quercus in central Europe. Both of them form extensive forests in areas stretching from the Mediterranean Basin to the Scandinavian Peninsula and from Portugal to the western part of Russia. Phylogenetically they are closely related and both belong to the section Quercus. In spite of their different ecological requirements, they often occur in sympatry and natural hybridization between them has been widely reported. A vast amount of scientific works has dealt with differentiation and hybridization between them. Both Quercus petraea and Q. robur have been multifunctional tree species with a high economical, ecological, esthetical and cultural significance. Not only have they been valuable for their hard and durable wood, but their acorns have been used in animal husbandry, their bark has been used for tanning, while medicaments have been extracted from their leaves, fruits and bark. Nowadays, they are mostly demanded for their wood, which is used for construction purposes, furniture, ship building and for making wine barrels (Aas 2008a). Moreover, they play a significant ecological role, since oak stands show an increased biodiversity in comparison to other forest tree species of central Europe, like beech, spruce and fir (Ellenberg 1996).
3.3.1. Quercus petraea (Matt.) Liebl Quercus petraea is a large deciduous tree up to 40 m. It is morphologically very similar to Q. robur from which it can be distinguished by its long leaf petioles and its stalkless acorns (Aas 2008a). Its main distribution range is characterized by a temperate climate with an oceanic to sub Mediterranean influence. It grows on shallow to moderately deep, dry to moist, well drained soils on various types of geological substrate. It is light demanding, but can tolerate medium shading in young age. Many forests of Q. petraea in central Europe historically grew under strong anthropogenous influence. Coppicing has been practiced for many centuries resulting in a dominance Q. petraea in sites where the European beech ( Fagus sylvatica ) would be more competitive under natural conditions. However, during the last 200 years, many oak rich stands have been turned into conifer stands, thus leading to losses of the range of Q. petraea (Aas 2008a). A decline in oak forests (both Q. petraea and Q. robur ) has been observed during the 20 th century, which was attributed to an interaction of abiotic (e.g. frost and drought) and biotic factors (e.g. fungal infections and browsing by row deer Thomas et al. 2002).
3. Literature review 11
In future forestry, Quercus petraea is expected to play an important role not only due to its economical significance, but also due to its adaptability to drought. An increased frequency of exceptionally dry summers is expected to give Q. petraea a competitive advantage in comparison to other deciduous forest tree species of Central Europe (Leuzinger et al. 2005). Thus, under scenarios of global warming, detailed ecological, physiological, silvicultural and genetic studies extended to its whole distribution range and covering its whole ecological spectrum are a prerequisite for its rational use in Central European forests of the future.
3.3.2. Quercus robur L. Quercus robur is a large deciduous tree up to 40 m. In contrast to Q. petraea , its leaves are nearly sessile or with very short petioles, while its acorns are pedunculate (Aas 2008a). Its distribution range covers much the same area as that of Q. petraea . However, compared to Q. petraea , Q. robur can tolerate more pronounced continental climates and is thus extended further east into European Russia (Ellenberg 1996). In addition, its habitat lies mostly on nutrient rich, acidic soils where it can tolerate waterlogging. Additionally, it can be found on very dry highly calcareous or acidic sites. In less extreme sites it is replaced by more shade intolerant species like Q. petraea and Fagus sylvatica . Its silvicultural history and historical uses are similar to Q. petraea . As an ecologically ample forest tree species with a special economical importance, Q. robur , as Q. petraea , is of particular interest for future forestry, given the ongoing global warming.
3.3.3. Differentiation and hybridization between Q. petraea and Q. robur The habitats of Q. petraea and Q. robur are delimited by their different ecological requirements. The main limiting factor is water drainage, which is a prerequisite for the establishment of Q. petraea . Quercus robur can cope well with water logging and thus dominates in periodically flooded areas, being not competent enough outside these areas (Aas 2008a). In intermediate habitats the two species coexist in sympatric stands where hybridization takes place (Bacilieri et al. 1995). The first documentation of hybrid forms dates back to the beginning of 19 th century (Gardiner 1970). Since then, a large body of scientific research has dealt with differentiation and hybridization between Q. petraea and Q. robur rendering them to model species for studying interspecific gene flow in an evolutionary context. Initially, studies of natural hybridization were mostly based on morphological data. Due to a high morphological similarity between the two species, a clear separation between them and their hybrids based on simple observations has not been easy and led to a controversy about the intensity and extensity of natural hybridization. Several authorities stated that ‘free’ and ‘wide’ introgression between the two species exists (review in Gardiner 1970). This opinion was supported by the first cytological observations which revealed no karyotypic differences between Q. petraea and Q. robur (Hoeg 1929). The first attempts to quantify introgression between them were made by carrying statistic analyses in natural populations mainly based on leaf morphometric data. Large scale studies carried out in Ireland, Scotland, Sweden and Yugoslavia which supported increasing introgression in the North (Krahl Urban 1951, Cousens 1962, Cousens 1963, Cousens 1965). Since the late seventies, more advanced multivariate methods have been used for the study of introgression between the two species (Rushton 1979). Multivariate analyses
12 3. Literature review
of morphological traits are still used today as an ‘easy to use’ tool for discriminating between Q. petraea , Q. robur and their hybrids (Kremer et al. 2002). The introduction of molecular markers has opened new perspectives in the study of differentiation and hybridization between Q. petraea and Q. robur since the early nineties. Kremer et al. (1991) revealed for first time the high degree of cpDNA sharing versus a species specific nuclear variation between Q. petraea and Q. robur . These first results gave the motivation for large scale multipopulation phylogeographic studies. In the species complex of Q. petraea , Q. pubescens and Q. robur , it was shown that chlorotypes are regionally distributed and do not correlate with species (Petit et al. 1993, Petit et al. 1997, Olalde et al. 2002). These results support that cpDNA geographic distribution reflects the postglacial recolonization of Central Europe, whereas subsequent interspecific gene flow has led to an exchange of chlorotypes among species. Strong evidence towards this view was provided by a large scale study including 2600 populations of eight oak species belonging to the section across Europe Quercus (Petit et al. 2002). First, unique chlorotypes and higher cpDNA diversity were observed in refugial areas, showing that the sources of the Quartenary migration were located there. Only a fraction of these chlorotypes was observed in non refugial areas. Second, a longitudinal gradient of cpDNA lineages in Central Europe reflects the northward recolonization routes from the three main refugia, the Iberian, the Italian and the Balkan Peninsulas. On the other hand, nuclear genetic variation mostly follows a species specific pattern. The first evidence of this interspecific differentiation arose from isoenzyme studies. However, low variability and the lack of diagnostic loci restricted the discriminative power of these markers. Nevertheless, although allele frequency differences between the species were small, these were significant at a fraction of loci (Kremer et al. 1991, Bacilieri et al. 1995) and constant over large geographic areas (Zanetto et al. 1994, Gömöry et al. 2001), supporting that species maintain their genetic identities throughout their distribution range. The introduction of hypervariable nuclear DNA markers and the development of computationally intensive assignment methods led to a better resolving power of molecular methods. High and statistically significant genetic differentiation between Q. petraea and Q. robur across their distribution range was revealed by analyses based on multilocus studies with SSR markers (Streiff et al. 1998, Muir et al. 2000, Muir and Schlötterer 2005). However, these results were again based on allele frequency differences among the studied populations. No diagnostic loci between the two species (i.e. uniquely amplified in one of two species) were found. Moreover, a considerable amount of genetic variation was shared between the two species (Scotti Saintagne et al. 2004a). Both common ancestry and interspecific gene flow have been discussed as the reasons for this pattern (Muir and Schlötterer 2005, Lexer et al. 2006). Genetic analyses including adult trees and progenies in mixed stands provided direct evidence about levels of interspecific gene flow between Q. petraea and Q. robur under natural conditions. This has been proved either by analyzing allele frequencies in pollen clouds (Bacilieri et al. 1993, 1996), or based on parental assignments of progenies (Streiff et al. 1999, Curtu et al. 2009). At the level of adult trees, molecular data supported the presence of a limited number of genetically intermediate individuals in sympatry (Curtu et al. 2007, Gugerli et al. 2007, 2008) agreeing with previous studies based on morphology. A directionality of the interspecific gene flow has been also shown with Q. robur being more often pollinated by Q. petraea than the reciprocal (Bacilieri et al. 1993, 1996, Curtu et al. 2009, Jensen et al. 2009). This is in agreement with differential success of controlled crosses between the two species, showing that pollination of Q. robur mother trees with
3. Literature review 13
pollen from Q. petraea by far was more successful than the opposite (Aas 1988, Rushton 1977). It has also been revealed that the rate and direction of interspecific pollen flow can vary depending on the spatial distribution of the potential father trees. For instance, Streiff et al. (1999) found an excess of Q. robur pollen donors in a progeny array of a Q. petraea mother tree, which was attributed to the dominance of Q. robur adults surrounding this tree. In a more recent study, Lepais et al. (2009) stress the impact of species relative abundance on hybridization rate and introgression directionality. In addition, paternity assignment revealed increased interspecific crossings in northern latitudes (Jensen et al. 2009) agreeing with earlier morphological studies which supported more extensive introgression there (Olsson 1975, Rushton 1979). The abundant molecular data significantly improved the stand of knowledge about differentiation and hybridization between Q. petraea and Q. robur . They have also provided ‘feed for thought’ in the discussions about the underlying evolutionary mechanisms. A focal point of these discussions has been the role of natural selection in the maintenance of the genetic identities of the two species. An important outcome of the molecular studies is that the taxonomic component of differentiation is not equally expressed across the genome. A limited proportion of loci accounts for interspecific differentiation (Bodénès et al. 1997, Kremer et al. 2002, Scotti Saintagne et al. 2004a, Curtu et al. 2007). In a model based analysis, Scotti Saintagne et al. (2004a) compared genome wide differentiation between Q. petraea and Q. robur at 389 loci against the neutral expectation – corresponding lack of natural selection – and found only 12% of outlier loci. These loci, in spite of being per se neutral, might be hitchhiked representing genome regions where divergent selection accounts for species differentiation. On the contrary, the rest of the genome might be permeable and thus subjected to homogenizing gene flow. This hypothesis agrees with modern species concepts pointing the gene as the main unit of speciation and assuming porous genomes (Wu 2001, Nosil et al. 2007, Lexer and Widmer 2008). Less is known about genetic variation at genome areas of Q. petraea and Q. robur which do not display the traces of divergent selection. In these genomic regions, gene flow may result in an exchange of neutral or mutually beneficial genetic variants in what has been named ‘adaptive introgression’ (Lexer and Widmer 2008). This phenomenon has been already observed in other interfertile plant species groups (e.g. in Helianthus spp.; (Kane et al. 2009). In oaks, a few studies have focused on the comparison of interspecific versus intraspecific genetic differentiation using data from ecologically divergent sites. Bodénès et al. (1997) described one gene locus showing a high genetic variation among different regions, but very low interspecific introgression at the local level. In addition, observations about genetic variation along ecological clines have been made within species, showing that adaptation influences a limited portion of the genome (Alberto et al. 2010). The extensive cpDNA sharing, as well as the direct molecular evidence of hybridization between Q. petraea and Q. robur support that the exchange of genetic variants through gene flow is possible. However, the influence degree of interspecific gene flow as an evolutionary factor is still debated (Muir and Schlötterer 2005, Lexer et al. 2006). More studies of ‘neutral’ and adaptive introgression within and between the two species using multipopulation approaches in a large scale are needed, in order to better understand the consequences of natural hybridization on their genomes.
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3.4. Quercus alnifolia , Q. coccifera and Q. infectoria ssp. veneris in Cyprus: Species distribution and ecological requirements. The island of Cyprus along with Southern Asia Minor is placed within one of the ten most significant biodiversity hotspots of the Mediterranean Basin (Medail and Quezel 1997). The highly diverse geological substrate and the variable relief of the island result in a rich flora and vegetation, as well as high levels of endemism (Tsintides et al. 2002). The genus Quercus is represented by three species on the island, Quercus alnifolia and Q. coccifera , both evergreen, and the semi deciduous Q. infectoria . Quercus alnifolia is a narrow endemic restricted to the Troodos Mountains on the central and south western part of Cyprus, where it is abundant. Quercus coccifera is a common element of various sclerophyllous communities, whereas Q. infectoria has a more restricted distribution range mainly depending on water availability (Meikle 1977, Tsintides et al. 2002). All three species are absent from natural associations of the central plain due to its increased aridity. According to recent molecular evidence, the first two species belong to the section Cerris, as this was initially defined by Manos et al. (1999) and are closely related the Mediterranean ‘ilicoid’ oak species Quercus ilex and Q. aucheri (Denk and Grimm 2010). The phylogenetic status of Quercus infectoria was disputed until very recently (Meikle 1977, Schirone and Spada 2001). According to molecular evidence published in the last years, the species should be placed into section Quercus along with Q. petraea , Q. robur , Q. pubescens and other deciduous oak species of Europe, since it shows a strong genetic affinity with these species (Denk and Grimm 2010).
3.4.1. Quercus alnifolia Poech. Quercus alnifolia is an evergreen shrub or a much branched wide crowned small tree up to 10 m, exceptionally reaching 14 m under optimal conditions (Meikle 1977, Knopf 2006). It is characterized by its dark green glabrous leaves with their golden tomentous lower surface. It grows exclusively on the Troodos Mountains where it is confined to ultrabasic igneous rocks of the ophiolite complex, in altitudes ranging from 400 to 1800 m. Its whole habitat is characterized by a Mediterranean climate with increasing winter cold and decreasing summer aridity at the higher altitudes. At the lower altitudes it is restricted to the moister sites and it is gradually replaced by more drought tolerant species (Knopf 2008). Its upper altitudinal limit is characterized by relatively harsh winters with temperatures dropping to 15°C and snow cover lasting 3 4 months (Tsintides et al. 2002). Its total area of distribution is 23 700 ha (Esser 1996). Within its distribution range, Q. alnifolia plays an important ecological role. It occurs in dry habitats, where it is associated with pines ( Pinus brutia or P. nigra ) or, less often, forms high maquis in mesic habitats characterized by deep forest soils with mull humus (Barbero and Quezel 1979). Moreover, it colonizes loose diabasic screes contributing to soil stabilization. Quercus alnifolia can readily regenerate vegetatively after fire or felling through coppicing. Besides its significant role in soil protection against erosion, its fruits offer an excellent food source to the local fauna. In contrast to Q. coccifera , it is not used as a food source for grazing animals. In earlier times its hard wood has been widely used in the past for construction of tools, chairs and parquet floors. Nowadays, it is still used for production of high quality charcoal (Knopf 2008).
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3.4.2. Quercus coccifera L. Quercus coccifera is a dominant element of the sclerophyllous communities throughout the Mediterranean Basin. It is an evergreen shrub or small tree up to 10 m, occasionally attaining large heights up to 20 m (Meikle 1977, Chatziphilippidis 2006). Its leaves are leathery with serrate margins and glabrous or thinly stellate pubescent lower surface. In Cyprus, it can be found in a wide variety of ecosystems occurring in altitudes from near sea level up to 1400 m. It grows both on limestone and igneous rocks and can occur under drier site conditions than Q. alnifolia (Tsintides et al. 2002). It can be found more often as an understory element of pine forests ( Pinus brutia ). It also occurs in association with Q. infectoria on deeper soils on calcareous geological substrate in the south western part of Cyprus, whereas dense maquis communities where Q. coccifera is dominant are not common in Cyprus (Barbero and Quezel 1979). It is not an important source of wood, but it plays an important ecological role. As Q. alnifolia , it possesses a high ability of vegetative regeneration and it is tolerant against overgrazing and fire (Chatziphilippidis 2006). Besides its importance for animal grazing (mainly goats), its acorns are consumed by the local wild fauna. Additionally, it offers protection against soil erosion with its dense foliage. In earlier times, it was famed for the crimson dye obtained from the scale insect ( Kermes vermilio ) infesting the twigs (Meikle 1977). The subspecies calliprinos , cited by several authors, is characterized by larger dimensions of individuals and occurs in the eastern Mediterranean including Cyprus. In contrast, the subspecies coccifera grows in the western part of the Mediterranean and mostly attains shrubby dimensions. However, neither a clear cut geographic restriction, nor diagnostic morphological (Bussotti and Grossoni 1997) or molecular traits (Denk and Grimm 2010) support this intraspecific separation.
3.4.3. Quercus infectoria ssp. veneris A. Kern Quercus infectoria ssp. veneris is a semi deciduous robust tree attaining 10 m height. In Cyprus, it grows in altitudes from sea level up to 1500 m. Its distribution is mostly marginal and restricted to small fragmented populations along streams or at the borders of cultivated land. These are believed to be remnants of extended woodlands formerly covering the southern part of the Troodos Mountains (Meikle 1977, Christou 2001). From the ecological point of view, Quercus infectoria ssp. veneris plays an important role by forming a special phytosociological association (Anagyro phoetidae Quercetum infectoriae) characterized by a rich flora and developing deep forest soils (Barbéro and Quézel 1979). However, these sites are often used agriculturally due to their high fertility, thus threatening Quercus infectoria ssp. veneris with limitation and fragmentation of its habitat. For this reason, it is currently under protection and its importance is mainly ecological and esthetic. In earlier times it has been used for timber production, dyes, acorns, and medical extracts, whereas its acorns were used for animal husbandry (Christou 2001).
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3.4.4. Differentiation and hybridization between Q. alnifolia and Q. coccifera The distribution ranges of Q. alnifolia and Q. coccifera are overlapping in wide areas in the igneous rock formations of the Troodos Mountains in altitudes between 400 and 1400 m. In these areas, the two species occur in sympatry and sometimes form mixed stands. Individuals with intermediate morphology are known to local people, among others because they were preferentially used for grazing by goats in past. However, differentiation and hybridization between the two species have been only recently investigated scientifically. A botanic description of hybrid individuals was published by Hand (2006). The first analyses of morphological differentiation between the two species were carried out with use of leaf morphometric traits. Distinct morphology was supported by statistically significant differences of specific leaf dimensional characters. Besides some sparse designated hybrids, no indications of extensive introgression could be provided (Neophytou et al. 2000, Neophytou et al. 2007). This result, together with an observed rarity of intermediate individuals (Hand 2006), suggests that hybridization between the two species may be occasional. However, segregation of the parental phenotypes in backcrossed individuals can result in underestimation of genetic introgression (Anderson 1948, Rushton 1993). An additional finding based on leaf morphology is a reduced variability of Q. alnifolia in comparison to Q. coccifera (Neophytou et al. 2000). At the molecular level, there are only sparse data about differentiation between Q. alnifolia and Q. coccifera and these mostly focus on phylogenetic relationships. According to isoenzyme, chloroplast DNA and nuclear DNA studies, the two species show affinity to other evergreen members of the section Cerris (Toumi and Lumaret 2001, Lumaret et al. 2002, Denk and Grimm 2010). Previously, Q. alnifolia had been classified to the ‘Cerris group’ (along with Q. cerris and Q. suber ) and Q. coccifera to the ‘ilex group’ (along with Q. ilex ), both being subclades of the section Cerris, based on morphological traits (Camus 1934). According to recent molecular data, both species are classified to the ‘ilex group’. At the population level, a reduced variability of Q. alnifolia in comparison to Q. coccifera was inferred from an analysis of seven isoenzyme loci (Toumi and Lumaret 2001). On the other hand, all alloenzymes observed in Q. alnifolia were also present in Q. coccifera , thus not precluding that the two species are able to exchange their genetic variants through hybridization. In other evergreen species of the section Cerris, it has been shown that hybridization rates vary depending on phylogenetic relationships. For instance, limited hybridization has been reported between Q. ilex and Q. suber (Mir et al. 2009), which belong to different groups of the section Cerris (‘Ilex group’ and ‘Cerris group’ respectively; Denk and Grimm 2010). Post pollination reproductive barriers hamper pollination of Q. suber from Q. ilex (Boavida et al. 2001). However, this limited hybridization was sufficient for an exchange of cpDNA haplotypes. Quercus suber often possesses cpDNA lineages typical for Q. ilex , while the opposite has been much more rarely observed, thus reflecting the aforementioned directionality of hybridization (Belahbib et al. 2001, Jimenez et al. 2004, Mir et al. 2009). On the other hand, hybridization between Q. ilex and Q. coccifera , both belonging to the ‘ilex group’ is more common as supported by both nuclear and chloroplast DNA admixture (Jimenez et al. 2004, Ortego and Bonal 2010).
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A recent study indicates that nuclear genetic differentiation among species in the section Cerris is more pronounced in comparison to the section Quercus. Based on rRNA markers, the ‘ilex group’ is the best resolved with several species specific mutants, while among white oaks classification at the species level is not possible (Denk and Grimm 2010). A higher molecular differentiation may be connected with a more limited hybridization in comparison to the section Quercus. In the case of Q. alnifolia , a distinct ITS sequence from 5S rRNA was found compared to all its Mediterranean counterparts. On the other hand, Quercus coccifera shares some of its alleles with Q. ilex and Q. aucheri . However, inferences about hybridization rates cannot be drawn from most phylogenetic studies, since individual sampling is mostly very limited. Further studies based on highly variable nuclear and chloroplast DNA loci and on larger sample sizes are required, in order to provide insights into differentiation and hybridization between Q. alnifolia and Q. coccifera .
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4. Accomplished research In this chapter, results of the research are summarized following the order of the scientific objectives. 4.1. Detecting interspecific and geographic differentiation in two interfertile oak species ( Quercus petraea (Matt.) Liebl. and Q. robur L.) using small sets of microsatellite markers (Manuscript I) The results of the research conducted in relation to the first objective of this study were published in the manuscript entitled ‘Detecting interspecific and geographic differentiation patterns in two interfertile oak species ( Quercus petraea (Matt.) Liebl. and Q. robur L.) using small sets of microsatellite markers’ (Manuscript I). In the following chapters, the used methodology and the main results are summarized and discussed briefly.
4.1.1. Methodology In order to survey interspecific and geographic patterns of genetic variation within and between Q. petraea and Q. robur , pure autochthonous populations of the two species from Central Europe and the Balkan Peninsula were sampled. Three geographic areas, South Western Germany, Central Bulgaria and Northern Greece, were chosen along an ecological gradient with increasing aridity towards south. From each area, one population of each Q. petraea and Q. robur were sampled. Multilocus genotypic data from hypervariable nuclear DNA microsatellites were used to carry out population genetic analyses. Diversity and differentiation were assessed at the population level for each locus. Loci with high genetic differentiation (i.e. a high F ST value) between species were characterized as ‘species discriminant’. Similarly, loci with high intraspecific differentiation among provenances, but lower interspecific differentiation were characterized as ‘provenance discriminant’. Locus specific F ST s were tested for deviation from the neutral expectation both within and between species. Finally, a Bayesian analysis of genetic structures was carried out using all loci jointly (Structure; Pritchard et al. 2000), as well as by treating ‘species’ and ‘provenance discriminant’ loci separately.
4.1.2. Results and discussion Among a total of 20 SSR loci tested, 14 displayed Hardy Weinberg Equilibria which were generally constant among all studied populations. Six other loci were dismissed, since they presented heterozygote deficits due to ‘null’ alleles. Three ‘species discriminant’ loci – QrZAG30, 96 and 112 – were detected. They exhibited high frequency of one allele and very low diversity in one species, whereas they remained highly variable in the other.
Locus QrZAG96 was an ‘outlier’ possessing a significantly higher F ST value the expected one under selectively neutral conditions. The other two ‘species discriminant’ loci (QrZAG96 and 112) possessed nearly significant values. In addition to the three ‘species discriminant’ loci, five ‘provenance discriminant’ loci – QpZAG1/5, 15 and 110,
QrZAG87 and 101 – were found. These loci showed a higher F ST value among 4. Accomplished research 19
populations of the same species, than between the two species in general. An examination of allele frequencies of these loci revealed frequency gradients, often with a predominant allele in both species at the one edge of our ecological gradient. Interspecific differentiation at these loci was significantly lower than expected under selectively neutral conditions at the interspecific level, as revealed by neutrality tests. By performing a model based Bayesian cluster analysis on all used loci, the two species could be well resolved by setting two modelled subpopulations (K=2), which corresponded to the uppermost hierarchical level of structure. A meaningful clustering was also received by setting four modelled subpopulations (K=4). Within species substructures were revealed corresponding to the German populations on one hand and to the Balkan populations (Greek and Bulgarian) on the other. By restrictively using the same method with the ‘species discriminant’ loci, taxonomic clustering was even more distinctive, whereas geographic differentiation was weaker. Results were remarkably different when the ‘provenance discriminant’ loci were used. Two derived clusters were depicted; one corresponding to both Q. petraea and Q. robur populations from Germany and another including the Balkan populations (Greece and Bulgaria). A further clustering to species could not be achieved. Finally, high degree of individual admixture and loose structure mostly resolving species was observed for the remaining six loci. The aforementioned results are supportive of a directional selection acting at ‘species discriminant’ loci and accounting for maintenance of species integrity. In general, selection for one adaptive trait leads to gene fixation. As a result of genetic hitchhiking, allelic frequencies at neighbouring neutral marker loci are also influenced (Andolfatto 2001). In our case, one allele presented a very high frequency in Q. robur at locus QrZAG96, while this locus remained variable in Q. petraea . According to a QTL association study, this locus resides within a genomic area associated with a morphological QTL (petiole length as a ratio of the total leaf and petiole length), which is strongly discriminative between Q. petraea and Q. robur (Kremer et al. 2002, Saintagne et al. 2004). On the other hand, loci QrZAG30 and QrZAG112 were highly variable in Q. robur , whereas in Q. petraea one allele occurred in high frequency. Directional selection and hitchhiking effects in Q. petraea might have resulted to this pattern. Differential adaptation along an ecological gradient might have contributed to the formation of common genetic structures between Q. petraea and Q. robur at the five ‘provenance discriminant’ loci. Clinal variation in oaks has been observed among others for bud burst, growth, stem form and frost hardiness (Kremer et al., 2002). Interestingly, a QTL association study reveals that locus QrZAG87 resides in a genome region coding for timing of bud burst (Scotti Saintagne et al. 2004b). Moreover, Porth et al. (2005) could map a putative osmotic stress modulated gene (1T21) only 3,1cM apart from locus QrZAG101. These adaptive traits are expected to follow diversity patterns corresponding to differential ecological conditions, with bud burst being influenced by photoperiod and osmotic stress being more intensive with increasing aridity. The fact that interspecific population pairs from ecologically equivalent areas in our study show common genetic structures makes the hypothesis of adaptive introgression (common adaptation in presence of interspecific gene flow) plausible. A respective common structure was not observed for the six remaining loci (neither ‘species’ nor ‘provenance discriminant’) which demonstrated loose structures and high admixture, probably representing selectively neutral genome regions.
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4.1.3. Conclusion By using a multilocus approach genetic structures among regions and between species were studied. The ‘null’ hypothesis of genetic differentiation absence either at the taxonomic or the geographical level was disproved. However, diverse patterns of genetic differentiation were revealed depending on the marker set used. Three ‘species discriminant’ loci displayed genetic structures between species but not among regions, expressing the taxonomic factor of differentiation. Five ‘provenance discriminant’ loci showed mainly geographic variation, being locally shared between species at the regional scale. The remaining six loci were loosely structured between the two species. Individual genetic admixture based on these loci was high. From the evolutionary point of view, these results fit speciation theories suggesting that directional selection at a limited number of genes accounts for maintenance of species integrity, affecting adjacent genomic regions, while the rest of the genome can be exchanged through interspecific gene flow (Wu 2001, Nosil et al. 2007, Lexer and Widmer 2008). ‘Species discriminant’ loci from the present study might represent such genomic regions. On the other hand, the common geographic structures of the ‘provenance discriminant’ loci suggest adaptive introgression and may carry the genetic imprint of common adaptations of Q. petraea and Q. robur along the studied ecological gradient. The remaining six SSR loci seem to express “permeable” genome regions subjected to interspecific gene flow, which is supported by the high degree of individual and population admixture. 4.2. Interfertile oaks in an island environment. I. Contrasting patterns of nuclear and chloroplast DNA differentiation between Quercus alnifolia and Q. coccifera in Cyprus (Manuscript II)
4.2.1. Methodology Interspecific differentiation between Q. alnifolia and Q. coccifera was investigated by sampling several populations across each species distribution range. First, one large sample per species (96 individuals each) was collected from a pure population. Second, a mixed stand including both species (207 individuals of Q. alnifolia and 66 individuals of Q. coccifera ) and four individuals with intermediate morphology were sampled. Third, six further smaller samples (5 12 individuals) were collected from 12 additional phenotypically pure stands of each species in a greater geographic scale. The large samples were used for an estimation of genetic variation parameters and an analysis of molecular variation (AMOVA; Excoffier et al. 1992). A model based Bayesian procedure (Structure; Pritchard et al. 2000) was carried out using all samples, in order to detect genetic structures and assign individuals to groups. Finally, cpDNA differentiation among populations was calculated based on Slatkin’s R ST (Slatkin 1995) and significance of spatial genetic barriers was assessed by bootstrapping.
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4.2.2. Results and discussion Results showed a significant differentiation between Q. alnifolia and Q. coccifera in terms of nuclear microsatellite variation. Yet this differentiation was due to allele frequency differences. No locus was found with a strictly diagnostic species specific pattern. Differentiation between species varied from locus to locus. Two loci – QrZAG11 and QrZAG112 – were highly discriminant. In particular, at locus QrZAG11, one allele presented a very high frequency in Q. alnifolia , whereas in Q. coccifera a high diversity was observed. At locus QrZAG112, both species possessed a different characteristic allele in a high frequency. According to AMOVA, the largest percentage of genetic variation was found within populations and between species. Variation among populations and within species was very low and non significant in most cases. On the contrary, interspecific differentiation was high and significant at all analyzed loci. By performing a model based Bayesian cluster analysis based on all loci used (Structure; Pritchard et al. 2000), the two species could be well distinguished by setting two modelled subpopulations (K=2). No additional clustering was received by increasing the number of modelled subpopulations, showing homogeneity within species. High membership proportions and low number of admixed individuals indicated limited genetic introgression, either in pure populations or in sympatry. In contrast to Q. alnifolia and Q. coccifera in Cyprus, an increased occurrence of introgressed genotypes has been observed in sympatry among closely related white oaks in continental Europe (Curtu et al. 2007, Gugerli et al. 2008, Salvini et al. 2009). Opposite to nuclear microsatellite markers, chloroplast DNA haplotypes were largely shared between the two species and presented a regional distribution. Common chlorotypes were often observed between neighbouring Q. alnifolia and Q. coccifera populations. Chloroplast DNA sharing at the regional level may be the result of past colonization and historic genetic introgression events. In general, cpDNA variation in Mediterranean sclerophyllous oaks reflects very ancient migration and introgression events, since it remained unaffected by the Quartenary glaciation (Jimenez et al. 2004, López de Heredia et al. 2007). Ancestral lineages are concentrated in the eastern part of the region is in agreement with the hypothesis of an eastern origin of these species and a westward colonization during the Tertiary (Zhu 1993, Lumaret et al. 2002, Lumaret et al. 2005).
4.2.3. Conclusion In the present publication, genetic variation among regions and between species was tested against the ‘null’ hypothesis of absence of genetic differentiation either at the taxonomic or the geographical level. Highly significant interspecific differentiation between Q. alnifolia and Q. coccifera was shown by an analysis of nuclear microsatellite loci. This differentiation was pronounced in pure populations, as well as in sympatry indicating low levels of interspecific gene flow. Within species, differentiation was lower and non significant at most loci. In general, it was shown that hybridization between Q. alnifolia and Q. coccifera does not lead to high levels of genetic introgression at least in the nuclear genome, whilst populations within species are not significantly structured. On the other hand, cpDNA haplotypes were shared between species at the regional level. A higher degree of cpDNA sharing between the two species might be the result of historic
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hybridization events. The fact that common structures are shared at the regional scale supports this hypothesis. 4.3. Interfertile oaks in an island environment. II. Limited hybridization between Quercus alnifolia Poech and Q. coccifera L. in a mixed stand (Manuscript III)
4.3.1. Methodology Hybridization dynamics between Q. alnifolia and Q. coccifera in sympatry were investigated by focusing on the mixed stand of the previous study. Adult trees included individuals phenotypically assigned to the two parental species, as well a limited number of available intermediate forms. Furthermore, acorns were collected from nine mother trees, phenotypically assigned either to the two parental species of being morphologically intermediate. The adult trees of the stand were analysed by a principal component analysis (PCA) and a multiple discriminant analysis (MDA) of leaf morphometric characters, a factorial correspondence analysis (FCA) of genotypic data from nuclear microsatellite loci and a calculation of introgression index based on cpDNA haplotypes according to Belahbib et al. (2001). Progenies were genotyped using the same nuclear microsatellite loci. Male gamete heterogeneity and differentiation among progeny arrays were studied with use of a TwoGener analysis (Smouse et al. 2001). Finally, male gametic contributions (pollen clouds) were analyzed by means of a Structure analysis (Pritchard et al. 2000), in order to detect genetic structures and assign them to species groups.
4.3.2. Results and discussion As revealed by the multivariate analyses of leaf morphometric traits, Q. alnifolia and Q. coccifera were morphologically well circumscribed. By using the first principal component from the PCA, which explained 62,0% of the variation, designated Q. alnifolia and Q. coccifera could be well separated. Moreover, individuals assigned to the parental species in the field returned by 100% to their species group after discriminant analysis. The intermediate state of the four designated hybrids collected from the stand was verified by both multivariate methods used. Multivariate analysis of leaf morphometric traits has been proved to be an efficient tool to distinguish between Q. alnifolia , Q. coccifera and their hybrids. However, due to segregation of the parental phenotypes in the second and in higher generations, backcrossed individuals and genetic introgression in oaks may not be detectable based only on morphology (Anderson 1948, Rushton 1993, Jensen et al. 1993). Analysis of nuclear and chloroplast microsatellites in the adult individuals of the stand provided additional evidence towards a low degree of genetic introgression between Q. alnifolia and Q. coccifera . The median position of the four pinpointed hybrids was verified by both nuclear and chloroplast DNA analyses. In contrast to their distribution in a large scale, chloroplast genomes demonstrated a very low introgression in this mixed stand, which indicated a low interbreeding of the two species on this site. Furthermore, Q. alnifolia formed a markedly more condensed group on the scatter diagram of FCA, which may attest its lower genetic variation in comparison to Q. coccifera (Manuscript III). Similar results were found in previous studies with isoenzymes (Toumi and Lumaret 2001), but also with leaf morphological traits (Neophytou et al. 2007). This was partially attributed
4. Accomplished research 23
to genetic drift in Q. alnifolia , due to its restricted distribution area (Toumi and Lumaret 2001). Genetic analyses of progeny arrays further supported that interspecific crossings between Q. alnifolia and Q. coccifera are rare. Pollen clouds shared the same gene pool as their maternal trees. Interspecific differentiation among pollen clouds was always high and significant in contrast to the intraspecific one. Only five offspring in a total of 176 among Q. coccifera mother trees might have arisen from interspecific crosses. Four of them were observed in the same progeny array. No evidence about interspecific crossings in the opposite direction could be provided. Interestingly, the hybrid mother tree that was employed in this analysis was predominantly pollinated by Q. alnifolia paternal trees. This directionality may be due to differences in flowering phenology. Flowering in Quercus alnifolia and intermediates occurs with a delay in comparison to Q. coccifera (Hand 2006). Given that the two species are protandrous, a higher degree of fertilization of Q. coccifera and hybrid female flowers with Q. alnifolia pollen is expected than the reciprocal (it is more likely that female flowers of Q. coccifera are receptive during maturation of male flowers of Q. alnifolia than the opposite). Other factors resulting in this directionality may be the relative abundance of Q. alnifolia in the study stand, as well as post pollination reproductive barriers.
4.3.3. Conclusion Exceptionally low levels of interspecific gene flow in sympatry were supported by various different approaches followed in Manuscript III. Quercus alnifolia and Q. coccifera demonstrated separate morphological identities and distinctive genetic diversity patterns. Only four intermediate individuals among a total of 277 adult trees possessed a mixed morphological and molecular identities. The very low degree of cpDNA sharing between adult trees of the two species provided additional support towards limited hybridization and introgression. This is further supported by analysis of pollen clouds which showed that levels of successful interspecific pollinations were very low. In all cases of interspecific crosses Q. alnifolia acted as the pollen donor. Moreover, this directionality was observed in the pollen cloud of an intermediate mother tree. Male gametes of this progeny originated predominantly from Q. alnifolia . Pre or post pollination reproductive barriers may have resulted in this pattern. 4.4. Conservation of nuclear SSR loci reveals high affinity of Quercus infectoria ssp. veneris A. Kern (Fagaceae) to section Robur (Manuscript IV)
4.4.1. Methodology Cross amplification of nuclear microsatellite loci was tested in this study as a means for studying its taxonomic identity in comparison to Q. petraea and Q. robur on one hand and Q. alnifolia and Q. coccifera on the other. Conservation of 16 nuclear microsatellite loci originally developed in various white oak species (Quercus macrocarpa (Dow et al. 1995), Q. petraea (Kampfer et al. 1998), Q. robur (Steinkellner et al. 1997) and Q. myrsinifolia (Isagi
24 4. Accomplished research
and Suhandono 1997) was tested in a natural population of Q. infectoria ssp. veneris from Cyprus. Moreover, sequences of allelic variants from Q. alnifolia and Q. coccifera were compared to those of Q. infectoria ssp. veneris . In particular, allele sequences of equally sized alleles at locus QpZAG9 were analyzed in all three species. Homoplastic allele variants at this locus between oaks belonging to the sections Quercus and Cerris had been described in a previous study (Curtu et al. 2004). A comparison of the results from the three species of Cypriot provenance to published sequences was attempted as well.
4.4.2. Results and discussion All tested microsatellite loci were successfully amplified in Q. infectoria ssp. veneris and showed diploid patterns. They were polymorphic and fragment sizes matched those initially measured in the source species. Population genetics parameters also provide some preliminary indication, that this species kept high levels of genetic diversity, in spite of long time fragmentation and limitation of its habitats in Cyprus. Taking into account that the tested loci belong to 10 out of 12 genomic chromosomes and provided that genetic variation is evenly dispersed in the genome, it could be postulated that this variability might be genome wide. The degree of cross amplification using microsatellite primers has been shown to correlate with the phylogenetic affinity of the species (Steinkellner et al. 1997, Soto et al. 2003). Overlapping allele sizes among Q. infectoria ssp. veneris , Q. alnifolia and Q. coccifera at locus QpZAG9 was due to homoplasy. Quercus infectoria ssp. veneris showed three additional insertions in the microsatellite flanking regions, whilst the repetitive sequence region was shorter. On the other hand, the amplified sequences of equally sized alleles in Q. alnifolia and Q. coccifera demonstrated exactly the same insertions and deletions (indels). Homoplasy at the same locus has been reported in a comparison between Q. cerris versus four other species of the section Quercus in a natural population in Romania (Curtu et al. 2004). The sequence of Q. infectoria ssp. veneris showed an indel pattern identical to Q. robur . On the other hand, sequences of Q. alnifolia and Q. coccifera did not absolutely match the sequence of Q. cerris as reported in Curtu et al. (2004). This suggests a longer time of divergence, between the two sclerophyllous Cypriot oaks and Q. cerris , which allowed accumulation of mutations. In a recent study, Denk and Grimm (2010) provide evidence that groups ‘Ilex’, including Q. alnifolia , Q. aucheri , Q. coccifera and Q. ilex , and ‘Cerris’, including Q. cerris and Q. suber , are paraphyletic. This is in agreement with the results from the present study.
4.4.3. Conclusion Amplification patterns and diversity of the tested microsatellite loci, as well as allele sequence data at locus QpZAG9 support a high affinity of Q. infectoria ssp. veneris to section Quercus (where Q. petraea and Q. robur belong). All data present a very distinct evolutionary history in comparison to section Cerris (where Q. alnifolia and Q. coccifera belong). Furthermore, sequential similarity of Q. alnifolia and Q. coccifera supports the high phylogenetic relatedness of these species, which is in agreement with the incomplete reproductive isolation between them. Both locus diversity patterns and allele sequences reflect major genomic rearrangements and show the great phylogenetic distance between Q. infectoria ssp. veneris on one hand and Q. alnifolia and Q. coccifera on the other. Based on
4. Accomplished research 25
these results, effective gene flow between these distinct groups, at least under natural conditions, should be precluded. 4.5. Phylogenetic relationships among the study species based on chloroplast DNA haplotypes
4.5.1. Methodology In this last chapter, a comparison is made among chlorotypes (chloroplast DNA haplotypes) from all species studied. The analysis was based on seven cpDNA microsatellite loci. Each chlorotype was defined as a unique combination of the SSR allele lengths at the analyzed loci. A detailed description of the analysis method and a citation of the used loci are given in Manuscript II. Quercus alnifolia and Q. coccifera , chlorotypic data are also included in Manuscript II. In the case of Q. infectoria ssp. veneris , Q. petraea and Q. robur further unpublished data from the same loci are presented. For this purpose, a subsample was taken systematically from each one of the species populations described in Manuscripts I and III. Mutational steps among operational taxonomic units (OTUs) were calculated assuming the stepwise mutation model (SMM) by the Arlequin software (Excoffier et al. 2005). Results were used to construct an unrooted minimum spanning tree by employing the SplitsTree software (Huson and Bryant 2006). It was sought to resolve phylogenetic relationships among study oaks of section Cerris ( Q. alnifolia and Q. coccifera ) on one hand and study oaks of section Quercus ( Q. petraea , Q. robur and Q. infectoria ssp. veneris ) on the other.
4.5.2. Results and discussion In total, 20 chlorotypes were identified. Twelve chlorotypes occurred in Q. alnifolia and Q. coccifera and eight in Q. infectoria ssp. veneris , Q. petraea and Q. robur (Table 4.1). Two main lineages separated by four mutational steps could be recognized, one representing the two sclerophyllous oaks of Cyprus, belonging to section Cerris, and one representing the remaining oak species of section Quercus (Figure 4.1). Chlorotype sharing was observed within both lineages. The wide cpDNA sharing between Q. alnifolia and Q. coccifera and their geographic distribution in Cyprus was discussed in Manuscript II. Within the oaks of section Quercus, two related groups could be recognized. Chlorotypes 21, 22, 23 and 28 form a distinct clade. They were found in both Q. petraea and Q. robur and they were met in all analyzed regions, namely Greece, Bulgaria and Germany (Table 4.1). In contrast, chlorotypes 24, 25, 26 and 27 are confined to the Balkan (Greek and Bulgarian) populations of these species. This is in agreement with large scale phylogenetic studies showing that only a fraction of the refugial cpDNA variation migrated towards Central Europe during post ice age recolonization (Petit et al. 2002). Occurrence of chlorotype 24 in the Cypriot population of Q. infectoria ssp. veneris (the only one chlorotype observed there) further supported the affinity of this species to section Quercus and its distinctness in comparison to Q. alnifolia and Q. coccifera . Furthermore, this observation indicates a common evolutionary history of white oaks between Southern Balkans and the Eastern Mediterranean.
26 4. Accomplished research
Table 4.1 – Chloroplast DNA haplotypes. The name of each chlorotype, allele lengths at each SSR locus, species and region are given. A = Q. alnifolia , C = Q. coccifera , I = Q. infectoria ssp. veneris , P = Q. petraea , R = Q. robur . Chlorotype dt1 dt3 cd7 kk3 ccmp3 cd4 ccmp2 Species Region 1 81 119 83 95 115 95 235 A, C CY 2 81 119 84 95 115 95 235 A, C CY 3 81 120 83 95 115 93 234 A CY 4 81 120 83 95 115 94 234 A, C CY 5 81 120 84 95 115 93 234 C CY 6 81 120 84 95 115 94 234 A, C CY 7 81 120 84 95 115 95 234 A, C CY 8 82 119 83 94 115 93 235 C CY 9 82 119 84 94 115 93 235 A, C CY 10 82 120 83 95 115 94 234 A CY 11 82 120 84 95 115 94 234 A CY 12 82 120 84 95 115 93 234 C CY 21 78 122 83 95 116 93 233 R DE 22 79 122 83 95 116 92 233 P GR, BG, DE 23 79 122 83 95 116 93 233 P BG 24 80 120 83 95 116 91 233 I, R CY, GR 25 80 120 83 95 116 92 233 R GR 26 80 120 83 96 116 93 233 R BG 27 80 120 83 96 116 94 233 R BG 28 80 122 83 95 116 92 233 P DE
4.5.3. Conclusions Two monophyletic groups are revealed by the construction of an unrooted minimum spanning tree of haplotypes based on chloroplast DNA SSRs. The collocation of Q. alnifolia and Q. coccifera within the same phylogenetic clade and the high degree of cpDNA sharing supports their common membership to the section Cerris and particularly to the group ‘Ilex’ along with other sclerophyllous Mediterranean species. In contrast, the species Q. infectoria ssp. veneris , Q. petraea and Q. robur all belong to a distinct lineage compared to Q. alnifolia and Q. coccifera supporting their common membership to the section Quercus. Moreover, cpDNA sharing between the Cypriot population of Q. infectoria ssp. veneris and the Greek population of Q. robur underlines the close relationship of the former species to section Quercus.
4. Accomplished research 27
Figure 4.1 – Unrooted minimum spanning tree of chlorotypes observed. Distance is measured in mutational steps.
28
5. General discussion Diverse aspects of genetic differentiation and its underlying evolutionary mechanisms in oaks were elucidated in the present study. A special focus was given on hybridization, a widely distributed phenomenon in the genus Quercus . Its role in shaping genetic variation between species was investigated by examining two specific paradigms of oak species arising from drastically different environments and possessing distinct evolutionary histories. A synergy of interspecific gene flow and adaptation was shown to form and shape genetic differentiation within and between the euryoecious Q. petraea and Q. robur (section Quercus; Manuscript I). On the other hand, in the insular environment of Cyprus, hybridization was shown to be restricted and to have limited influence on interspecific nuclear genetic and phenotypic (in terms of leaf morphometrics) variation patterns between the indigenous Q. alnifolia and Q. coccifera (section Cerris). However, hybridization might have happened recurrently throughout their long co existence on the island as supported by common chloroplast DNA structures at the regional level (Manuscripts II, III). Regarding the third indigenous oak species of Cyprus, Q. infectoria ssp. veneris , a completely different phylogenetic identity and an affinity to the white oaks (section Quercus), including Q. petraea and Q. robur , was evidenced by both chloroplast and nuclear DNA data. Based on this result, natural hybridization of this species with Q. alnifolia and Q. coccifera should be excluded (Manuscript IV, Chapter 4.5). In both pairs of interfertile oaks investigated in the present study, genetic differentiation varies (a) between nuclear and organelle genomes and (b) across nuclear genomes. In the case of Q. alnifolia and Q. coccifera , sharing of the main chloroplast DNA (cpDNA) haplotypes was revealed by analysis of multipopulation data. The main chlorotypes were not only found in both species, but they formed spatial geographic structures as well. These were shared between the species at the regional level (Manuscript II). This finding supports the hypothesis of past hybridization rather than common ancestry. Since, according to geological and paleobotanical evidence, uplift of Cyprus in the Mediterranean succeeded speciation of both taxa (Robertson 1977, Palamarev 1989), the most plausible explanation for the common cpDNA structures is exchange through interbreeding. Besides cytoplasmic introgression, hybridization was shown to result in homogenizing introgression varying from locus to locus within the nuclear genomes of interfertile oaks. In the paradigm of Q. petraea and Q. robur in Europe (Manuscript I), regional structures shared by the two species were revealed by a series of ‘provenance discriminant’ loci. Two conspecific clusters corresponding to two distinct ecological regions were formed by an analysis of these loci and no taxonomic separation could be resolved using these particular loci, which again provides evidence of introgressive hybridization. On the other hand, evidence about low levels of interspecific introgression in the paradigm of Q. alnifolia and Q. coccifera was provided by a large scale multipopulation approach followed in Manuscript II. An equally high interspecific differentiation either between pure or sympatric populations was revealed either at the single locus level or by considering all loci simultaneously. Individuals with admixed coancestry were also sparse. A closer insight into interspecific gene flow was gained by performing fine scale analysis of hybridization between Q. alnifolia and Q. coccifera in sympatry (Manuscript III). It involved two generation levels, adult trees and their progenies (acorn embryos), in order to quantify interspecific matings and to infer their directionality. Results indicate that 5. General discussion 29
hybridization between these species is a rare phenomenon. Within a total of 277 trees, only four intermediate individuals were found, whose hybrid origin was supported by morphological traits, as well as by nuclear and chloroplast DNA markers. Besides these scattered hybrids, further genetic introgression could not be ascertained among adult trees in this stand. Sharing of chloroplast DNA in the mixed stand was very limited and genotypes of individuals possessing shared chlorotypes were not admixed. The low rates of hybridization were endorsed by the results from analysis of acorn embryos, which revealed very low rates of interspecific matings (Manuscript III). Moreover, an asymmetry was observed with Q. alnifolia always acting as pollen donor in the rare cases of interspecific pollinations, but also in the progeny array of a hybrid maternal tree. Results of the present study suggest that reproductive barriers play a significant role in preventing gene flow between Q. alnifolia and Q. coccifera . Prezygotic reproductive barriers may involve asynchronous flowering. This is supported by directionality of hybridization with Q. alnifolia acting preferentially as pollen donor. Flowering in Q. alnifolia occurs in average 1 2 weeks later than in Q. coccifera (Knopf 2008). Given protandry in these oak species, this flowering pattern may rather facilitate pollination of Q. coccifera by Q. alnifolia than the opposite. This hypothesis was postulated in the case of the Mediterranean species Q. ilex and Q. suber where a unidirectional hybridization with Q. suber being the paternal parent was attributed to its delayed anthesis (Belahbib et al. 2001, Jimenez et al. 2004, Varela et al. 2008). Among the seldom interspecific pollination cases of Q. coccifera by Q. alnifolia , all except one were observed in one certain mother tree. This may indicate that intraspecific variation of anthesis timing may lead to a rare and limited flowering period overlapping of the two species and in these cases hybrids are generated. One of the four hybrid trees was observed close to this particular Q. coccifera mother tree. The genotype and chlorotype of this hybrid show that it arose probably through a past interspecific pollination event of this – potentially older aged – mother tree by Q. alnifolia pollen. Different levels of hybridization among mother trees, along with a variation of flowering phenology within each species have also been reported in the species complex of Q. petraea and Q. robur (Streiff et al. 1999) and of Q. ilex and Q. suber (Varela et al. 2008). Pre and post zygotic reproductive barriers due to physiological incompatibilities may additionally restrict interspecific gene flow and influence its directionality. In the case of Q. ilex and Q. suber , pollen pistil interactions and abortions of female flowers and immature acorns shortly after pollination were reported to prevent pollination of the latter species by the former (Boavida et al. 2001, Varela et al. 2008). Complex reproductive cycle patterns exist in Q. coccifera , including biennial and annual acorn maturation, with the former occurring predominantly. Variation among reproductive cycle patterns has been found within and among individuals and has been shown to depend on weather conditions during the growing period (Bianco and Schirone 1985). The same phenomenon has been described in Q. suber , another Mediterranean oak species of section Cerris (Boavida et al. 1999, 2001; Díaz Fernández et al. 2004), whereas Q. ilex has a typical annual reproductive cycle (Bellarosa et al. 1990). These biological differences have been shown to restrict hybridization between Q. ilex and Q. suber (Varela et al. 2008) and may account for limited hybridization observed between Q. coccifera and Q. ilex as well (Ortego and Bonal 2010). In Q. alnifolia , annual acorn maturation is assumed (Hand 2006); however, detailed observations comparable to Q. coccifera and Q. suber are not available. This biological difference may set an additional barrier against hybridization between Q. alnifolia and Q. coccifera .
30 5. General discussion
The present study provides evidence that interbreeding frequency between Q. alnifolia and Q. coccifera is low. Moreover, occurrence of morphological intermediates is fairly sparse in Cyprus, although the two species often grow in sympatry. The presence of the observed hybrids along with some other exemplars around Kampos village (Hand 2006, Neophytou et al. 2007) may be due to particular ecological conditions there. Reduction of mate recognition due to environmental stress has also been reported as a factor that reinforces hybridization in oaks (Williams et al. 2001). The study stand may represent such a disturbed environment, quite proximal to a rural community where strong anthropogenic influence has been reported. The remnants of previous agricultural use are still visible and grazing by goats was practiced as recently as 20 30 years ago (A. Christou 1, personal communication). Based on the hypothesis mentioned above, disturbed environmental conditions in the studied stand may have increased the degree of successful interspecific matings allowing the limited hybridization observed. In the previous paragraphs factors have been discussed that may have enhanced or prevented interspecific matings between Q. alnifolia and Q. coccifera . Nevertheless, the degree of realized gene flow between two interfertile species depends on further seed germination, survival and fitness of hybrids. In the case of Q. alnifolia and Q. coccifera , adult hybrids were fertile and produced large amounts of seeds. However, introgression of the chloroplast genomes among adult trees was very limited in sympatry (Manuscript III). If backcrosses were successful, given a sufficient number of generations, chloroplast DNA introgression would be expected. For instance, the directionality of interspecific mating would result in chlorotype capture by Q. coccifera by Q. alnifolia . Nonetheless, results from the mixed stand do not support such an introgression pattern. It could therefore be hypothesized that acorns from backcrossing fathered by Q. alnifolia have limited or no germination success. Alternatively, the two species may have not coexisted for a long time on this site. Given that Q. coccifera is preferred as a pasture species in comparison to Q. alnifolia , anthropogenic introduction of the former in the proximity of the rural community adjacent to the study site in relatively recent times cannot be ruled out. In case the species contact is recent, the time span of their sympatric coexistence may not have been enough to allow cpDNA exchanges and genetic introgression in general. The rarity of interbreeding and the fact that regeneration through seeds is limited in the two species (Chatziphilippidis 2006, Knopf 2006) suggest that a relatively long time interval is required for an introgression of their chloroplast genomes to occur. Adaptability to the site conditions is a further important factor that can limit interbreeding and can influence the directionality of backcrosses. Ecological differentiation has been shown to set an effective barrier to hybridization. Often hybridization is well restricted to intermediate habitats (Anderson 1948, Muller 1952, Burger 1975, Petit et al. 2004). Quercus alnifolia and Q. coccifera are partially ecologically differentiated, with the first possessing a narrower habitat confined to volcanic rocks of the Troodos Mountains and the second having a wider distribution and being independent of geological formation and more drought tolerant (Knopf 2008). Sympatric populations mostly occur in the lower areas of the distribution range of Q. alnifolia , where this species seems to be less competent due to increasing drought. In the higher elevations of the Troodos Mountains, Q. alnifolia becomes more dominant and increasingly forms pure stands. The area of ecological overlapping is fairly wide and sympatric occurrence is not infrequent (Neophytou et al. 2000). However, hybrid swarms
1 Andreas K. Christou Research Section Forestry Department, Ministry of Agriculture, Natural Resources and Environment, Cyprus
5. General discussion 31
have not been observed in overlapping distribution areas and intermediate forms are very rare. Thus, it can be stated that interspecific hybridization is rather impaired by reproductive than by ecological barriers. In contrast to the above, in the Q. petraea – Q. robur complex, there is evidence that ecological differentiation plays a significant role as a barrier against a widespread establishment of hybrids. Regarding their habitat requirements, the two species are well circumscribed. Quercus petraea is drought resistant, does not tolerate water logging and can afford a moderate degree of shading. Quercus robur can cope well with flooding, but has a more pronounced pioneer character being light demanding (Bacilieri et al. 1995, Aas 1998). As shown by controlled crosses and studies in natural stands, reproductive barriers between the two species are generally weak and anthesis is synchronous (Bacilieri et al. 1995, Bacilieri et al. 1996, Steinhoff 1998). Natural selection has been shown to favour the ‘resurrection’ of the parental species through backcrosses within a few generations (Petit et al. 2004). During this natural process, neutral or mutually beneficial genetic variants may be transferred between the two taxa. Low genetic differentiation at the majority of nuclear loci (Bodénès et al. 1997, Lexer et al. 2006, Scotti Saintagne et al. 2004a) and common cpDNA structures, both in fine (Petit et al. 1997, Petit et al. 2002) and in large scale (Petit et al. 2002), support this model. ‘Manuscript I’ aimed to provide insights into the differential effects of this process among a number of hypervariable marker loci along divergent macro ecological conditions. The 14 analyzed SSR loci in Q. petraea and Q. robur displayed contrasting patterns of differentiation. Three of them sharply resolved the two species (species discriminant loci) and six of them were loosely structured between the two species, whereas five others presented geographic structures which were shared between the two species at the regional level (provenance discriminant loci). The three ‘species discriminant’ loci might represent genomic regions that encode genes responsible for the adaptive profiles of the two species. Stebbins (1950), by observing hybridization in several oak pairs, noticed that both artificial and natural hybrids segregate sharply. He raised the hypothesis that the number of genes by which species of oaks differ from each other should be very small. Scotti Saintagne et al. (2004a), in a recent molecular study, could provide molecular evidence that a limited portion of the genome accounts for interspecific differentiation between Q. petraea and Q. robur , pointing towards natural selection to account for the re emergence of the parental species. Highly and significantly differentiated markers could be located on nine out of twelve chromosomes. However, only 12% of the 389 tested markers were characterized as ‘outliers’ due to the limited size of hitchhiking regions. The three ‘species discriminant’ loci of the present study might represent genome regions affected by selection of genes accounting for species integrity. In all three cases a significant decrease of variation in one species versus a diverse pattern in the other was observed pointing to selection. One of these loci lies within a QTL coding for a leaf morphological trait, which is diagnostic between the two species (Saintagne et al. 2004). It is noteworthy that genetic variation pattern of the three ‘species discriminant’ loci of the present study is stable across all study populations. Genetic differentiation patterns of the remaining loci (i.e. apart from ‘species discriminant’) suggest high levels of introgression between Q. petraea and Q. robur . On one hand, six loci presented loose structures corresponding to the two species. Based on these loci, membership proportions of the populations to their species cluster were low and the number of genetically admixed individuals was high. On the other hand, a completely different pattern of genetic structures was revealed by using five ‘provenance
32 5. General discussion
specific’ loci. A separate analysis of these loci supported no species structures. It resulted in a clustering of two provenances, whereby the two species did not form additional structures within each provenance. The fact that differentiation between the Central European populations and the Balkan populations was high might be due to differential adaptations along an ecological cline. Petit et al. (2003) support that exchange of adaptive variation between Q. petraea and Q. robur through backcrosses is possible and is expected to increase their adaptability. Several traits present clinal adaptations along ecological gradients in these two oak species. These include bud burst, growth and stem form, and frost hardiness (Kremer et al. 2002, Alberto et al. 2010). ‘Provenance discriminant’ loci partly reside within genomic regions coding for timing of bud burst and for osmotic stress, traits that are expected to vary between Central Europe and the Balkan due to decreasing photoperiodicity and increasing drought respectively (Manuscript I). By and large, results from the present study support that interspecific gene flow between Q. petraea and Q. robur is limited by ecological rather than reproductive barriers. Presence of weak reproductive barriers and absence of reproductive isolation between the two species is further attested by the genetic differentiation pattern of ‘provenance discriminant’ markers. The common structures observed at these loci indicate a higher connectivity between heterospecific populations within a provenance, than within the same species and across provenances. A possible adaptive introgression may have enhanced the formation of these common structures. On the other hand, only a minority of loci (21%) were highly discriminant between the two species, which is in agreement with available molecular data from the last two decades (Bodénès et al. 1997, Kremer et al. 2002, Scotti Saintagne et al. 2004a, Curtu et al. 2007). Quercus petraea and Q. robur are a paradigm of interfertile species keeping their identities by occupying different ‘adaptive peaks’, according to ‘ecological speciation’ (Van Valen 1976). Species differentiation is expressed by a minimal number of phenotypic and genomic adaptations, while the rest of the genome might be open to neutral and adaptive introgression. This pattern of speciation, assuming the gene as the unit of speciation, has been widely discussed in last years (Wu 2001, Nosil et al. 2007, Lexer and Widmer 2008) and has been supported with molecular markers in several genera (e.g. Helianthus ; Yatabe et al. 2007, Silene ; Minder et al. 2007; Serapias ; Belluschi et al. 2010). Finally, within the scope of establishing phylogenetic relationships, both interfertile oak pairs of the study and Q. infectoria ssp. veneris from Cyprus were analyzed. A clear separation between Q. alnifolia and Q. coccifera on the one hand, and Q. infectoria ssp. veneris , Q. petraea and Q. robur on the other was evident. These relationships were supported both by evolutionary conservation of nuclear microsatellites and by chloroplast DNA haplotypes and reflect the distinct evolutionary histories of these species. Evolutionary distance has been shown to result in decreased locus conservation in oak and other multispecific plant genera (Steinkellner et al. 1997, Whitton et al. 1997, Soto et al. 2003). Among Q. infectoria ssp. veneris , Q. petraea and Q. robur all tested microsatellite loci were amplifiable and possessed common allele size ranges. By contrast, in Q. alnifolia and Q. coccifera , a very limited number of SSR markers were transferable (the loci included in Manuscripts II and III) and still in these cases even sized alleles were found to be non homologous in comparison to the other species (Manuscript IV). In terms of chloroplast DNA variation, two distinct clades verified the phylogenetic distance between the two different taxonomic groups. Within each clade, sharing of chlorotypes points introgressive hybridization or recent common ancestry, or a combination of both.
5. General discussion 33
The aforementioned results strongly support the phylogenetic distinctness of the two resolved groups ( Q. alnifolia and Q. coccifera vs. Q. infectoria ssp. veneris , Q. petraea and Q. robur ). A very recent phylogenetic study about western Eurasian oaks based on ribosomal DNA markers agrees with the aforementioned pattern and additionally supports a membership of Q. alnifolia and Q. coccifera to the group ‘Ilex’ of the section Cerris and that of the other species to the section Quercus along with all European white oaks and their North American counterparts (Denk and Grimm 2010). An early vicariance between in two sections at least since the beginning of the Oligocene, about 33,5 million years ago, is supported by paleobotanical data (Axelrod 1983, Tiffney and Manchester 2001). Separate evolution since then might have resulted in major genomic rearrangements, which are expressed by the low degree of locus conservation. In the paradigm of the well studied sunflowers ( Helianthus spp.), another angiosperm genus characterized by high levels of hybridization among species, extensive chromosomal repatterning between phylogenetically remote species has been shown to result in sterility barriers between the species (Lai et al. 2005). Similar rearrangements have been shown between the closely related genera Quercus and Castanea , by using highly variable SSRs (Barreneche et al. 2004). Given that time of separate evolution between the sections Cerris and Quercus is substantial (at least since the late Eocene – about 35 million years ago; Tiffney and Manchester 2001), significant genomic rearrangements may contribute to sterility between the two sections.
34
6. Conclusions and outlook Genetic differentiation and hybridization within the two complexes of interfertile oak species studied in this Thesis presented some common features, but also important differences. On the one hand, the euryoecious continental species Q. petraea and Q. robur were shown to share substantial portions of their genetic variation. Results suggest that interspecific gene flow plays a significant role in their evolution. The revealed common genetic structures in ecologically similar locations are suggestive of adaptive introgression between the two species. On the other hand, rare hybridization in the insular sclerophyllous Q. alnifolia and Q. coccifera is supported, which results in a limited degree of genetic introgression between the two species. Nuclear interspecific genetic variation was high both between pure and sympatric populations, whilst chloroplast DNA introgression in sympatry was also very limited. Low frequency of interspecific pollinations suggests that reproductive barriers may restrict gene flow between them. Finally, a phylogenetic distinctness between the two oak complexes mentioned above is supported by nuclear and chloroplast variation and underlines the completely divergent evolutionary histories of them. The third oak species of Cyprus, the semi deciduous Q. infectoria ssp. veneris , shows a markedly close affinity to Q. petraea and Q. robur , thus pointing to its high evolutionary divergence and genetic isolation in comparison to Q. alnifolia and Q. coccifera . Results from the current study, besides their evolutionary relevance, may set the starting point for scientific and practice oriented applications involving the five included species. The identification of ‘provenance discriminant’ markers in Q. petraea and Q. robur could be a useful tool for characterization of individual trees, populations and reproductive material (certification of both reproductive material and logs). As a consequence of the ongoing climatic change, a shift of climatic zones is already observed, forcing species to migrate. Migratory responses even faster than the maximum post glacial rates might be required if the present global warming continues with the same speed (Pearson and Dawson 2005, Aitken et al. 2008, Vitasse et al. 2009). Connectivity of populations and establishment of corridors allowing species movement northwards and towards higher altitudes are important measures towards a sustainable conservation of forest tree species (Lindenmayer et al. 2000). Connectivity in terms of adaptive variation may be enhanced by high levels of interspecific gene flow resulting in a flexible ecological reaction. In addition, under scenarios of rapid global warming, facilitated gene flow of preadapted alleles (assisted migration) from warmer climates may be necessary to promote adaptation at the leading edge of migration (Aitken et al. 2008, Savolainen et al. 2007). Identification of adaptive clines is a prerequisite for the implementation of conservation strategies. ‘Provenance discriminant’ microsatellite loci from the present study show variation along an ecological gradient and suggest adaptation processes. These loci may be an ‘easy to use’ tool for the recognition of such clinal variation. Inclusion of additional sites and refugial areas will refine the resolution of the results. Moreover, marker assisted study of genomic regions under selection could complement QTL studies for adaptive traits. The increasing availability of EST derived microsatellite markers in oaks introduces a new perspective towards this study area. Regarding Q. alnifolia and Q. coccifera , novel results were provided about their genetic differentiation and hybridization patterns. Although a limited number of loci from other oak species were usable, highly discriminant markers were found. The utility of these markers for further population genetic studies within and between the two species could 6. Conclusions and outlook 35
assist conservation policies. The recent development of EST derived hypervariable microsatellites which are highly conserved among different taxonomic groups (Ueno et al. 2008) may increase the potential of population genetic studies. Study of reproductive barriers between the two species should be a priority in future studies. Controlled crosses and study of the fertilization process would elucidate the underlying mechanisms restricting interspecific gene flow. An additional point of interest is the extension of fine scale studies in more mixed stands under varying ecological conditions. This could provide clues about mechanisms of interspecific gene flow between the two species. It would further elucidate the particular fine scale vs. landscape scale distribution of haplotypes revealed by the present study, as well as a potential adaptive significance of hybridization leading to criteria for in situ conservation strategies. This applies especially for Q. alnifolia which is one of the most notable endemic species of Cyprus. Furthermore, given that the Mediterranean climatic zone is expected to migrate northwards (Giorgi and Lionello 2008), new habitats may be created in Europe, which could be suitable for these two species. Knowledge of their genetic resources and patterns of hybridization is a prerequisite for their potential use out of their current habitats. An even higher need for conservation in Cyprus exists for Q. infectoria ssp. veneris . The populations of this species are small and fragmented and its main habitat is largely used for agriculture (Christou 2001). The study of evolutionary conservation of hypervariable SSR loci (Manuscript IV) provides an efficient tool for assessment of its genetic resources and detection of inbreeding and spatial genetic barriers. Additionally, given its high wood quality it may be a potential candidate for use in European forestry. It is of particular interest to study possible drought adaptations and underlying QTLs, given climatic aridity in its distribution area in Cyprus. Its high phylogenetic affinity to European deciduous oak species of the section Quercus suggests that it should be interfertile with the central European Q. petraea and Q. robur . Genetic introgression from xeric oak species into Q. petraea and Q. robur in southern areas of their distribution ranges under natural conditions has been reported and resulted in a series of intermediate phenotypic forms which had been wrongly recognized as separate species (e.g. Q. pedunculiflora , Q. virgiliana , Q. longipes and Q. erucifolia ; Kleinschmit 1993). It is entirely plausible that hybridization serves as a mechanism of adaptive trait transfer in these cases. In a similar way, adaptive traits from southern provenances and species may be transferred through controlled interspecific and intraspecific crosses with the two Central European species. Oaks are among the tree species whose ecological and economical importance in Central Europe is expected to increase in light of global warming. Whilst a retreat of other forest tree species like beech, spruce and fir from their current distribution areas is predicted, oaks may profit from these changes and occupy new sites (Iverson and Prasad 2001). There is evidence that during its evolutionary history, the genus Quercus behaved opportunistically and rapidly evolved in periods of climatic deterioration (Axelrod 1983, Manos et al. 1999, Manos and Stanford 2001, Tiffney and Manchester 2001). As revealed by the geographic distribution of their chloroplast DNA lineages and by fossils, Mediterranean oaks persisted in their current geographic regions without large extinctions at least throughout the late Miocene and Pleistocene (in the last 30 million years) through periods of extreme drought, cooling and warming (Jimenez et al. 2004, Magri et al. 2007, López de Heredia et al. 2007). Hybridization has played a significant role throughout this long evolutionary history as a means of gene exchange. Facing an ongoing climate change it is important to improve our knowledge about tree species that have been proved to be true lords of evolution, such as oaks. The present study of
36 6. Conclusions and outlook
genetic differentiation and hybridization among oak species with divergent ecological and evolutionary profiles aimed to make a contribution in this area of research.
37
7. Summary Oaks ( Quercus L.) constitute one of the most species rich genera of woody angiosperms in the Northern Hemisphere. Oak species are adapted to a large variety of ecosystems and play an important ecological and economical role in many regions. Hybridization in the genus is widely distributed and its role in the evolution of oaks has been increasingly recognized. Recent molecular evidence supports substantial exchanges of genetic variation among different taxonomic units. In the present thesis, species differentiation and interspecific gene flow was studied in two paradigms of oak species with divergent ecological and evolutionary profiles. The first objective was to study interspecific and geographic patterns of genetic variation among very distinct environments in the interfertile Q. petraea and Q. robur , two continental oak species of section Quercus with a large distribution range and analogous ecological amplitude. For this purpose, pure populations of these species were sampled from three regions, South western Germany, Central Bulgaria and Northern Greece, along an ecological gradient with increasing aridity towards south. A multilocus approach based on nuclear microsatellites (SSRs) was followed. Diversity patterns differed among the used loci. By using a group of three ‘species discriminant’ loci species genetic structures could be well resolved. By contrast, five ‘provenance discriminant’ loci were characterized by a high genetic differentiation among geographic regions, whereas they displayed common genetic structures within regions. Six further loci included in the analyses showed loose species structures and high levels of introgression between Q. petraea and Q. robur . This differentiation pattern suggests a combination of gene flow and natural selection, which form genetic variation within and between the two species. ‘Species discriminant’ loci might represent genome regions affected by directional selection maintaining species integrity. ‘Provenance discriminant’ loci might represent genome regions with high interspecific gene flow and common adaptive patterns to local environmental factors. These findings agree with the notion that ecological barriers account for maintenance of species identity in this oak complex. The second objective of the thesis was to investigate the interspecific gene flow and genetic differentiation within and between Q. alnifolia and Q. coccifera , two Mediterranean oak species of section Cerris growing in the restrictive insular environment of Cyprus. Firstly, a large scale approach was followed in order to assess genetic differentiation within and between species. Large samples were taken from a mixed and one pure population of each species in order to analyze nuclear genetic variation and to infer diversity measures based on SSR markers. In addition, smaller samples were taken from six further pure populations in order to study chloroplast DNA differentiation and spatial structuring. A high and significant interspecific differentiation and a limited genetic introgression were revealed by nuclear SSRs, either among pure or sympatric populations. On the contrary, chloroplast DNA haplotypes were shared between the two species and formed common spatial structures at the regional level. In a second approach, insights into interspecific gene flow were gained by performing further analyses within the mixed stand. Multivariate analyses of leaf morphometric traits and nuclear SSR loci, as well as an analysis of chloroplast DNA was performed in all adult trees of the stand, in order to investigate genetic introgression at a fine scale. In addition, acorns were sampled from known maternal trees representing the two species and intermediate forms, in order to study gene flow. By subtracting the maternal 38 7. Summary
contribution, a genetic characterization of the pollen cloud which fertilized each tree was made. Regarding adult trees, results further supported a very limited genetic introgression. The two species were both morphologically and genetically distinct and, in contrast to the results from the multipopulation study, chloroplast DNA introgression was also very limited. On the other hand, the intermediate status of four putative hybrids (1,4% among all adult trees) was supported by both morphological and genetic data. Regarding progenies (acorn embryos), evidence about interspecific pollinations was rare. In particular, no interspecific pollinations were detected in Q. alnifolia . In Q. coccifera , rare pollination from Q. alnifolia could be inferred (2,8% among all male gametes) and these were mostly observed in a particular mother tree. Additionally, the hybrid mother tree was mostly pollinated by Q. alnifolia . Results from both large and fine scale approaches show that interspecific hybridization between Q. alnifolia and Q. coccifera in Cyprus is rare and genetic introgression limited. This is particularly evident in sympatry, where, besides the four designated hybrids, intermediate individuals were not supported either by leaf morphometric traits, or by genetic data. Although chloroplast DNA haplotypes were shared in a landscape scale, chloroplast DNA introgression within the mixed stand was very low. Additionally, interspecific crossings, as revealed by the analysis of pollen clouds, were scarce and were observed only in one direction. Therefore, reproductive barriers (pre or post pollination) might account for the limited genetic introgression between the two species. On the other hand, regional interspecific sharing of chloroplast DNA can be attributed to seldom historical events of hybridization and backcrossing. The third objective of the thesis was to resolve phylogenetic relationships among all study species. Quercus infectoria ssp. veneris , the third oak species growing on Cyprus was also included in this analysis, in order to provide molecular evidence about its membership to the section Quercus. A clear separation between Q. alnifolia and Q. coccifera on the one hand, and Q. infectoria ssp. veneris, Q. petraea and Q. robur on the other was supported by both nuclear SSR conservation and chloroplast DNA haplotypes. Among Q. infectoria ssp. veneris , Q. petraea and Q. robur all tested microsatellite loci could be cross amplified and their allele sizes occurred in the same range. By contrast, in Q. alnifolia and Q. coccifera , a very limited number of these loci, originally described in oaks of the section Quercus, were conserved. Additionally, allele sequencing showed size homoplasy at one locus between the two aforementioned groups. Finally, based on chloroplast DNA SSRs, these groups were assigned to two distinct clades. Within each clade, haplotypes were often shared among species. In summary, results from the phylogenetic analysis support the membership of Q. infectoria ssp. veneris to section Quercus along with Q. petraea and Q. robur , whereas Q. alnifolia and Q. coccifera should be placed within the section Cerris and in particular in the group ‘ilex’ together with other evergreen oaks of the Mediterranean Basin.
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8. Zusammenfassung Die Eichen ( Quercus L.) stellen eine der artenreichsten Gattungen der Angiospermen auf der nördlichen Halbkugel dar. Eichenarten sind an eine große Vielfalt von Ökosystemen angepasst und in vielen Regionen spielen sie eine wichtige ökologische und wirtschaftliche Rolle. Hybridisierung ist weit verbreitet innerhalb der Gattung und ihre Bedeutung für die Evolution der Eiche wird zunehmend anerkannt. Neue Erkenntnisse basierend auf molekulargenetischen Methoden weisen darauf hin, dass ein bedeutsamer Austausch genetischer Variation zwischen verschiedenen taxonomischen Einheiten durch Hybridisierung stattfindet. In der vorliegenden Arbeit wurde Artdifferenzierung und Genfluss von Eichenarten an zwei Beispielen mit verschiedenen ökologischen und evolutionären Profilen erforscht. Das erste Ziel der Arbeit bestand in der Untersuchung von interspezifischer und geographischer genetischer Variationsmuster zwischen ökologisch differenzierten Regionen in Q. petraea und Q. robur , zweier kontinentaler Eichenarten der Sektion Quercus mit einem großen Verbreitungsareal und einer entsprechenden ökologischen Amplitude. Zu diesem Zweck wurden reine Populationen dieser Arten aus drei Regionen – Südwestdeutschland, Zentralbulgarien und Nordgriechenland – entlang eines ökologischen Gradienten beprobt. Die genetische Diversität dieser Populationen wurde basierend auf mehreren Mikrosatellitenloci (SSR) analysiert. Verschiedene Diversitätsmuster wurden nachgewiesen, je nachdem welche SSR loci verwendet wurden. Durch die Verwendung von drei „artunterscheidenden“ Loci konnten artspezifische genetische Strukturen nachgewiesen werden. Im Gegesatz dazu zeigten fünf weitere „herkunftsunterscheidende“ Loci eine hohe genetische Differenzierung zwischen geographischen Regionen, wobei sie innerhalb einer Region keine genetischen Strukturen zwischen Arten aufzeigten. Sechs weitere Loci zeigten nur in geringem Ausmaß genetische Strukturen und darüber hinaus einen hohen Grad genetischer Introgression zwischen Q. petraea und Q. robur . Dieses Ergebnis lässt sich durch eine Kombination von interspezifischem Genfluss und natürlicher Selektion erklären, die die genetische Differenzierung innerhalb und zwischen den beiden Arten prägen. „Artunterscheidende“ Loci könnten Genombereiche vertreten, die von einer direktionalen Selektion beeinflusst werden und die für den Erhalt der Artintegrität sorgen. „Herkunftsunterscheidende“ Loci könnten Genombereiche mit hohem interspezifischem Genfluss und gemeinsamen Anpassungen von Arten an lokale bzw. regionale ökologische Bedingungen vertreten. Diese Ergebnisse weisen darauf hin, dass an diesem Beispiel von Eichenarten ökologische Barrieren für die Artunterscheidung der untersuchten Eichenarten sorgen. Das zweite Ziel dieser Arbeit war die Ermittlung interspezifischen Genflusses und genetischer Differenzierung innerhalb und zwischen Q. alnifolia und Q. coccifera – zwei mediterraner Eichenarten der Sektion Cerris, die sich auf der geographisch eingeschränkten insularen Umgebung von Zypern verbreiten. Als erstes wurde eine weiträumige Analyse durchgeführt, um die genetische Differenzierung innerhalb und zwischen den Arten zu erfassen. Zwei Rein und ein Mischbestand wurden intensiv beprobt, um eine Analyse der nuklearen genetischen Variation durchzuführen und Parameter der genetischen Diversität zu ermitteln. Zusätzlich wurden geringere Stichproben in sechs weiteren reinen Populationen jeder Art durchgeführt, um die Variation der Chloroplasten DNA und die räumliche Verteilung dieser Variation zu untersuchen. Sowohl zwischen reinen als auch zwischen sympatrischen Populationen konnten eine hohe und signifikante genetische Differenzierung und eine geringe 40 8. Zusammenfassung
genetische Introgression zwischen den Arten mithilfe von nuklearen Mikrosatelliten nachgewiesen werden. Im Gegensatz dazu zeigten die untersuchten Artein ein gemeinsames Vorkommen von Chloroplasten DNA Haplotypen geteilt zwischen den Arten und sowie gemeinsame räumliche genetische Strukturen. Darüber hinaus wurde anhand von weiteren Analysen in dem Mischbestand Einblick in den Genfluss zwischen den Arten gewonnen. Multivariatanalysen von blattmorphometrischen Merkmalen und nuklearen Mikrosatelliten wurden in allen Altbäumen im Mischbestand durchgeführt, um die genetische Introgression auf einer kleineren räumlichen Ebene intensiv zu untersuchen. Darüber hinaus wurden Eicheln von bekannten Mutterbäumen aus beiden Arten als auch einem Individuum mit intermediären Phänotyp beerntet, um den Genfluss zu erforschen. Dadurch, dass der mütterliche Beitrag an jedes Eichelembryo bekannt war, konnten anhand der väterlichen Beiträge die Pollenwolken charakterisiert werden. Bezüglich der Altbäume konnte keine wesentliche genetische Introgression nachgewiesen werden. Die beiden Arten waren sowohl morphologisch als auch genetisch unterschlich und im Gegensatz zu der weiträumigen Analyse war die Chloroplasten DNA Introgression auch sehr begrenzt. Auf der anderen Seite wurden vier Individuen unter allen Altbäumen (1,4%), die als Zwischenformen im Gelände bezeichnet worden waren, nach den Multivariatanalysen sowohl morphologisch als auch genetisch als Hybride eingestuft. Was die Nachkommen (Eichelnembryonen) betrifft, waren interspezifische Paarungen äußerst selten (2,8% der männlichen Gameten). Insbesondere wurden unter den Mutterbäumen in Q. alnifolia keine interspezifischen Paarungen nachgewiesen. In Q. coccifera wurden einige seltene Paarungen gezeigt, die vorwiegend auf einen Mutterbaum zurückzuführen waren. Der Hybridmutterbaum war größtenteils von Q. alnifolia Pollen befruchtet. Sowohl Ergebnisse aus der weiträumigen Untersuchung als auch aus dem Mischbestand weisen darauf hin, dass interspezifische Hybridisierung zwischen Q. alnifolia und Q. coccifera selten und genetische Introgression gering sind. Dies ist äußerst eindeutig in dem Mischbestand, in dem abgesehen von vier aufgezeichneten Hybridformen keine signifikante genetische Introgression gezeigt werden konnte. Untersuchung der Chloroplasten DNA zeigte, dass die genetische Introgression in dem Mischbestand äußerst gering war, obwohl gemeinsame genetische Strukturen zwischen den Arten nach der weiträumigen Untersuchung nachgewiesen wurden. Darüber hinaus waren interspezifische Paarungen sehr selten und auf eine Richtung beschränkt, wie aus den Pollenwolkenanalysen zu entnehmen ist. Daher kann vermutet werden, dass reproduktive Barrieren (vor oder nach der Bestäubung) die genetische Introgression zwischen den beiden Arten einschränken. Andererseits könnten gemeinsame Chloroplasten DNA Strukturen auf der regionalen Skala auf seltene historische Hybridisierungs und Rückkreuzungsereignisse zurückgeführt werden. Das dritte Ziel der vorliegenden Arbeit bestand darin, eine phylogenetische Analyse zwischen allen Arten durchzuführen. In dieser Analyse wurde auch Quercus infectoria ssp. veneris , die dritte Eichenart, die auf Zypern vorkommt, mit einbezogen, um ihre Zugehörigkeit zu der Sektion Quercus mit molekularen Markern zu untersuchen. Sowohl eine Analyse der evolutionären Konservierung von nuklearen Mikrosatellitenloci als auch eine Analyse der Chloroplasten DNA Haplotypen zeigten eine klare Trennung zwischen Q. alnifolia und Q. coccifera auf der einen Seite und Q. infectoria ssp. veneris , Q. petraea und Q. robur auf der anderen. Zwischen Q. infectoria ssp. veneris , Q. petraea und Q. robur konnten alle getesteten Mikrosatellitenloci amplifiziert werden und besaßen die gleichen Allellängenbereiche. Dem entgegen waren war in Q. alnifolia und Q. coccifera nur ein
8. Zusammenfassung 41
geringer Anteil dieser Loci konserviert, die ursprünglich in Eichenarten der Sektion Quercus entwickelt wurden. Darüber hinaus wurde an einem Locus mittels DNA Sequenzierung eine Allellängenhomoplasie zwischen den o. g. Gruppen nachgewiesen. Schließlich waren diese Gruppen basierend auf Chloroplasten DNA Mikrosatelliten in zwei verschiedenen phylogenetischen Zweigen eingestuft. Insgesamt unterstützen die Ergebnisse der phylogenetischen Analyse einerseits die Zugehörigkeit von Q. infectoria ssp. veneris zu der Sektion Quercus zusammen mit Q. petraea und Q. robur , und andererseits die Zugehörigkeit von Q. alnifolia zusammen mit Q. coccifera und anderen immergrünen Eichenarten des Mittelmeerraums zu der Sektion Cerris und inbesondere zu der Gruppe „Ilex“.
42
9. Περίληψη Οι δρύες ( Quercus L.) αποτελούν ένα από τα πιο ποικίλα ως προς τον αριθ ό ειδών γένη ξυλωδών αγγειοσπέρ ων στο Βόρειο Η ισφαίριο. Τα διάφορα είδη του γένους παρουσιάζουν προσαρ ογές σε ία εγάλη ποικιλία οικοσυστη άτων και παίζουν ένα ση αντικό οικολογικό και οικονο ικό ρόλο σε πολλές περιοχές. Ο υβριδισ ός εντός του γένους είναι ευρέως διαδεδο ένος και ο ρόλος του στην εξέλιξη των δρυών αναγνωρίζεται όλο και περισσότερο. Πρόσφατα δεδο ένα από οριακές γενετικές αναλύσεις υποστηρίζουν ότι έσω του υβριδισ ού λα βάνουν χώρα ση αντικές ανταλλαγές γενετικής ποικιλότητας εταξύ διάφορων ταξινο ικών ονάδων. Στην παρούσα διδακτορική διατριβή ελετάται η γενετική ποικιλότητα και η γονιδιακή ροή εταξύ των ειδών σε δύο παραδείγ ατα αλληλοδιασταυρού ενων ειδών δρυός από διαφορετικά οικολογικά περιβάλλοντα και ε διαφορετική εξελικτική ιστορία. Ο πρώτος στόχος της παρούσας διατριβής είναι η διερεύνηση της γενετικής ποικιλότητας σε επίπεδο είδους και σε επίπεδο γεωγραφικής περιοχής εταξύ διακριτών περιβαλλοντικών περιοχών στα αλληλοδιασταυρού ενα είδη Q. petraea και Q. robur . Τα είδη αυτά παρουσιάζουν εγάλες περιοχές εξάπλωσης και ανάλογο περιβαλλοντικό εύρος. Για το σκοπό αυτό λήφθηκαν δείγ ατα α ιγών πληθυσ ών του είδους από τρεις περιοχές, τη νοτιοδυτική Γερ ανία, την κεντρική Βουλγαρία και τη Βόρεια Ελλάδα κατά ήκος ενός οικολογικού κλινούς ε αυξανό ενη ξηρασία προς τα νότια. Οι γενετικές αναλύσεις βασίστηκαν σε πυρηνικούς ικροδορυφόρους (SSRs). Η γενετική ποικιλότητα εντός και εταξύ των ειδών ήταν διαφορετικά δο η ένη εταξύ των διάφορων γονιδιακών θέσεων. Ένας ξεκάθαρος διαχωρισ ός γενετικών δο ών εταξύ ειδών επιτεύχθηκε ε βάση τρεις «διαφοριστικές στο επίπεδο είδους γονιδιακές θέσεις». Αντίθετα, πέντε «διαφοριστικές στο επίπεδο προέλευσης γονιδιακές θέσεις» χαρακτηρίστηκαν από ία έντονη διαφοροποίηση εταξύ γεωγραφικών περιοχών, παρουσίασαν ό ως κοινή γενετική ποικιλότητα εταξύ ειδών στο τοπικό επίπεδο. Τέλος, έτρια διαφοροποίηση εταξύ των ειδών και ψηλά επίπεδα γονιδιακής εισδοχής βρέθηκαν ε βάση έξι επιπλέον γονιδιακές θέσεις που συ περιλήφθηκαν στις αναλύσεις. Αυτές οι διαφορές στο πρότυπο της διαφοροποίησης εταξύ διαφορετικών ο άδων γονιδιακών θέσεων συνηγορούν σε ία αλληλεπίδραση γονιδιακής ροής και φυσικής επιλογής. Οι εν «διαφοριστικές στο επίπεδο είδους γονιδιακές θέσεις» πιθανώς αντιπροσωπεύουν περιοχές του γενώ ατος οι οποίες επηρεάζονται από κατευθυντήρια επιλογή που συντείνει στη διατήρηση της ταυτότητας των ειδών. Στις δε «διαφοριστικές στο επίπεδο προέλευσης γονιδιακές θέσεις» η γενετική ποικιλότητα έχει πιθανώς δια ορφωθεί από τη εταξύ ειδών ροή γονιδίων και κοινές προσαρ ογές σε τοπικούς περιβαλλοντικούς παράγοντες. Τα ευρή ατα αυτής της ανάλυσης υποστηρίζουν την άποψη ότι η ακεραιότητα της Q. petraea και της Q. robur ως δύο ξεχωριστά είδη διατηρείται έσα από οικολογικούς φραγ ούς παρά τα υψηλά επίπεδα διεισδυτικού υβριδισ ού εταξύ τους. Ο δεύτερος στόχος της παρούσας διατριβής ήταν η διερεύνηση της γονιδιακής ροής και της γενετικής διαφοροποίησης εντός και εταξύ της Q. alnifolia και της Q. coccifera , δύο εσογειακών ειδών του τ ή ατος Cerris τα οποία φύονται στο περιορισ ένο νησιωτικό περιβάλλον της Κύπρου. Σε ια πρώτη προσέγγιση λήφθηκαν εγάλα δείγ ατα από ένα α ιγή πληθυσ ό από κάθε είδος και ένα ικτό πληθυσ ό εταξύ τους ε σκοπό την ανάλυση της γενετικής ποικιλότητας εντός και εταξύ των ειδών και τον υπολογισ ό διάφορων παρα έτρων γενετικής παραλλακτικότητας των πληθυσ ών ε τη χρήση πυρηνικών ικροδορυφόρων. Επιπλέον λήφθηκαν ικρότερα δείγ ατα από έξι επιπρόσθετους α ιγείς πληθυσ ούς κάθε είδους ε σκοπό τη ελέτη της γενετικής 9. Περίληψη 43
διαφοροποίησης και των γεωγραφικών δο ών του χλωροπλαστικού DNA. Τα αποτελέσ ατα ε βάση τους πυρηνικούς ικροδορυφόρους καταδεικνύουν ση αντική διαφοροποίηση και χα ηλά επίπεδα γονιδιακής εισδοχής εταξύ ειδών, είτε εταξύ α ιγών, είτε εταξύ συ πατρικών πληθυσ ών. Αντίθετα, οι απλότυποι του χλωροπλαστικού DNA βρέθηκαν να συνυπάρχουν στα δύο είδη και να σχη ατίζουν κοινές χωρικές δο ές εταξύ γειτονικών πληθυσ ών. Σε ια δεύτερη προσέγγιση πραγ ατοποιήθηκε σε βάθος ανάλυση της γονιδιακής ροής εταξύ ειδών έσα στη ικτή συστάδα. Πολυ εταβλητές αναλύσεις διεξήχθηκαν αφενός ε βάση ορφο ετρικά γνωρίσ ατα του φύλλου και αφετέρου ε βάση την ποικιλότητα σε γονιδιακές θέσεις πυρηνικών ικροδορυφόρων. Στις αναλύσεις αυτές συ περιλήφθηκαν όλα τα ενήλικα δέντρα της συστάδας. Επίσης, ε σκοπό τη διερεύνηση της γονιδιακής ροής λήφθηκαν δείγ ατα βαλανιδιών από προεπιλεγ ένα ητρικά δέντρα από τα δύο πατρικά είδη και ένα εν δυνά ει υβρίδιο. Αφαιρώντας τη ητρική συνεισφορά στο έ βρυο επιχειρήθηκε ένας γενετικός χαρακτηρισ ός του νέφους γύρης που επικονίασε κάθε ητρικό δέντρο. Σ’ ό,τι αφορά τα ενήλικα δέντρα, τα αποτελέσ ατα υποστηρίζουν πολύ περιορισ ένη γενετική εισδοχή. Τα δύο είδη αποτελούν ξεχωριστές οντότητες, τόσο στο ορφολογικό, όσο και στο γενετικό επίπεδο. Σε αντίθεση ε τις συγκρίσεις σε επίπεδο πληθυσ ού, η κατ’ άτο ο γενετική εισδοχή του χλωροπλαστικού DNA είναι πολύ περιορισ ένη. Από την άλλη, ο ενδιά εσος χαρακτήρας τεσσάρων εν δυνά ει υβριδίων (1,4% των ενηλίκων), τα οποία είχαν χαρακτηριστεί στο πεδίο, επιβεβαιώθηκε τόσο ε βάση ορφολογικά, όσο και ε βάση γενετικά δεδο ένα, υποστηρίζοντας ότι οι διασταυρώσεις εταξύ των ειδών πορούν να είναι επιτυχείς. Σ’ ό,τι αφορά τους απογόνους (έ βρυα βαλανιδιών), οι διασταυρώσεις εταξύ των δύο ειδών ήταν ιδιαίτερα σπάνιες. Στην περίπτωση της Q. alnifolia κα ία διασταύρωση εταξύ των ειδών δε διαγνώστηκε. Ανά εσα στα ητρικά δέντρα της Q. coccifera διασταυρώσεις εταξύ των ειδών διαγνώστηκαν σε σπάνιες περιπτώσεις (2,8% των αρσενικών γα ετών), οι οποίες περιορίζονταν κυρίως σε ένα συγκεκρι ένο ητρικό δέντρο. Επιπλέον, η γενετική ανάλυση κατέδειξε ότι το ητρικό δέντρο ε χαρακτηριστικά υβριδίου επικονιάστηκε ως επί το πλείστον από πατρικά άτο α Q. alnifolia . Τα αποτελέσ ατα τόσο από τις αναλύσεις εταξύ πλυθυσ ών σε εγάλη κλί ακα, όσο και από τα κατ’ άτο ο αποτελέσ ατα από τη ικτή συστάδα συγκλίνουν στο ότι ο υβριδισ ός εταξύ Q. alnifolia και Q. coccifera στην Κύπρο είναι πολύ περιορισ ένος. Αυτό είναι ιδιαίτερα φανερό σε συ πατρία, όπου πέρα από τα τέσσερα ενήλικα εν δυνά ει υβρίδια, τα αποτελέσ ατα δεν κατέδειξαν άτο α ε ενδιά εσα χαρακτηριστικά, είτε στη βάση ορφο ετρικών χαρακτηριστικών του φύλλου, είτε στη βάση γενετικών δεδο ένων. Στο επίπεδο του χλωροπλαστικού DNA, παρόλο που σε εγάλη κλί ακα οι απλότυποι ε φανίζονται από κοινού και στα δύο είδη, η κατ’ άτο ο γενετική εισδοχή στη ικτή συστάδα ήταν πολύ περιορισ ένη. Επίσης, οι διασταυρώσεις εταξύ των ειδών ήταν σπάνιες και περιορίστηκαν σε ία κατεύθυνση, όπως καταδείχθηκε από την ανάλυση των νεφών γύρης. ς εκ τούτου συ περαίνεται ότι αναπαραγωγικοί φραγ οί (πριν ή ετά την επικονίαση) έχουν ως αποτέλεσ α τον περιορισ ό της γενετικής εισδοχής εταξύ των δύο ειδών. Από την άλλη, σπάνια επεισόδια υβριδισ ού και αναδιασταυρώσεων στο παρελθόν εξηγούν τις κοινές χωρικές γενετικές δο ές χλωροπλαστικού DNA. Ο τρίτος στόχος της ανά χείρας διατριβής ήταν η εξακρίβωση των φυλογενετικών σχέσεων εταξύ των υπό ελέτη ειδών. Στη φυλογενετική ανάλυση συ περιλήφθηκε και το τρίτο είδος δρυός που φύεται στην Κύπρο, η Q. infectoria ssp. veneris ε σκοπό την εξέταση της φυλογενετικής του ταυτότητας και της πιθανής του συγγένειας ε τις δρύες του τ ή ατος Quercus. Τα αποτελέσ ατα, τόσο ε βάση τη ενίσχυση πυρηνικών ικροδορυφόρων, όσο
44 9. Περίληψη
και ε βάση τους απλότυπους του χλωροπλαστικού DNA, καταδεικνύουν ια ξεκάθαρη διαφοροποίηση εταξύ ενός κλάδου αποτελού ενου από την Q. alnifolia και την Q. coccifera αφενός και αφετέρου ενός κλάδου που περιλα βάνει την Q. infectoria ssp. veneris , Q. petraea και Q. robur . Μεταξύ των τριών τελευταίων ειδών επιτεύχθηκε ενίσχυση όλων των δοκι ασ ένων θέσεων ικροδορυφόρων, ειδικά αναπτυγ ένων σε διάφορα είδη του τ ή ατος Quercus, και τα εγέθη των αλληλο όρφων κυ άνθηκαν στο ίδιο εύρος. Εν αντιθέσει, στην Q. alnifolia και Q. coccifera , ανά εσα στις δοκι ασ ένες θέσεις ικροδορυφόρων όνο ένας πολύ περιορισ ένος αριθ ός έδωσε ενίσχυση. Επίσης, ανάλυση αλληλουχιών σε συγκεκρι ένη θέση ικροδορυφόρου κατέδειξε ο οπλασία στο έγεθος εταξύ των προαναφερθεισών ο άδων. Τέλος, ε βάση τους απλότυπους του χλωροπλαστικού DNA, οι δύο αυτές ο άδες κατανε ήθηκαν σε δύο διακριτούς φυλογενετικούς κλάδους. Γενικά τα αποτελέσ ατα της φυλογενετικής ανάλυσης υποστηρίζουν την ένταξη της Q. infectoria ssp. veneris στο τ ή α Quercus αζί ε τις Q. petraea και Q. robur . Η φυλογενετική συγγένεια της Q. alnifolia ε την Q. coccifera , όπως καταφαίνεται από τα δεδο ένα της παρούσας εργασίας υποστηρίζει την ένταξη των δύο αυτών ειδών στο τ ή α Cerris και ειδικά στην ο άδα ‘Ilex’, στην οποία ανήκει η Q. coccifera και άλλες αείφυλλες εσογειακές δρύες.
45
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11. Acknowledgments This thesis was financially supported by a DAAD scholarship and a scholarship according to the State Law on Graduate Funding. The laboratory equipment was financed by the FVA in the framework of the project entitled “Artbestimmung und Hybridisierung von verschiedenen Eichen Arten aus Mitteleuropa”. A special thank is owed to all those who supported me during this project in many respects – scientifically, technically and mentally. I want to express my gratitude to Prof. Dr. Siegfried Fink for giving me the opportunity to accomplish my thesis in the fascinating study area of evolutionary genetics. I am grateful for his supervision and support to my application for a scholarship according to the State Law on Graduate Funding. I am grateful to my second supervisor, Prof. Aravanopoulos, who provided his scientific mentoring with a high motivation and enthusiasm throughout my thesis, in spite of strong geographic barriers between us! His help and his mental support were invaluable. I would like to thank Dr. Katerina Dounavi for her contribution to the preparation and implementation of the study concept of my thesis and for giving me the opportunity to work at the FVA and get known with the working area of molecular genetics. I am indebted to Dr. Eberhard Aldinger for making the implementation of my proposal possible by approving the financial support of the laboratory equipment. A great and warm thank is owed to Dr. Aki Höltken, who offered his scientific help and advice with a relaxed and human way during his period of employment at the FVA. I am thankful to Prof. Dr. Albert Reif for all his hints and his enthusiasm in sharing his knowledge in the field and elsewhere. I would like to acknowledge the help of Mr. Richard Haas for introducing me to laboratory work. I am grateful to all my colleagues not only for their help, but also for the pleasant atmosphere during my work as a doctorate student at the FVA. I owe a special thank на мой приятел Radko Babakov, for his help in lab and in sample collections and for being a very kind and very honest friend. A great “eskerrik asko” is dedicated to Elizabeth Macho Carretero for helping in the lab and giving me mental support with empathy and humour. I express my cordial thank to Sara Virseda and Barbara Madariaga, who worked hard to carry out part of the project analyses. It was a pleasure for me to work with them during their diploma theses which were based on these analyses. I express my gratitude to Ariane Lorenz and Mari Carmen Dacasa Rüdinger for her steady support, especially during difficult times in the laboratory. A great thank is owed to Dr. Henning Wildhagen for offering his help in the – always critical – last hours before printing this manuscript. I am also grateful to Thomas Seliger for some hints in lab and about the present text. A special thank belongs to Christine Schumacher for her help and very kind attitute throughout my stay at the FVA and especially in these last moments shortly before this manuscript goes to press. In any case, I cannot omit from the acknowledgments Nour Alhammoud who not only worked by my side, but also contributed a lot to the nice atmosphere in the laboratory team with an oriental temperament, partially reminding me my loved homeland. Herrmann Schott and Klaus Winkler are greatly acknowledged for their assistance during field collections in Germany and their help to improve my language skills in the allemanisch language! I am indebted to Petros Anastasiou, Zacharias Triftarides, Savvas Protopapas and other colleagues of the Department of Forests of Cyprus personnel, for their assistance during plant collections in steep slopes and under the harsh conditions of 11. Acknowledgments 57
the Cypriot summer. I express my special gratitude to Dr. Andreas Christou, head of the research department of the Cypriot Department of Forests, for his invaluable advice throughout my doctorate thesis. I thank Dr. Costas Kadis for some scientific hints and his help together in field work together with Constantinos Kounnamas. I would like to express – also in written form – a great thank to all those people who mentally stood by my side during this important period of my education and my life. A great thank is owed to Peter Frey for being steadily by my side sharing good and bad moments and common dreams as well. It is difficult to express my gratitude in words. I express my gratitude to my old study colleagues and good friends Nikos Alexandris, Dimitris Samaras and Fotis Xystrakis, with whom I shared many common reflections during almost the whole time of my academic experience since entering the University of Thessaloniki more than ten years ago. I cannot omit my friends Constantinos Kounnamas and Adonis Panayides from the acknowledgments. Although geographic distance among us was always big, mental distance never grew large and we always shared funny and difficult moments of our lives. Finally, I express a great thank to my loved parents, who have always been the most important backing during difficult moments and conflicts and could always express a word of motivation in work and life like nobody else could do.
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12. Publications Manuscript I: C. Neophytou, F.A. Aravanopoulos, S. Fink, A. Dounavi 2010. Detecting interspecific and geographic differentiation patterns in two interfertile oak species ( Quercus petraea (Matt.) Liebl. and Q. robur L.) using small sets of microsatellite markers. Forest Ecology and Management. 259 : 2026–2035 Manuscript II: C. Neophytou, A. Dounavi, S. Fink, F.A. Aravanopoulos 2010. Interfertile oaks in an island environment. I. Contrasting patterns of nuclear and chloroplast DNA differentiation between Quercus alnifolia and Q. coccifera in Cyprus. European Journal of Forest Research. DOI: 10.1007/s10342 010 0442 8. Manuscript III: C. Neophytou, F.A. Aravanopoulos, S. Fink, A. Dounavi 2011. Interfertile oaks in an island environment. II. Limited hybridization between Quercus alnifolia Poech and Q. coccifera L. in a mixed stand. European Journal of Forest Research. DOI: 10.1007/s10342 010 0454 4. Manuscript IV: C. Neophytou, A. Dounavi & F.A. Aravanopoulos 2008. Conservation of nuclear SSR loci reveals high affinity of Quercus infectoria ssp. veneris A. Kern (Fagaceae) to section Robur. Plant Molecular Biology Reporter 26 : 133 141.