Cent. Eur. J. Biol.• 6(4) • 2011 • 597-605 DOI: 10.2478/s11535-011-0034-8

Central European Journal of Biology

Genetic differences within natural and planted stands of Quercus petraea

Research Article

Jiří Dostálek*, Tomáš Frantík, Miroslava Lukášová

Silva Tarouca Research Institute for Landscape and Ornamental Gardening, CZ-252 43 Průhonice, Czech Republic Received 15 February 2011; Accepted 31 March 2011

Abstract: Five sessile oak [Quercus petraea (Matt.) Liebl.] stands from the Czech Republic were studied to learn about the impact of different types of forest management regimes on the genetic differences among tree populations and on population structures. One population had not been markedly affected by human activity, two populations represented unplanted stands that were extensively managed for a long period of time using the coppice system, and two populations were planted stands. Approximately 100 trees from each stand were mapped and subsequently genotyped using 10 nuclear microsatellite loci. We determined the spatial genetic structure of each population and the genetic differentiation among the populations. We found that: (i) the populations were genetically differentiated, but the differences between the unplanted and planted stands were not markedly significant; (ii) the genetic differentiation among the populations depended on the geographical distribution of the populations; (iii) within unplanted stands, a strong spatial genetic structure was seen; and (iv) within planted stands, no spatial genetic structure was observed. Our findings implies that the analysis of spatial genetic structure of the sessile oak forest stand can help reveal and determine its origin.

Keywords: Quercus petraea • Genetic structure within a population • Genetic differences among populations • Microsatellites • Forest management © Versita Sp. z o.o.

1. Introduction To help protect the diversity of woody forest species, it is important to determine how forest management Long-term human activity in European forests has influences the genetic variability of these species. influenced the genetic diversity of forest ecosystems In this study we examined sessile oak, Quercus [1] including forest trees [2]. Modified environmental petraea, a woody species of high economic value conditions and forest structure, as well as the selective commonly found in the Czech Republic and in most of removal of trees during thinning and harvesting, can all Europe. Recently, a number of genetic studies have been potentially change the genetic structure of a forest [3,4]. performed, as genetic markers allow quick surveys of Selectively favoring special phenotypes may strongly variation within and between populations [7-9]. However, influence adaptive capability and economically important most of these studies aimed to assess the genetic traits [3]. Artificial regeneration by means of sowing or characteristics of natural or semi-natural populations. intentional planting often results in changes in the genetic Studies concerning genetic structure and genetic composition of the target species [5]. We can anticipate variability at the population level were performed by that a narrow selection of seed-producing trees will several authors [10-13]. Finkeldey [11] analyzed 17 result in lower genetic variability in planted forest stands. isoenzyme loci to reveal that differentiation among Intensive forest management can also disturb the initial populations within a species is very low, reflecting the genetic structure of a population due to the planting of geographical location of populations to a certain extent. genotypes of alien origin within a species, i.e., seedlings Bruschi et al. [14] also noticed a slight correlation between from remote geographic regions within a country or genetic distance and geographic distance among from abroad. This can even result in an outbreeding populations; they evaluated the diversity of several depression as described, for example, by Woessner [6]. populations using both morphological and molecular

* E-mail: [email protected] 597 Genetic differences within natural and planted stands of Quercus petraea

markers. Using microsatellites, a low or not significant management schemes. Stands 1, 2 and 3 are located genetic differentiation among populations of Q. petraea in the Křivoklátsko Protected Landscape Area, and was reported in France [15,16], Belgium [17], Ireland stands 4 and 5 are located in the Podyjí National Park [18], Denmark [19], and the Czech Republic [20]. Similar (Figure 1). results were also reported by Bakker et al. [12]. Stand 1. Brdatka Nature Reserve (50º 02´ 56´´ N, When analyzing the genetic structure of populations 13º 53´24´´): This locality represents a natural forest that using enzyme-coding loci, Zanetto and Kremer [21] has not been influenced by human activity. The habitat observed significant differences in the frequencies is located above the Berounka River on an extremely of alleles in oak populations. Streiff et al. [10] used jagged slope with a south to southeastern orientation isozymes and six microsatelite loci to study the diversity and occasional rock outcrops. In this area, the oak and fine-scale genetic structure within a native oak population forms a layered dwarf thermophilous oak forest containing Quercus robur and Q. petraea in the forest that is typical of a wild service-oak forest (Sorbo northwest of France. Their study demonstrated that the torminalis-Quercetum; Svoboda ex Blažková 1962). stand had a significant but low spatial genetic structure The dominant Q. petraea cover comprises 25%–50% of that was greater for Q. petraea than for Q. robur. Cottrell the tree layer, and the admixture of Pinus sylvestris L. et al. [13] compared by means of microsatellites the cover is 5%–25% (for details see [23]). genetic structures of two British oak woods containing Stand 2. Červený Kříž Nature Reserve (49º 59´ 30´´ N, both Q. robur and Q. petraea. One stand contained 13º 55´47´´): This locality represents a natural stand that has unplanted natural woods managed by coppicing, while been managed for a long time using the coppice system. the second stand was influenced by human-mediated This habitat is located in a flat area. The oak population in planting. Significant spatial genetic structure was found the area is part of a thermophilous cinquefoils-oak forest in both stands, with the most substantial spatial genetic (Potentillo albae-Quercetum Libbert 1933). structure occurring in unplanted forests. In general, The tree layer is composed almost exclusively of these studies point to high genetic diversity in the stands oak, with a cover of 75–100%. Individuals of Carpinus that were investigated. This diversity was attributed to betulus L., Tilia cordata Mill., and Sorbus torminalis (L.) the high level of outcrossing that occurs in Quercus Crantz can be found only sporadically in this stand. A spp., particularly Q. petraea. shrub layer is absent in this community primarily due to In this study, we paid close attention to the wild animal activity (for details see [23]). relationship between genetic characteristics and Stand 3. Křivoklát (50º 23´ 88´´ N, 13º 52´ 53´´): management intensity and to the level of autochthony This locality represents a stand of Q. petraea planted of the Q. petraea populations. An autochtonous stand approximately eighty years ago on municipal land in the is a conspecific stand that has spontaneously arisen at town of Křivoklát. The tree layer is composed primarily a given location. The high correlation between spatial of planted Q. Petraea with an admixture of C. betulus, genetic structure and forest management regime for Fagus sylvatica L., Larix decidua Mill., and P. sylvestris. Fagus sylvatica was mentioned by Gregorius and Stand 4. Lipina (48º 52´ 45´´ N, 16º 01´ 13´´): The Kownatzki [22]. In our study, we examined five stands locality in the Podyjí National Park represents a semi- of Q. petraea in Czech Republic. We tested whether natural unplanted stand on a slope of approximately there were any differences in genetic characteristics 20º inclination that was extensively utilized by coppice among the populations that could be attributed to the management, at least for the past 500 years [24]. From various management regimes. The aim of this study 1951 to 1991, only sanitation cutting was carried out. was to answer the following questions: First, are there Since 1991, the year when the Podyjí National Park any genetic differences between natural and planted was established, the stand has been preserved without stands? Second, does the type of forest management intervention. The oak population here comprises part regime influence the genetic structure of the stands? of the wild service-oak forest with S. torminalis and Third, is there existing relation between the origin of the Vincetoxicum hirundinaria Med. (Sorbo torminalis- stand and the genetic structure of the population? Quercetum Svoboda ex Blažková 1962) transformed into an acidophilous woodrush oak forest with Festuca ovina L. (Luzulo albidae-Quercetum Hilitzer 1932). Oak 2. Experimental Procedures is dominant in the tree layer, comprising 70–80% of the total canopy layer. The admixture consists of Tilia 2.1 Localities platyphyllos Scop., T. cordata Mill., Carpinus betulus L., We choose five populations of Quercus petraea Acer campestre L., Fraxinus excelsior L., and Sorbus (Matt.) Liebl. that are managed using different forest aucuparia L. (for details see [24]).

598 J. Dostálek et al.

Figure 1. Location of the studied sessile oak (Quercus petraea) vegetation in the Křivoklátsko Protected Landscape Area and Podyjí National Park: 1–Brdatka National Nature Reserve (natural stand), 2–Červený kříž Nature Reserve (unplanted stand managed with the coppice system), 3–Křivoklát (planted stand in the vicinity of the town), 4–Lipina (unplanted stand managed with the coppice system), 5–Podmolí (planted stand).

Stand 5. Podmolí, stand No. 55De2v (48º 49´ 49´´ N, method. Topcon Hiper+ GPS equipment was used, and 15º 57´ 37´´): This locality represents a stand planted GPS measurement was carried out on connecting points approximately 12 years ago. The stand is composed of at the same time with a minimum observation time of young trees where planted oak (Q. petraea) prevails with 30 min. This technology allowed individual tree positions the admixture of C. betulus, Salix caprea L., Populus to be determined with accuracy of ±0.3 m. tremula L., and Betula pendula Roth. 2.3 DNA analysis 2.2 Sampling of plant material Young leaves were frozen to -80°C and lyophylized. DNA In July 2008 and 2009, several healthy leaves were was extracted from the dry material using the DNeasy collected from each of approximately 100 individuals Plant Mini Kit (Qiagen). Microsatellite analysis was registered on a spatial grid where the closest trees were employed to genotype the individuals, and the primers an average of 5 m apart. Sampling area of individual for the amplification of polymorphic short sequence stands was about 1 000 m2, containing approximately repeats (SSR) of the genus Quercus were adopted from 120–150 trees. Samples in coppice stands were collected the literature. The loci that were previously developed with consideration to avoid clonality. Individual trees were for Q. petraea were ssrQpZAG9, ssrQpZAG16, mapped using the polar method from the temporarily ssrQpZAG36, ssrQpZAG104, ssrQpZAG108, and stabilized tops of polygonometric traverses using the ssrQpZAG110 [25]. The loci developed for Q. robur universal Leica TC307 theodolite (accuracy of direction included ssrQrZAG11 and ssrQrZAG90 [26]. Finally, the measuring 2 mgon, accuracy of length measuring loci developed for Q. macrocarpa included MSQ4 and 2 mm + 2 ppm). The polygonometric traverses (each MSQ13 [27]. in one locality) were fixed between connecting points The PCRs were done separately for each locus. determined using GPS receivers by the rapid static DNA fragments were amplified by fluorescent-labeled

599 Genetic differences within natural and planted stands of Quercus petraea

primer pairs (reverse primers labeled with FAM, the coefficient of Loiselle et al. [31]; however, it was VIC, NED and PET dyes at the 5’-end). The mixture sensitive to presence of alleles with low frequencies

contained 8 ng of total DNA, 2 mM MgCl2, 100 μM (less than 5%). Its value decreased with decreasing of each dNTP, 0.2 μM of each primer, 0.32 U of Taq number of samples. Kinship coefficient calculated polymerase (Fermentas) and 1 x reaction buffer in a according to Loiselle et al. [31] had higher dispersion total volume of 10 μl. The PCR conditions included a variance if compared to the coefficient of Ritland [30], preliminary denaturation for 4 min at 94°C followed by 30 but it was resistant to the incidence of low frequent cycles of denaturation for 45 s at 94°C, primer annealing alleles. Its values did not decrease significantly with for 45 s at 50°C, DNA synthesis for 30 s at 72°C, and decreasing number of samples. Therefore we choose a final extension for 10 min at 72°C. The fluorescently to use the coefficient based on Loiselleet al. [31] for the labeled PCR products were electrophoretically evaluation of our data. separated in the ABI 310 automatic genetic analyser (Applied Biosystems). The alleles were detected using GeneMapper software (Applied Biosystems). 3. Results

2.4 Data processing The basic genetic characteristics of the study populations Genetic differentiation among populations was tested are summarized in Table 1. In all populations, the values

by Arlequin 3.11 [28]. Differentiation between all pairs of of the Wright´s fixation coefficientsIS (F ) were low. All

populations was evaluated using FST statistics (AMOVA values differed from the Hardy-Weinberg equilibrium. procedure, 110 permutations). In order to graphically However, they were very close to zero, indicating a very display differences between the populations, Genotype small proportion of inbreeding. Assignment Procedure was used. In addition, the individual populations were evaluated using Wright`s 3.1 Genetic differences

fixation index ISF to evaluate deviations from the Hardy- Genetic differentiation among all study populations were Weinberg Equilibrium, which allowed us to estimate statistically significant; however, the index of genetic

relevance of inbreeding (AMOVA procedure, 1000 differentiation FST was low (Table 2), rarely exceeding permutations). 0.05, which indicates a low level of genetic divergence The spatial genetic structure of the populations was [32]. Pairwise genetic differentiation depended on

assessed using SPAGeDi [29]. Genetic similarity of all geographic distances. The lowest FST value was found pairs of individuals within the population was expressed between the unplanted Lipina stand and the planted using the kinship coefficient [30,31]. The matrix of locality in Podmolí, with a distance of approximately these pairwise similarities was then correlated with the 1 km (Figure 2). Very small differences were observed matrix of their spatial distances. In principle, the spatial between the natural Brdatka stand and the planted genetic structure (SGS) of a population is indicated by a Křivoklát stand, which were located approximately 2 km

statistically significant decrease in genetic similarity that (Figure 3). Conversely, the FST value found between corresponds to the spatial distance between individuals. Brdatka and Červený Kříž was higher (their distance The kinship coefficient calculated according to Ritland is approximately 7 km). The greatest genetic distances [30] had a lower dispersion variance if compared to were found between populations from the Křivoklátsko

Natural Coppice system Planted

Locality Brdatka Červený kříž Lipina Křivoklát Podmolí

n 104 104 96 104 96

A 18.0 17.3 19.1 19.1 19.4

Ho 0.8212 0.8001 0.8239 0.8135 0.7855

He 0.8739 0.8575 0.8742 0.8822 0.8777

FIS 0.0570 0.0429 0.0341 0.0427 0.0959 P 0.0001 0.001 0.01 0.00001 0.0001

Table 1. Diversity statistics and Wright´s fixation coefficient of five studied populations of Quercus petraea.

A – number of alleles; Ho – observed heterozygosity; He – expected heterozygosity; FIS – Wright coefficient of fixation; P – level of significance

600 J. Dostálek et al.

Brdatka Červený kříž Křivoklát Lipina (Natural) (Coppice system) (Planted) (Coppice system) Červený kříž 0.0178 (Coppice system) Křivoklát 0.0105 0.0155 (Planted) Lipina 0.0339 0.0509 0.0277 (Coppice system) Podmolí 0.0408 0.0462 0.0313 0.0065 (Planted)

Table 2. Genetic differentiation (FST) between the studied populations of Quercus petraea. All values are significant at P=0.0001.

-80 -60 -40 -20 0 -150 -100 -50 0 0 0

-20

-50 -40 Podmolí Křivoklát -60 -100

-80

-100 -150 Lipina Brdatka Lipina Podmolí Brdatka Křivoklát

Figure 2. Assignment of individual Quercus petraea genotypes to Figure 3. Assignment of individual Quercus petraea genotypes to the Lipina stand (unplanted stand, under the coppice the Brdatka stand (natural stand) and the Křivoklát stand management system) and the Podmolí stand (planted (planted stand). In case of two outliers 6 loci were not stand). possible to amplify.

-100 -80 -60 -40 -20 0 Protected Landscape Area (Brdatka, Červený kříž, and 0 Křivoklát) with populations from the Podyjí National Park (including the Lipina and Podmolí localities), which are approximately 190 km apart. There was a -20 distinct separation of multilocus genotypes from the Červený Kříž and Lipina populations (Figure 4). Similar -40 results were obtained when comparing other pairs of geographically-distant populations. Lipina -60 3.2 Spatial genetic structure The genetic structure of the study populations is described in Figure 5. In Brdatka, a distinct spatial -80 genetic structure was detected (Figure 5a). The genetic similarity of individuals was negatively correlated -100 (P=0.02) with their spatial distance. The trees growing Červený kříž up to 8 m apart were genetically more similar than on Červený kříž Lipina average within the entire population. A genetic structure was also observed in Červený Figure 4. Assignment of individual Quercus petraea genotypes kříž and Lipina (both coppice system stands). There to the Červený Kříž and Lipina stands (both unplanted stands were managed under the coppice management was a highly–significant correlation (P=0.0001) between system).

601 Genetic differences within natural and planted stands of Quercus petraea

A 0,025 C 0,025

0,020 0,020

0,015 0,015

0,010 0,010

0,005 0,005

0,000 0,000 Genetic similarity Genetic -0,005 similarity Genetic -0,005

-0,010 -0,010

-0,015 -0,015

-0,020 -0,020 5 8 15 25 40 100 5 8 15 25 40 100 Distance class (m) Distance class (m)

B 0,025 D 0,025 0,020 0,020

0,015 0,015

0,010 0,010

0,005 0,005

0,000 0,000 Genetic similarity Genetic Genetic similarity Genetic -0,005 -0,005

-0,010 -0,010

-0,015 -0,015

-0,020 -0,020 5 8 15 25 40 100 5 8 15 25 40 100 Distance class (m) Distance class (m)

E 0,025 0,020

0,015

0,010

0,005

0,000

Genetic similarity Genetic -0,005

-0,010

-0,015

-0,020 5 8 15 25 40 100 Distance class (m)

Figure 5. Relationship between genetic similarity and spatial distance between individuals within study populations of Quercus petraea. Dashed lines indicate 95% confidence limits under the null hypothesis (no spatial genetic structure) obtained after 1000 permutations of the multilocus genotypes. a) Brdatka (natural stand); b) Červený kříž (unplanted stand under coppice management); c) Lipina (unplanted stand under coppice management) – value for distance class ≤ 5 m is 0.09; the out-of-range value on the 5 m distance class is 0.09; d) Křivoklát (planted stand); e) Podmolí (planted stand).

602 J. Dostálek et al.

the decrease in genetic similarity between individuals population. For Q. petraea, Streiff et al. [10] showed and increasing spatial distance between them. Only that there was a markedly decrease in genetic structure trees growing within 15 m of each other (Červený kříž) when individual trees were more than 40 m apart. Cottrell and within almost 8 m of each other (Lipina) showed et al. [13] showed that there was a genetic relationship a significant genetic relationship. On the contrary, in between individuals of Q. petraea growing up to 80 m the planted Křivoklát stand and in the Podmolí locality, apart and for Q. robur growing up to 160 m apart. In no relationship between genetic affinity and spatial contrast, Redkina et al. [33] used allozyme analysis to distance between individual trees was seen. The kinship detect significant genetic similarity between Q. robur coefficient of the closest trees was not significantly trees that were growing up to 12 m apart. Finally, Sork higher than the average similarity within the entire et al. [34] found significant near-neighbor autocorelation population. in Q. rubra trees growing 5 m apart, and Montalvo et al. [35] observed similar results in Q. chrysolepis Liebm. trees growing up to 4 m apart. 4. Discussion Regarding the characteristics of the stands, we found spatial genetic structures at the shortest distances 4.1 Genetic differentiation in habitats where the crowns were not very patulous. We observed a low genetic differentiation among the Significant genetic similarity was proven when trees populations of Q. petraea, which was similarly observed were separated by a distance of approximately 4-times in other studies [18-20]. Our results showed that genetic the length of the average radius of the crowns. We found differences among studied stands were statistically this phenomenon in all of the stands with established significant; however, the index of genetic differentiation spatial genetic structure (i.e., in Brdatka, Lipina, the

FST was low in all cases. We observed differences average radius of the crowns was approximately 2 m in the genetic affinity of populations depending on and in Červený kříž, the average radius of the crown their geographical distance – an increase in distance was about 3 m). We therefore postulate that the crown between populations coincident with a decrease in proportion plays an important role in closed stands genetic distance. These spatial differences were more where pollen and seed dispersal is limited. In more open substantial than the differences that were revealed from stands where individual trees are not so close together, a comparison of the stands under diverse management we expect the dispersal of both pollen and seeds to regimes. This confirms the data of Bodénès et al. [15] have a stronger effect. and Bruschi et al. [14]. Based on our results, we can assume that planting material obtained from a nearby 4.3 Sampling design area was used to produce the new forest. To determine the minimum number of individuals In general, all of the populations that we studied needed to specify the genetic structure of a population, were slightly inbred. Most of them showed low FIS values we compared the kinship coefficients for 100, 50, and (0.0341–0.0570, P=0.01–0.0001). Only the stand 30 individuals. The analysis of 50 individuals was still planted twelve years ago in the Podmolí locality had sufficient to determine the spatial genetic structure in higher FIS values (0.0959, P=0.0001), which suggests natural and semi-natural populations, but the statistical a more significant degree of inbreeding. Similar results, significance of the correlation was negatively affected. including a case in which there was a more significant If 100 individuals were included in the calculation, the excess of homozygotes in one locality, were also found spatial genetic structure was revealed with a higher by Cottrell et al. [13]. A very low deviation from the plausibility and infallibility. When analyzing only 30 Hardy-Weinberg equilibrium towards inbreeding was individuals, the genetic structure was not proven. also seen by Finkeldey [11] and Bakker et al. [12]. Moreover, we did not find significant spatial genetic structure within the trees growing at distances greater 4.2 Spatial genetic structure than 15 m. Thus, in case of less amount of individuals We observed a spatial genetic structure within both sampled, we recommend to choose spatially-close the natural Brdatka stand and the stands that were individuals, i.e., to collect samples from individuals extensively managed for a long period of time using growing just in a segment of the population. On the the coppice system (Červený kříž and Lipina). Genetic contrary, to delineate genetic differences between similarity between the trees growing up to 8 m apart (in populations, it is advisable to collect samples evenly the Brdatka and Lipina localities) or up to 15 m apart (in from the entire population. The analysis of 30 individuals the Červený kříž locality) was statistically significantly from each population was sufficient to prove the genetic higher than the average similarity within the respective differences between populations.

603 Genetic differences within natural and planted stands of Quercus petraea

4.4 Informativeness of markers differentiation between populations corresponded to In this study, ZAG110 and ZAG104 were the most the geographic distances between localities; (iii) in suitable loci for determining the genetic structure natural, unplanted stands, the spatial genetic structure of natural and semi-natural populations. These loci was documented; and (iv) in planted stands, there was gave significant negative correlation between genetic no spatial genetic structure. Therefore, the analysis of similarity and distance of individuals. To determine the spatial genetic structure of the sessile oak forest stand genetic differentiation between populations, MSQ13 and can help reveal and determine its origin. ZAG11 were the most appropriate loci. The total number of loci used for analysis can be less than 10 if the most appropriate markers are selected (i.e., approximately 5). Acknowledgements However, the proper loci used to determine the genetic structure of the population and the genetic differences We wish to thank Pavel Moucha from the Křivoklátsko between populations did not overlap. Landscape Protected Area and Petr Vančura from Podyjí National Park for their logistic support of our field work. Zdeněk Lukeš from Czech Technical University 5. Conclusions in Prague mapped the positions of individual trees. We are also grateful to Roman Businský, Jana Doudová We can make the following conclusions based on all of and Markéta Pospíšková for field assistance. This our analyses: (i) all studied populations were genetically project was supported by grant no. MZP0002707301 differentiated, but there were no major effects that can be and by the Ministry of the Environment of the Czech attributed to different management regimes; (ii) genetic Republic.

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