Inflow of seeds through the forest edge: evidence from seed bank and vegetation patterns

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1 Plant Ecology :1-17 Springer 2005 Inflow of seeds through the forest edge: evidence from seed bank and vegetation patterns Rebecca Devlaeminck*, Beatrijs Bossuyt and Martin Hermy Laboratory for Forest, Nature and Landscape Research; University of Leuven; Vital Decosterstraat 102; B 3000 Leuven; Belgium; *Author for correspondence ( rebecca.devlaeminck@agr.kuleuven.ac.be, fax: ) Received 4 July 2003; accepted in revised form 29 January 2004 Key words: Ancient forest, edge effect, seed inflow, seed longevity Abstract To determine the influence of the proximity of a forest edge on seed bank composition and diversity, we performed a seed bank sampling at ancient deciduous forests bordering intensive arable fields. Also vegetation patterns were taken into account. We hypothised that forest edges may facilitate the entrance of diaspores of invasive species into the forest and the successive incorporation of these species in the forest seed bank. We noticed a substantial influence of the proximity of an edge on seed bank composition at as well the forested side of the edge as the field side. The forest edge zone was limited to 3 m into the forest and the field edge zone extended 3 m into the field. The seed bank samples of field and forest edge are characterised by a higher species diversity and seed density and a higher similarity between seed bank and vegetation, compared to field or forest samples. The forest edges contains fewer pioneer species in comparison with the forest interior and more competitive species and species of edges and clearings compared with field and forest samples. The seed longevity index increases towards the forest interior. We can conclude from our data that the forest and edge seed bank are composed by both seeds from recent dispersal processes and local seed set and by seeds originating from past vegetation on the site. Near the edge, actual seed input seems of primal importance. Further towards the forest interior seed input decreases and long-living seeds of past vegetation become more important. Ancient forest edges thus act as a barrier for seeds of species of the surrounding arable field. Introduction Intensive agricultural land use, clear-cut and urbanisation have led to changes in landscape structure and increasing forest fragmentation in many parts of the world. As a consequence, the remaining forest patches have become more isolated and patch size has decreased e.g., Jacquemyn et al Furthermore the amount of area under influence of the proximity of an edge has strongly increased. Small forest fragments have indeed a high edge to interior ratio and are consequently largely influenced by edge conditions Laurance and Yensen Through the edge an important flux of matter, energy and species is mediated Ryszkowski 1992; Wiens A distinction can be made between natural abiotic, antropogenic abiotic, direct biotic and indirect biotic fluxes. Natural abiotic gradients comprise the different aspects of variation in microclimate between forest edge and forest interior. In general one can conclude that the forest edges exhibit higher values of light, air and soil temperature, wind speed and cloud water deposition e.g., Chen et al. 1995; Cadenasso et al. 1997; Weathers et al Relative humidity and soil/litter moisture increase with increasing distance into the forest e.g., Chen et al. 1995; Gehlhausen et al Especially in areas of

2 2 marked agricultural and industrial activity important antropogenic abiotic fluxes such as the drift of agrochemical products and atmospheric deposition, have been observed. Penetration distances of these abiotic variables highly differ and depend on edge structure, physiognomy, orientation and age, forest type, disturbance regime, management of forest and matrix, successional state of forest and matrix, landscape characteristics, season, climate and the variable under consideration e.g., Chen et al. 1992; Cadenasso et al Most empirical studies found penetration distances of less than 150 m Laurance 2000, but further distances have also been reported Chen et al As a result of the biotic flux of diaspores from the landscape matrix into the forest edge, forest edges may facilitate the entrance of invasive and exotic species into the forest Peterson and Carson 1996; Cadenasso and Pickett This may be due to the altered microclimatic conditions at the forest edge, the higher probability of disturbance of an edge and the open structure of the vegetation in young edges. If diaspores of non-forest species can penetrate the forest interior and establish populations in the vegetation there, they may have a profound influence on the forest ecosystem. The microclimatic, soil and other abiotic gradients directly influence various biotic aspects such as structure of the vegetation, distribution of species, sensitivity to alien invasions, plant reproduction, growth and mortality e.g., Matlack 1994; Gehlhausen et al. 2000; Honnay et al This results in indirect influences due to changes in interactions between species, such as competition. Consequently, species richness and composition change along the gradient from the landscape matrix over forest edge to forest interior. In general species richness declines from the edge into the forest interior e.g., Matlack Shade intolerant and competitive or ruderal species occur at the edge but are often absent or scarce at the forest interior, while interior-oriented species have a lower importance at the edge Honnay et al Matlack 1994 found edge-oriented species within 5 m from the forest edge, some up to 40 m. Recently, there is an increasing knowledge concerning the composition of forest seed banks e.g., Dougall and Dodd 1997; Bossuyt and Hermy 2001; Bossuyt et al. 2002, but we found no previous research on seed banks over forest edges. However, many forest species have short-lived seeds and do not form a persistent seed bank, while a lot of arable weeds, on the contrary, produce long-lived seeds Thompson and Grime 1979; Bossuyt and Hermy So, it can be expected that the latter become incorporated in the forest edge and probably forest interior seed bank. Therefore, we investigated in this study seed bank and vegetation patterns along transects from forest interior into arable fields. The main objectives are: 1 to determine the width of the edge zone, in which the proximity of a forest edge influences seed bank composition, 2 to assess quantitative and qualitative differences in seed bank characteristics related to distance from the edge and 3 to determine whether non-forest species can penetrate the forest seed bank through the edge. Methods Study area For this study, we selected 14 ancient forest edges on silt or silty soil adjacent to intensively used arable land and not separated from it by a road, hedge or similar element Table 1. Ancient forest is defined as forest that has been continuously forested since 1770 date of the oldest available map up to today. All forest stands are classified as Milio-Fagetum communities sensu Noirfalise 1984 and are covered with mature, mixed deciduous forest mainly with Fagus sylvatica, Quercus robur and Acer pseudoplatanus. As with most ancient forests, the vegetation is characterised by a pronounced vernal phase with Anemone nemorosa, Convallaria majalis, Hyacinthoides non-scripta and Polygonatum multiflorum. The edges were as straight as possible and at least 150 m long. All edges were sharply delineated, as farmers cultivate the adjacent fields almost up to the first trees. Seven edges had a southern aspect; the other seven edges were north facing. Data collection Within every edge a transect was established perpendicular to the forest edge Figure 1. The transects extended from 40 m into the farmland to 50 m into the forest, distances measured from the field/forest boundary. The border between arable land and forest was objectively located based on the presence or absence of a ploughed layer. A vegetation survey was made in blocks of 3 40 m at 10 distances along

3 3 Table 1. Characteristics of the surveyed transects Transect Study area Location Area ha Edge aspect Data Vegetation Seed bank Soil Microclimate 1 Bassegem forest Kaster 5.73 SE 2 Bassegem forest NW 3 Bertem forest Bertem SW 4 Bertem forest N 5 Grevens/Eiken forest Bertem 94.7 SSW 6 Grevens/Eiken forest NW 7 Hei forest Kortenaken 65 SE 8 Hei forest NE 9 Helle forest Kampenhout 130 SE 10 Helle forest NE 11 Lembeek forest Lembeek N 12 Meerdaal forest Hamme-Mille SE 13 Moorsel forest Moorsel 84.9 NE 14 Spek forest Veltem-Beisem 25.5 S this transect in which five 3 m 3 m plots were randomly selected and sampled. Three blocks at the field side of the field/forest margin and 7 blocks in the forest were sampled. This resulted in 50 plots per edge and an overall total of 700 plots. Cover of all present vascular species in the plots, including tree seedlings up to 1.5 m, was estimated using the decimal cover scale Londo The survey was executed in spring 2002, when spring vegetation was present, and repeated in September Data of both surveys were combined into one dataset whereby for each species the highest cover was used. Microclimate variables were measured in August 2002, during a period of stable, bright weather, between and h. A mobile weather station Skye Datahog 2; Skye Instruments Ltd., Powys, UK was used to measure photosynthetically active radiation PAR, nm, W/m 2, soil and air temperature C and relative air humidity %. The PAR was measured with a Solar Hog Sensor SDL 5000 series solar cell PSU. With the exception of soil temperature, all variables were measured at 30 cm above ground level. Forest overstorey density was measured using a spherical densiometer Lemmon Every measurement was repeated 5 times in every plot, except the forest overstorey density, which was measured 8 times per block. As microclimatic variables exhibit highly variable temporal patterns Chen et al they were expressed as fractions of the open field 40 m into the field values. Seven edges three north facing and four south facing were selected for seed bank sampling Table 1. At every distance, we randomly picked one of the five plots in which the vegetation was surveyed and established therein a 1m 1m subplot in which seed bank sampling was executed. Soil sampling for seed bank research was carried out in March 2002 and repeated in September This repeated sampling increases the probability of finding species with different germination requirements as the seeds in the spring samples have undergone a stratification period during winter while the autumn samples have experienced a dry period. In every subplot, 30 samples were taken at random with an auger of 3.5 cm diameter down to 20 cm depth, after removing the litter layer. Every forest sample was divided in two subsamples, of 0 10 cm depth and of cm depth, to be able to take the temporal dynamic of the seed bank into account e.g., Bonis and Lepart 1994, Thompson et al In every plot the samples were pooled per soil layer, resulting in two samples in every plot. Arable field samples were not subdivided as ploughing activities have mixed the soil layers. The samples were subsequently sieved according to the method of Ter Heerdt et al. 1996, which was recommended for estimating a forest seed bank Bossuyt et al The samples were washed through a course 4 mm mesh width and a fine 0.2 mm mesh width sieve to remove debris, root fragments and coarse and fine soil material. The concentrated samples were subsequently spread out into a layer of about 2 mm depth in seed trays filled with sterilised potting soil. A layer of expanded clay granules on the bottom of the trays prevented the

4 4 Figure 1. Layout of the sampling, showing a transect of ten 3 40 m blocks. Vegetation surveys were made in five at random plots 3 3m per block. In every block seed bank sampling was executed in a subplot 1 1m placed randomly in one of the plots. soil from waterlogging. The samples were randomly placed in a growing room with a light regime of 16 hours light and 8 hours darkness. Temperature was not controlled and ranged between 15 and 24 C. Three trays filled with sterilised soil were placed among the samples as a control for contamination. No seedlings emerged in these control trays. As soon as possible, the seedlings were identified, counted and removed. This was performed for four months until no further seedlings emerged. Then the samples were mixed and put in the growing room for another two months. Seedlings that could not be identified immediately were transplanted into pots to allow further growth. Sporophytes were not included in the seed bank data as, because of their small size, spores may be lost upon sieving of the samples. In the seven transects used for seed bank research, soil samples up to 5 cm depth after litter removal were taken for soil analysis. Five samples were taken per plot and pooled together resulting in one soil

5 5 sample per block. These samples were analysed for available ph KCl, available phosphate Egner-Riehm method, carbon content Walkley and Black method and nitrogen content Kjeldahl-Lauro method Hendrickx Data analysis To avoid statistical problems related to pseudoreplication, vegetation cover data of the five plots per block were averaged for each species, resulting for every species into one average cover value per distance for every edge. The same was done for microclimatic data. In a first explanatory phase, a Detrended Correspondence Analysis DCA was executed on the vegetation and seed bank data. For this analysis, vegetation and seed bank data were expressed as relative importance values. This is the abundance cover or number of seedlings of each species in each plot divided by the sum of the abundances of all species in that plot. The DCA-plot scores were subsequently correlated with distance to the forest edge, microclimatic and soil variables and soil layer in the case of seed bank data using a Kendall s tau test Siegel and Castellan As all variables lacked a normal distribution tested with a Kolmogorov- Smirnov test, a non-parametric statistical approach was used. Since for seed bank data the explanatory variables were all correlated with distance from the edge, a partial correlation was performed controlling for the effect of distance. To delineate an edge zone, we tested for significant differences between DCA scores of the plots with a Kruskal-Wallis test with multiple comparisons. Since a forest edge is defined as the zone where species richness and species diversity differ from the forest core Forman and Moore 1992, the edge zone was defined as that zone where DCA plot scores were significantly different from field and forest interior plots. This was done separate for seed bank and vegetation DCA scores. After defining a forest and field edge zone, differences in vegetation and seed bank characteristics between edge, field and forest zones were examined. Therefore, several characteristics of the seed bank and vegetation were calculated per block. To determine the contribution of species of the landscape matrix and the forest, the species occurring in vegetation and in seed bank were divided in socio-ecological groups based on the classification of Stieperaere and Fransen To reduce the number of groups, we combined species of forests and shrubs into one group. Species of trampled sites, pioneer species and species of bare ground were also combined. Species of edges, clearings and young plantations are described as species of edges and clearings. We also distinguished a group of ancient forest species based on the list of Honnay et al Next to this, the dispersal mode, dispersule weight class Kleyer 1995 and seed longevity index Thompson et al. 1997; Bekker et al. 1998a were determined for each species. For every plot the number of seedlings or the cover of species belonging to each socio-ecological group was summed and divided by the total number of seedlings of that sample or the total cover in that plot. So, we calculated the relative abundance of each socio-ecological group in the seed bank for each sample and in the vegetation for each plot. As Juncus bufonius L. accounts for 31.3% of the total number of seedlings and consequently had a strong influence on these values, this species was omitted in the analyses. The same was done for the dispersal mode and seed weight classes. Next to this, for each seed bank sample and vegetation plot, the mean seed longevity index was calculated, weighted by the number of seedlings or the cover for seed bank and vegetation data respectively. As the seed input by former vegetation, external seed input and local seed input determine seed bank composition, we assessed the potential contribution of the present vegetation through determining the similarity between seed bank and present vegetation. A Czekanowski similarity coefficient Kent and Coker 1995 was calculated for each plot based on relative performances of each species in the vegetation and in the seed bank. This was done for both the separate soil layers and for the combination of the two soil layers. All calculated variables were tested for differences between edge, field and forest zones using a Kruskal- Wallis test with multiple comparisons Siegel and Castellan Moreover, to explore patterns with distance, the variables were also correlated with distance from the field/forest margin Spearman Rank Correlation. A Wilcoxon signed rank test for related samples was used to determine differences between characteristics of the two soil layers of the seed bank data. Data analysis was performed using SPSS 10.0 SPSS Inc., 1999 and CANOCO for Windows 4.0

6 6 Ter Braak and Smilnauer Plant species nomenclature follows Lambinon et al Results Detrended Correspondence Analysis The first three axes of DCA analysis on seed bank data explained 17.4% of the variance of the plots. As expected these axes are significantly correlated with distance from the field/forest margin Table 2. After partial correlation controlling for distance, the first DCA-axis is significantly correlated with percentage photosynthetic active radiation, canopy cover, soil ph, carbon and nitrogen content of the soil. The second axis is correlated with photosynthetic active radiation, canopy cover, air and soil temperature, soil ph and soil nitrogen content. The third is correlated with photosynthetic active radiation, canopy cover, air and soil temperature. Considering the vegetation data, the first three DCA axes explain 16.5% of the total variance. Here also these axes are significantly correlated with distance from the field/forest margin Table 2. After correction for distance, the first axis is correlated with photosynthetic active radiation, soil temperature, soil ph and nitrogen content of the soil. The second axis is correlated with photosynthetic active radiation, relative humidity, soil ph and soil nitrogen content. The third axis is correlated with air and soil temperature, soil ph and photosynthetic active radiation. Significant differences obtained by Kruskal-Wallis tests on the DCA plot scores were used to divide the different seed bank samples into four groups: field plots at distances 40 and 20 m, field edge plots at distance 3 m, forest edge plots at distance 0 m and forest interior plots at distances 4 m up to 50 m Figure 2a. Kruskal-Wallis analysis on the DCA plot scores of vegetation data revealed the same distinction into four groups as based on the seed bank data Figure 2b. Size and diversity of the seed bank and diversity of the vegetation. In the spring seed bank analysis 5,874 seedlings of 71 species germinated while the autumn seed bank analysis produced 2,155 seedlings of 64 species, resulting in 8,029 seedlings of 86 species Appendix. This corresponded to a seed density of 2,515 seeds/ Table 2. Kendall s tau correlation coefficients of seed bank and vegetation DCA plot scores with site characteristics. Seed bank Vegetation Correlation Partial Correlation Correlation Partial Correlation n AX 1 AX 2 AX 3 AX 1 AX 2 AX 3 n AX 1 AX 2 AX 3 AX 1 AX 2 AX 3 Distance m *** *** 0.21** 0.26*** Photosynthetic active radiation *** ** * 0.26** *** 0.38*** 0.17* 0.18* 0.32*** 0.05 W/m 2 Canopy cover % * 0.22** 0.19** 0.17* 0.22** 0.19* * Relative humidity % *** *** 0.24** * 0.00 Air temperature C ** *** * Soil temperature C ** ** 0.23** *** ** 0.36*** * Soil ph *** 0.24*** *** 0.29*** *** 0.37*** 0.35*** 0.30*** 0.31*** 0.24** Carbon content % *** * *** 0.21** Nitrogen content mg/100 g *** 0.19* ** 0.23** *** 0.27** 0.24** 0.19** 0.19** 0.11 Phosphate content mg/100 g * n: number of seed bank samples, resp. vegetation plots, * 0.01 p 0.05, ** p 0.01, *** p 0.001

7 Figure 2. Scatterplot of the plotscores on the first two DCA axes, with field, edge and forest zones, based on vegetation data a and seed bank data b. 7

8 8 m 2. In the forest samples 6,616 seedlings were recorded of 77 species. The upper soil layer produced 3,874 seedlings of 69 species while the lower soil layer produced 2,742 seedlings of 61 species. In the arable field samples 1,413 seedlings of 47 species germinated. Seeds in the upper soil layer have a significantly lower Longevity Index compared to seeds in the lower soil layer, res and 0.80 p As the longevity of seeds is assumed to be related to seed size Bakker et al. 1996, Bekker et al. 1998a, we may expect differential distributions of small and large seeds with depth of burial. The seed bank is indeed dominated by very small seeded species. 64% of seeds in the upper soil layer and 75% of the lower soil layer are seeds with weight less than 0.2 mg. These very small seeds are significantly more abundant in the lower soil layer p Largerseeded species are more often encountered in the upper soil layer p The seed bank predominantly consists of pioneer species and of species of edges and clearings Table 3, the former group being more abundant in the lower soil layer p while the latter is more abundant in the upper soil layer p The most abundant species are Juncus bufonius L. 31% of total seedlings, Urtica dioica L. 19%, Juncus effusus L. 10%, Hypericum humifusum L. 6% and Betula species 4%. Inthe fourteen forest edges used for the vegetation survey, 162 plant species were encountered Appendix 1. In the seven edges used for seed bank sampling, 114 species were recorded in the herbaceous layer. 47 species were recorded in both vegetation and seed bank, although abundant species in the vegetation were not necessarily abundant in the seed bank and vice versa. This is reflected in the low similarity between vegetation and seed bank, with a Czekanowski similarity index that ranges between 0% and 50% with a median of 6%. Seed bank and vegetation characteristics in relation with distance from the forest/field margin The analysis of the relationship between seed bank data, respectively vegetation data and distance are presented in Table 3. Characteristics of seed bank and vegetation seem to differ in their correlation pattern with distance. A clear distinction can be made between the arable field and the forest interior seed bank concerning their ecological characteristics Table 3. Field samples are significantly richer in arable and tall herb vegetation, with predominantly species without specialised dispersal mechanism and with dispersule weight less than 0.5 mg. Forest interior samples, on the contrary, have significantly more forest species. Species with very small 0.2 mg or very large 10 mg dispersules are significantly more frequent in the forest than in the field seed bank. Total seed density and number of species in the seed bank are not significantly correlated with increasing distance into the forest. This contrasts with the significant increase in vegetation cover with distance from the edge. The seed bank samples of field and forest edge have a higher number of species and of seeds/m 2 compared to field or forest samples. The Czekanowski similarity index, which expresses the similarity between seed bank and vegetation data, is highest in the edge and decreases with distance from the field into the forest interior The average Longevity Index of seed bank samples increases significantly with distance from the field into the forest interior. The Longevity Index of vegetation data shows the opposite trend. The relative abundance of species with dispersule weight of 0.21 to 0.5 mg in the seed bank declines with distance into the forest while both species with light up to 0.2 mg and heavy more than 10 mg dispersules increase. Compared to the other samples, the seed bank at the forested edge is relatively rich in species with dispersule weight between 1.1 and 2 mg. In the vegetation, species with dispersules of less than 1 mg decline with distance into the forest. Large seeded species increase in the vegetation with increasing distance from the field/forest margin into the forest interior, although species with dispersules weighing 2.1 to 10 mg decrease. The dispersal mechanism differs with distance from the field into the forest. Species with anemochorous and endozoochorous dispersal become more frequent in the soil seed bank near the forest interior while the abundance of species without specified dispersal mechanism declines with distance towards the forest interior. Epizoochorous species are very abundant in the edge soil samples. Species composition of both seed bank and vegetation show a gradual change in socio-ecological groups over the gradient from arable field into forest interior. The frequency in the vegetation of grassland, arable field, pioneer species and tall herb vegetation declines with distance. In the seed bank field species and tall herb vegetation become less important with increasing distance into the forest. The opposite correlation is observed in both vegetation and seed bank for forest and shrub species and ancient forest

9 9 Table 3. Correlation with distance and differences between field, edge and forest zones of the ecological characteristics of seed bank and vegetation and of the environmental variables. Juncus bufonius was omitted. Seed bank Vegetation Distance 1 sig Field 2 Field Forest edge 2 Forest 2 sig Distance 1 sig Field 2 Field Forest edge 2 Forest 2 sig edge 2 edge 2 Number of species a 10.9ab 17b 9.92a *** a 13.58ab 19.46b 9.4a ** Number of seeds/m 2, resp. % vegetation cover a 3446b 4171b 1565a *** 0.18 * 28a 49ab 87b 52ab *** Czekanowski similarity index 0.39* 0.13ab 0.17ab 0.22a 0.03b * Longevity index 0.54 *** 0.72a 0.73a 0.73a 0.86b *** 0.74 *** 0.60a 0.55bc 0.49ab 0.16c *** Dispersule weight` 0.2 mg 0.31 * 0.38a 0.60ab 0.62ab 0.67b * 0.46 *** 0.17a 0.24a 0.23a 0.04b *** mg 0.45 *** 0.38a 0.21ab 0.17ab 0.04b * 0.72 *** 0.37a 0.23a 0.08a 0.00b *** mg a 0.05a 0.08a 0.15a 0.47 *** 0.23a 0.20ab 0.08ab 0.04b *** mg a 0.03ab 0.06b 0.00a ** 0.29 ** 0.14a 0.13a 0.11a 0.36a mg a 0.06a 0.03a 0.01a 0.47 *** 0.08a 0.08a 0.07a 0.01b *** 10 mg 0.56 *** 0.02a 0.05a 0.04ab 0.13b *** 0.60 *** 0.01a 0.10a 0.40b 0.55b *** Dispersal` Anemochorous 0.39 *** 0.12ab 0.07a 0.15ab 0.29b * a 0.25a 0.11a 0.19a Hydrochorous a 0.00a 0.00a 0.00a 0.46 *** 0.04a 0.03a 0.02a 0.00a Epizoochorous a 0.57b 0.60b 0.19ab ** a 0.22a 0.25a 0.35a Endozoochorous 0.56 *** 0.02a 0.04a 0.04ab 0.11b *** 0.36 *** 0.05a 0.07ac 0.32b 0.28bc *** Myrmecochorous a 0.00a 0.00a 0.01a 0.24 ** 0.04a 0.01a 0.02a 0.07a Unspecified 0.41 *** 0.73a 0.32b 0.21b 0.25b *** 0.58 *** 0.48a 0.42a 0.29ab 0.11b *** Socio-ecological group` Grassland a 0.07a 0.11a 0.13a 0.54 *** 0.13a 0.18a 0.06a 0.00b *** Arable field 0.33 ** 0.16a 0.11ab 0.08ab 0.08b * 0.75 *** 0.29a 0.11ab 0.02bc 0.00c *** Tall herb vegetation 0.42 *** 0.11a 0.07a 0.04ab 0.00b ** 0.56 ** 0.11a 0.05a 0.03ab 0.00b *** Pioneer a 0.21ab 0.12b 0.49a ** 0.74 *** 0.21a 0.19a 0.03ab 0.00b *** Edge and Clearing a 0.48b 0.57b 0.14ab *** a 0.18cb 0.29b 0.12ac *** Forest and Shrub 0.51 *** 0.05a 0.05a 0.06ab 0.24b ** 0.28 *** 0.08a 0.15ab 0.63b 0.30b *** Ancient forest 0.28 * 0.00a 0.00a 0.01a 0.00a 0.77 *** 0.02a 0.05ab 0.17b 0.57c *** Forest total 0.53 *** 0.05a 0.05a 0.07ab 0.25b ** 0.75 *** 0.10a 0.20ab 0.49b 0.88c *** Environmental variables Photosynthetic active radiation W/m *** 1a 0.17a 0.03ab 0.2b *** Canopy cover % 0.35 *** 0.16a 62.52b 83.39b 78.73b *** Relative humidity % 0.73 *** 1a 0.99a 1.01a 1.11b *** Air temperature C a 0.98a 0.95a 0.95a Soil temperature C 0.64 *** 1a 0.96a 0.94ab 0.78b ** Soil ph 0.85 *** 5.03a 4.81a 4.48ab 2.92b *** Soil carbon content % 0.76 *** 2.76a 5.00a 9.01ab 12.92b *** Soil nitrogen content mg/100 g 0.73 *** 38.59a ab bc c *** Soil phosphate content mg/100 g a 20.51a 14.52a 21.75a 1 Spearman-rank correlation coefficients; 2 Kruskal Wallis pairwise comparisons, mean values are given, differences are indicated with letters, the microclimatic variables were standardised with respect to open field value 1 ; sig: significance of the test, * 0.01 p 0.05, ** p 0.01, *** p 0.001; `Expressed as relative frequencies

10 10 species. The only clear distinctions between edge seed bank and field or forest seed bank point at significantly fewer pioneer species in the forest edge in comparison with the forest interior and a higher abundance of species of edges and clearings in the edge. Discussion Composition and depth distribution of the seed bank Seed bank densities in different studies vary substantially, as is indicated by comparison of other seed bank studies in ancient deciduous forests e.g., Brown and Oosterhuis 1981 spring sampling; 3,230 seeds/ m 2, Staaf et al spring sampling; 1,757 seeds/ m 2, Kjellsson 1992 spring sampling; 13,286 seeds/ m 2, or in arable fields e.g., Reuss et al repeated sampling; 6,436 seeds/m 2, Torresen and Skuterud 2002 autumn sampling with cold and dry treatment; 4,363 up to 16,961 seeds/m 2. The here observed densities are relatively low compared to these other studies. This is probably due to the autumn sampling which produced relatively few seedlings, so that the seedling density, calculated on combined sampling of spring and autumn, is lowered. The dominance of the small-seeded Juncus bufonius L. is rather surprising. Several other authors report high frequencies of Juncus spp., but in forests this concerns predominantly J. effusus L. Brown and Oosterhuys 1981; Kjellsson 1992; Bossuyt et al The negative correlation between seed size and seed number Roff 1982, and between seed size and seed longevity Hodkinson 1998, may explain the dominance of small seeded species, which are also more prevalent in the deeper soil layer. The dominant species groups in the seed bank are pioneer species and of species of edges and clearings Table 3, with the former being more abundant in the lower soil layer and the latter being more abundant in the upper soil layer. Other authors Bossuyt et al have also reported a high proportion of species of edges and clearings in the forest seed bank. The seeds in the deepest soil layer are the oldest seeds Thompson et al and most probably represent seeds of past succession stages when the forest edge may have been more open and the pioneer species could more easily penetrate into the forest. With closure of the forest edge, the influx of pioneer species in the forest decreased and the contribution of local seed input of species of edges and clearings becomes more important. Characteristics of the seed bank of field and forest edge Up to now, most authors only considered the forested side of the field-forest ecotone instead of the entire landscape gradient, although previous research has already pointed at the influence of the forest on the surrounding non-forested land on microclimate and consequently on vegetation e.g., Brothers and Springarn 1992; Cadenasso et al We found based on seed bank and vegetation composition a distinct edge zone, not only at the forested side of the field/ forest margin, but also at the field side. Here, the forest edge zone is extending from the margin to 3m into the forest, and a field edge zone from the margin to 3 m into the field. This value is lower than the depth of influence values upon vegetation reported in other deciduous forests. Honnay et al reported an edge zone ranging between 3 and 23 m depending on edge aspect. Other studies report edge zones of 10 to 60 m Matlack 1994; Gehlhausen et al Ancient forest edges as the ones studied here, have developed relatively closed side vegetation, which functions as a kind of shield and so reduces the penetration depth of edge effects. At younger edges, where the edge vegetation is less developed, edge effects penetrate further into the forest Matlack The seed bank samples of forest and field edge are characterised by a high species richness. This also holds for edge vegetation plots. Three possible mechanisms can be postulated. Firstly this reflects the high species richness of the edge vegetation. In the edge not only species occur from the two adjacent habitats, but also species that are characteristic for edge environments, which often form long living seeds Bossuyt et al. 2002, here reflected in the high abundance of species of edges and clearings in field and forest edge Table 3. Secondly the proximity of an edge is promoting growth and reproduction of several species as more resources can be attained near the edge Jules and Rathcke 1999, resulting in a high local seed production. In the edge the similarity between seed bank and vegetation is relatively high 17 to 22%, Table 3, which suggests an important seed input into the seed bank from the present vegetation. Thirdly the seed input from the adjacent landscape is concentrated at the edge as the ancient forest edges, used here, have developed a relatively closed struc-

11 11 ture which serves as an effective barrier to the flux of seeds Cadenasso and Pickett Moreover as animals respond to the landscape structure, their dispersal movements are not homogeneous in a fragmented landscape van Dorp and Kalkhoven For example roe deer show preference for areas close to the edge Tufto et al. 1996; Casaer This is in accordance with the high abundance of zoochorous species near the edge Table 3. In contrast to the edge samples, few seedlings germinated in the field and forest samples. Field samples have a lower seed density, as agricultural practices such as the use of herbicides and fertilisation decrease the number of mature seed-producing plants Hoffman et al. 1998; Scursoni et al Furthermore soil tilling leads to a depletion of the seed bank through bringing non-dormant seeds unable to germinate in deep soil to the surface Mulugeta and Stoltenberg The field seed bank is relatively similar to the established vegetation Cs 13%, Table 3. Beatty 1991 also reported that early successional stages have a high number of species equal in seed bank and vegetation. In the forest, seed production is low as forest species devote a relatively small amount of their energy to sexual reproduction. They form relatively large seeds adaptive for successful establishment in shaded environments Leishman and Westoby 1994; Hodkinson et al and under litter Eriksson The seeds usually are shortlived and so do not form a persistent seed bank Bekker et al. 1998b. Consequently the seeds present in the forest seed bank are almost exclusively originating from past or external seed input and from secondary succession after forest management. The divergence in species composition between seed bank and vegetation in late-successional vegetation has also been reported by other authors Thompson and Grime 1979; Beatty Penetration of non-forest species into the forest interior and vice versa The higher seed density at the field edge and forest edge compared to the forest interior suggests that the forest edge functions as a physical barrier for the dispersal of seeds. This is also confirmed by the increase in seed longevity index towards the forest interior. This pattern indicates that in the forest interior longliving seeds from past vegetation are most dominant and that recent external seed input is most important near the edge and declines with distance into the forest. The abundance of arable field, tall herb vegetation and species of edges and clearings in the vegetation is clearly lower in the forest interior than at the edge Table 3. The same pattern prevails in the seed bank, indicating that seeds of these species cannot be incorporated into the forest seed bank by seed inflow. The forest interior seed bank consists to a large extent of pioneer species, although these species now do not occur in the present vegetation. The abiotic conditions in the forest interior, especially the low light level and the thick litter layer, are unfavourable for germination and establishment of these species. Only when disturbances e.g., windthrow gaps, cutting occur, resulting in an increase in light intensity and litter decay, their germination requirements are fulfilled and a vigorous population can establish. The importance of the light level for vegetation patterns over the forest edge is demonstrated through its significance upon DCA analysis Table 2. As we found significant correlations between soil ph and soil nitrogen content with the DCA axes of vegetation data, we might assume that not only litter quantity and quality, but also soil chemical properties influence vegetation composition Honnay et al Through their influence on the vegetation, and subsequently on the local production of seeds, these variables also influence seed bank composition, as is indicated through their correlation with the DCA-scores of the seed bank samples Table 2. The influence of soil and air temperature on seed bank composition may also be due through their importance for vegetation composition. It is plausible that soil properties such as temperature, ph, carbon and nitrogen content not only influence seed bank composition through their effect on the vegetation, but also through affecting the preservation of seeds in the soil for a review: see Hilhorst and Karssen Hence, the seed bank is composed by both seeds from recent dispersal processes and by seeds originating from past vegetation on the site. The relative importance of these two processes varies with type of vegetation, successional stage and distance to the edge. Near the edge, actual seed input seems of primal importance. Further towards the forest interior, as seed input decreases, remnant seeds of past vegetation become more important. Significant differences in seed bank and vegetation composition occur at a distance of about 3 m from the forest margin. Our results correspond with those of other studies e.g., Brothers and Springarn 1992;

12 12 Gehlhausen et al. 2000; Honnay et al who reported that the presence of most invasive species in ancient forests is limited to 3 m from the forest edge. However, the studied edges are relatively closed and therefore probably function as efficient barriers for seed dispersal. This may explain the limited edge width. Conclusions We noticed a gradient in seed bank composition over a field-forest ecotone. The forest edge zone was limited to 3 m into the forest and the field edge zone extended 3 m into the field. We can conclude from our data that ancient forest edges are relatively impenetrable for species from the surrounding arable field as their frequencies decline sharply with distance from the forest/field margin. There are however few seeds that penetrate the edge and become incorporated in the forest seed bank. As long as no disturbance occurs in the stable forest environment, the germination requirements of these species are not fulfilled and they cannot impose negative influences on the forest vegetation. Acknowledgements We thank K. Michiel and T. Stijnen for assistance with fieldwork, J. Van Assche for assistance with determining the seedlings and the Forestry Administration Leuven, Natuurpunt vzw, the Université Libre de Bruxelles and private owners for permission for sampling in their forests. The research was supported financially by a Research Assistance Grant of the Funds for Scientific Research, Flanders FWO. Appendix Table A1. List of the relative abundances % of the species encountered upon vegetation sampling based upon percentage cover of each species and in seed bank samples based upon number of seedlings of each species. Seed bnk data are given according to soil layer cm. Seedbank Vegetation Forest Forest edge Field edge Field Forest Forest edge Field edge Field Acer campestre L Acer pseudoplantus L Achillea millefolium L Adoxa moschatellina L Aegopodium podagraria L Agrostis capillaris L Agrostis stolonifera L Ajuga reptans L Alnus glutinosa L. Gaertn Alopecurus myosuroides Huds Anagallia arvensis L Anemone nemorosa L Angelica sylvestris L Anthoxanthum odoratum L Arctium spp Arrhenatherum elatius L. Beauv Artemisia vulgaris L Arum maculatum L Athyrium felix-femina L. Roth Atriplex patula L Avena sativa L Betula spp Bidens cernua L Bromus hordeaceus L Bromus sterilis L

13 13 Table A1. Continued. Seedbank Vegetation Forest Forest edge Field edge Field Forest Forest edge Field edge Field Buddleja davidii Franch Campanula trachelium L Capsella bursa-pastoris L. Med Cardamine flexuosa With Cardamine hirsuta L Cardamine pratensis L Carex pilulifera L Carex remota L Carex sylvatica Huds Carpinus betulus L Castanea sativa Mill Centaurium erythraea Rafn Cerastium fontanum Baumg Chenopodium album L Chenopodium murale L Chenopodium polyspermum L Chrysanthemum segetum L Circaea alpina intermedia Ehrh Circaea lutetiana L Cirsium arvense L. Scop Cirsium palustre L. Scop Cirsium vulgare Savi. Ten Conium maculatum L Convallaria majalis L Conyza canadensis L. Cronq Corylus avellana L Crataegus monogyna Jacq Crepis capillaris L. Wallr Cytisus scoparius L. Link Dactylis glomerata L Daucus carota L Digitalis purpurea L Dryopteris carthusiana Vill. H.P Fuchs Dryopteris dilatata Hoffm. A. Gray Dryopteris filix-mas L. Scott Echinochloa crus-galli L. Beauv Elymus repens L. Gould Epilobium spp Fagus sylvatica L Fallopia convolvolus L. A. Löve Festuca pratensis Huds Festuca rubra L Fraxinus excelsior L Galeopsis tetrahit L Galinosaga parviflora Cav Galium aparine L Geranium dissectum L Geranium molle L Geranium robertianum L Geranium spp Geum urbanum L Glechoma hederacea L Gnaphalium uliginosum L

14 14 Table A1. Continued. Seedbank Vegetation Forest Forest edge Field edge Field Forest Forest edge Field edge Field Hedera helix L Heracleum sphondylium L Hieracium laevigatum Willd Holcus lanatus L Holcus mollis L Hordeum vulgarum L Hyacinthoides non-scripta L Chouard ex Rothm. Hypericum humifusum L Hypericum perforatum L Hypericum pulchrum L Ilex aquifolium L Juncus bufonius L Juncus effusus L Kickxia elatine L. Dum Lamium album L Lamium galeobdolon L. L Lamium purpureum L Lapsana communis L Lolium perenne L Lonicera periclymenum L Lotus corniculatus L Luzula multiflora Retz. Lej Luzula pilosa L. Willd Lysimachia nemorum L Maianthemum bifolium L F.W.Schmidt Matricaria spp Melandrium dioicum L. Coss. et Germ. Mentha arvensis L Mercurialis annua L Mercurialis perennis L Millium effusum L Misopates orontium L. Rafin Moehringia trinervia L. Clairv Myosotis arvensis L. Hill Narcissus pseudonarcissus L Oxalis acetosella L Oxalis corniculata L Phalaris canariensis L Phleum pratense L Plantago major L Poa annua L Poa nemoralis L Poa pratensis L Poa trivialis L Polygonatum multiflorum L. All Polygonum aviculare L Polygonum convolvulus L Polygonum hydropiper L Polygonum lapathifolium L Polygonum persicaria L Populus alba L

15 15 Table A1. Continued. Seedbank Vegetation Forest Forest edge Field edge Field Forest Forest edge Field edge Field Populus nigra L Potentilla anserina L Potentilla sterilis L. Garcke Primula elatior L. Hill Prunus serotina Ehrh Quercus robur L Quercus rubra L Ranunculus ficaria L Ranunculus repens L Raphanus raphanistrum L Ribes rubrum L Robinia pseudoacacia L Rubus fruticosus agg Rumex acetosa L Rumex obtusifolius L Sagina procumbens L Sambucus nigra L Scirpus setaceus L Scrophularia nodosa L Senecio vulgaris L Solanum dulcamara L Solanum nigrum L Sonchus arvensis L Sonchus oleraceus L Sorbus aucuparia L Spergula arvensis L Stachys arvensis L. L Stachys sylvatica L Stellaria holostea L Stellaria media L. Vill Symphytum offıcinale L Tanacetum vulgare L Taraxacum spp Teucrium scordonia L Tilia platyphyllos Scop Trifolium pratense L Trifolium repens L Triticum aestivum L Typha latifolia L Urtica dioica L Veronica arvensis L Veronica beccabunga L Veronica chamaedrys L Veronica hederifolia L Veronica offıcinalis L Veronica persica Poir Veronica serpyllifolia L Viburnum opulus L Vicia sativa L Vinca minor L Viola arvensis Murray Unidentified species