Abstract
Ecological restoration requires large-scale reintroductions of plants, but their genetic basis is a controversial issue. Formerly, non-local seed sourcing of naturally occurring herbaceous species was common practice. Here we test whether the genetic pattern of the earlier introduction of non-local seeds of Leucanthemum vulgare agg. (ox-eye daisy) can still be detected several years after the application and whether it differs from that of the regional gene pool. We collected leaf material of the ox-eye daisy in Central Germany on sites of indigenous populations (I) and those formerly restored with non-local seed sources (R). Genome sizes and population genetic pattern (AFLP) were analysed. Genome size estimates of most of the individuals studied suggest, that most ox-eye daisies in the region have similar genome sizes regardless of their origin, while individuals from two indigenous populations from the most northwestern part of the study area had lower 1C values. All populations were genetically diverse and the former use of non-local geno-types of the species could not be detected up to more than 8 years after the establishment of the populations. The results shows that a recommendation for restoration purposes is unequivocal, it can only be concluded that it will be best to use seeds that are local and/or similar to the sites intended for sowing.
INTRODUCTION
Ecological restoration projects aim at assisting the recovery of altered or damaged ecosystems (Nevill et al. 2016). Along roadside verges and on compensatory sites, this requires large-scale reintroductions of plants in the form of seeds or seedlings. In Germany, for example, more than 21,000 t of herbaceous perennial seeds were sown in the year 2015, with constantly rising demands at regional, national and global level (Elzenga et al. 2019, Gemeinholzer et al. 2020, Guerrant Jr and Kaye 2007, Mainz and Wieden 2019, Reiker et al. 2015). The genetic basis of seed or seedling sourcing for restoration is a controversial issue concerning (i) costs, (ii) seed source availability and (iii) genetic constituency of the seed sources, while the last point – if knowledge about it is available - affects both preceding ones. For many years non-local seed sourcing of naturally occurring herbaceous species was the main strategy for restoration purposes along roadside verges and on compensatory sites. This was mainly due to significantly lower prices and former unavailability of large quantities of local seeds (Broadhurst et al. 2015, Kettenring et al. 2014, Merritt and Dixon 2011). However, during the last decade, awareness of the importance of the genetic constituency of the restored population increased and local provenancing as seed-sourcing practice received wide attention (e.g. revised in Durka et al. 2017, Elzenga et al. 2019, Mainz and Wieden 2019, Mijnsbrugge et al. 2010, and Williams et al. 2014). Dependent on the magnitude and spatial scale of local seed sourcing, this is nonetheless also arguable as it might result in populations with restricted genetic diversity and limited adaptive potential (Aavik et al. 2014, Hufford and Mazer 2003). This situation led to the recommendation of alternative seed or seedling procurement strategies, such as composite provenancing by using provisional seed zones (Bower et al. 2014), or seed transfer zones (Bucharova et al. 2019, Durka et al. 2017), admixture provenancing, climate-adjusted provenancing, or predictive sourcing for climate change (Broadhurst et al. 2015, Havens et al. 2015) (revised in Breed et al. 2013, and Williams et al. 2014). All of these various measures aim to maintain the genetic variation for plant fitness in the short term and for the adaptation potential in the long term (Aavik and Helm 2018, Leimu and Fischer 2008).
In Germany, the usage of relaxed local provenances with pre-defined seed transfer zones has become mandatory for restoration project from 2020 onward as part of the Nature Projection and Landscape Conservation act (Schumacher and Werk 2010), §40 Abs. 4 BNatSchG 2010). This will decrease the non-local genotype introduction into the landscape; but will also lead to ten-fold higher prices for the usage of indigenous seeds (Mainz and Wieden 2019).
In this study, we compare the genetic diversity and structure of populations originating from former non-local seed mixtures, with indigenous populations from conservation areas, to formulate recommendation for restoration. In a previous study, we examined the population genetic patterns of Daucus carota L. on indigenous and restored sites with non-local seed sources more than 8 years after restoration measures (Reiker et al. 2015). We found no population genetic differences between indigenous and restored sites and concluded that there were no obvious objections to the use of D. carota’s 10 times cheaper, non-local seeds for restoration projects (Reiker et al. 2015). On the other hand, on the same sites Pimpinella saxifraga L. (Gemeinholzer et al. 2020) showed genetic differentiation between indigenous and restored sites. In the present study we investigate whether the population genetic pattern of the formerly non-local seed use of Leucanthemum vulgare agg., in the course of restoration projects at compensation sites still differ from that of the indigenous population in the region. Leucanthemum vulgare is a common and widespread herbaceous plant species that is widely used in seed mixtures for restoration purposes in Germany. We perform genome size measurements to analyse the degree of ploidy of different indigenous- and restored-populations and use AFLP (Vos et al. 1995) to identify population genetic patterns. The following hypothesis is thus tested: The earlier introduction of non-local genotypes in ecological restoration projects can be detected several years after application and therefore a clear recommendation can be given for the use of local seeds in restoration projects for this widespread, herbaceous, insect-pollinated species.
MATERIAL AND METHODS
Study species
Here we investigate the population genetic pattern of the ox-eye daisy, Leucanthemum vulgare agg. (Asteraceae), which comprises three species with three different ploidy levels in Central Europe: the diploid L. vulgare (Vaill.) Lam., the tetraploid L. ircutianum DC., and the hexaploid L. adustum (W. D. J. Koch) Gremli (Oberprieler et al. 2011, Wagenitz et al. 1987). Due to highly variable morphological characters, the three species are often treated as L. vulgare agg. (see Heywood 1976, Marchi 1982). Oberprieler et al. (2011) examined the central European L. vulgare agg. by molecular, morphological, and cytological methods and concluded that the infrageneric taxonomy can only unsatisfactorily be resolved, presumably as a consequence of reticulate evolution. Most of the AFLP markers in their analysis (Oberprieler et al. 2011) were shared among all three members of the L. vulgare agg. Thus, despite different ploidy levels, we use the name L. vulgare agg. The ox-eye daisy was already mentioned in the herbal books of the botanical renaissance of the 16th and 17th century, either as a common and widespread wild species (e.g. in Bauhin 2003: Pinax Theatri botanici 1623) or as an ornamental plant (e.g. in Besler 2003 Hortus Eystettensis 1613). Krausch (2003) reported on several taxa from L. vulgare agg. that were either described and depicted from all over Europe between the 16th and 19th century and cultivated in botanical gardens (e.g. in Berlin and Leipzig). He also mentioned breeding experiments by English, French and German plant breeders. These taxa, e.g. as garden refugees, may have contributed and still contribute to the heterogeneity of the species, making taxonomic treatment difficult.
The ox-eye daisies are perennial herbs with upright, simple or branched stems, up to 60 cm high (Wagenitz et al. 1987). The plants flower from May to October (Georgia 1942, Howarth and Williams 1968). L. vulgare agg. exhibits a predominant cross-pollinated breeding system which is promoted by protandrous flowering (Georgia 1942) although self-pollination may also occur (Grime et al. 2007, Knuth et al. 1906, Villard 1970). More than 100 different flower visiting species of Coleoptera, Hymenoptera, Diptera and Lepidoptera are known to visit the ox-eye daisies (Georgia 1942, Knuth et al. 1898, Knuth et al. 1906) with flight distances up to 3,000 meters, e.g. for the western honey bee (Apis mellifera) (Greenleaf et al. 2007, Rader et al. 2011). L. vulgare agg. reaches fruit maturity in the first year. Achene dispersal is predominantly epi- and endozoochorous (Georgia 1942, Olson 1997, Salisbury 1961) or autochorous, and McDougall et al. (2018) mentioned considerable spread by humans, e.g. by vehicles and hay transports.
Study region and sampling
The study region is in Central Germany (Central and South Hesse, WThuringia, NW Bavaria) within an area of approximately 200 × 200 km2. Road authorities provided information about the sites that had previously been restored with non-local seed material. These were either compensatory sites or road verges. However, since the latter are chronically disturbed environments, we only sampled the compensatory sites. We examined seven restored compensatory sites (R), which were restored between 1994 and 2009 and had different sizes between 0.4 to 1.6 ha (Table 1, Fig. 1). Prior to restoration all sites were arable land (corn, rapeseeds, and cereals are the most common crops in the region) typically ploughed once annually. The contribution of indigenous seeds via the soil seed bank or seed dispersal at the study sites cannot be excluded, but was most likely lower than the amount introduced via seed mixtures. The original seed mixtures comprised 0.3% L. vulgare agg. in 5% herbs and 95% grasses content, with a probable amount of 50 kg per hectare (stable mixture since the late 1980s, according to the FLL Bonn; Research Society for Landscape Development and Landscaping, personal communications). The origin of the seeds for the restoration is unknown and can no longer be traced today. At the time of restoration, however, seeds were often produced in Eastern or Southern Europe, while indigenous seed sources were not available in such large quantities at this time.
Sample sites of Leucanthemum vulgare agg. populations. Abbreviations: Pop = population code: I = indigenous populations, R = restored populations; location in accordance to the nearest village or town; Nature Conservation identification code (CIC) in accordance to the conservation policy of the European Union; the Council of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora Council Directive 92/43 / EWG. GS = Genome size (pg); AGS = Average genome size (pg); MAI = Molecular analysis: no. of individuals; He = after Lynch and Milligan (1994); S. E. He = standard error; HeØ = after rarefaction; Diff. = difference between He and HeØ rarefaction; A = average; Aa = Average all; Aaw = Average all without; AIw = average I without.
| Pop | Location | Lat (N) | Long (E) | CIC | NoI | GS | AGS | MAI | PLP | He | S.E. He | HeØ | Diff. |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| I01 | Biebertal | 50.641687 | 8.556461 | 5317-305 (FFH) | 3 | 20.58–21.72 | 21.12 | 13 | 87.5 | 0.278 | 0.010 | 0.273 | 0.005 |
| I02 | Kirchvers | 50.690680 | 8.579207 | 5317-301 (NSG) | 3 | 20.84–22.88 | 21.77 | 7 | 90.1 | 0.293 | 0.009 | 0.317 | -0.024 |
| I03 | Niederkleen | 50.480773 | 8.616436 | 5517-301 (FFH) | 3 | 19.94–21.90 | 20.95 | 13 | 88.2 | 0.297 | 0.010 | 0.309 | -0.012 |
| I04 | Eichsfeld | 51.220721 | 10.358348 | 4728-301 (FFH) | 3 | 10.16–12.14 | 11.37 | 13 | 86.3 | 0.282 | 0.010 | 0.289 | -0.007 |
| I05 | Berka vor dem Hainich | 51.039571 | 10.415881 | 4828-301 (NP) | 3 | 11.54–11.66 | 11.60 | 13 | 79.8 | 0.249 | 0.010 | 0.258 | -0.009 |
| I06 | Weinberg bei Steinau | 50.327097 | 9.450231 | 5622-302 (FFH) | 3 | 21.08–22.12 | 21.53 | 13 | 86.7 | 0.298 | 0.009 | 0.304 | -0.006 |
| I07 | Hungen | 50.467687 | 8.877661 | 378409 (VSG) | 3 | 21.12–21.58 | 21.37 | 13 | 73.8 | 0.268 | 0.010 | 0.282 | -0.014 |
| R01 | Friedberg 1 | 50.344786 | 8.735547 | 2009 | 3 | 20.50–22.12 | 21.39 | 13 | 74.1 | 0.220 | 0.010 | 0.225 | -0.005 |
| R02 | Friedberg 2 | 50.335239 | 8.733971 | 2009 | 3 | 21.48–22.48 | 21.85 | 13 | 87.1 | 0.270 | 0.009 | 0.269 | 0.001 |
| R03 | Bad Nauheim | 50.392491 | 8.725655 | 1996 | 3 | 21.16–21.64 | 21.47 | 11 | 85.2 | 0.268 | 0.010 | 0.280 | -0.012 |
| R04 | Ober Erlenbach 1a | 50.219039 | 8.679331 | 2004 | 3 | 21.10–21.50 | 21.34 | 13 | 84.8 | 0.255 | 0.009 | 0.266 | -0.011 |
| R05 | Ober Erlenbach 2a | 50.226271 | 8.701537 | 2004 | 3 | 21.28–21.44 | 21.37 | 13 | 82.5 | 0.267 | 0.010 | 0.284 | -0.017 |
| R06 | Herleshausen 1 | 50.995496 | 10.153041 | 2004 | 3 | 20.86–21.34 | 21.05 | 13 | 85.6 | 0.268 | 0.010 | 0.276 | -0.008 |
| R07 | Steinau an der Straße | 50.323347 | 9.446011 | 1994 | 3 | 20.88–21.60 | 21.13 | 12 | 84.8 | 0.277 | 0.010 | 0.281 | -0.004 |
| Aa | 19.86 | Aa | 84.0 | 0.271 | 0.280 | ||||||||
| Aaw I04, I05 | 21.36 | Aaw I04, I05 | 84.2 | 0.271 | 0.281 | ||||||||
| AI | 18.53 | AI | 84.6 | 0.281 | 0.290 | ||||||||
| AIw I04, I05 | 21.28 | AI I04, I05 | 85.26 | 0.286 | 0.297 | ||||||||
| A R | 21.37 | A R | 83.4 | 0.261 | 0.269 |
Map depicting the sampling sites in the study region in Central Germany (Hesse, Thuringia and Bavaria). I01-I07 are the sampled indigenous sites, depicted in blue, R01-R07 are the sampled restored sites (see also Table 1), depicted in red. The Venn diagrams next to the sampling sites show the genotypic affiliations of the AFLP analysis according to the structural analysis (Fig. 3) with two genetic clusters, shown in red and blue. Map was prepared with ArcGIS Pro (ArcGIS Pro 3.2.1., Esri Inc.)
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.8
For comparison, local genotypic diversity was investigated on seven indigenous sites (I) in the same region (Table 1, Fig. 1). I sites were selected with the support of the “Association of German wild plants and seeds producers’ e.V.”. All I sites have not been altered or re-sown during the last 60 years (Prasse et al. 2010). Permits have been obtained from the local nature conservation authorities. A distance of at least 9 km between I- and R-sites was kept as well as a minimum distance of 200 m to any other adjacent population of horticultural relatives (L. vulgare agg.) to avoid introgression (Posselt 2000, Prasse et al. 2010), however, the possibility of cross-breeding between wild and horticultural plants cannot be completely excluded. Within the populations, the minimum distance between the examined individuals was two metres in order to best represent the genetic diversity of the site-specific populations. In large populations, the individuals were random selected with the intention to achieve a wide range over the area. However, in a highly anthropogenic influenced landscape with mainly small isolated nature conservation areas, our approach seemed to be the best trade-off to define “regional species diversity”. In summer 2012 we took leaf samples from 13 individuals per Ior R-site respectively, which were immediately dried and stored in silica gel until further processing.
Genome size estimates
For each I- and R-site, the genome sizes of three randomly selected individuals of L. vulgare agg. were analysed. Pre-tests revealed silica dried leaf material to be suitable for 1C value measurements of the flow cytometric analyser Cy Flow (Partec GmbH, Germany). Vicia faba L. (Bennett and Smith 1991) served as reference (1C value ~13.33). The analysis was conducted following the manufacturers’ instructions. To determine the nuclear DNA content of each sample, the following formula, proposed by Wang et al. (2015), was used: standard 2C value (pg) = (sample peak mean / standard peak mean) * nuclear DNA content of the reference standard (Michaelson et al. 1991, Wang et al. 2015).
AFLP analysis
We aimed to analyse 13 individuals per site, but due to laboratory constraints, sometimes-lower results were obtained (Table 1). DNA was extracted with the DNeasy Plant mini Kit (Qiagen, Germany) according to the manufacturers recommendations. AFLP analyses were carried out according to the protocol of Oberprieler et al. (2011) who have already performed AFLP analyses in the L. vulgare agg. An initial primer screening with six different primer combinations and two individuals per different I- and R-sites was performed. The primers of the final analysis also correspond to those of Oberprieler et al. (2011): E35 / M62, E35 / M48 and E37 / M61. The selected primer combinations proved to be variable and informative (Table S1) and provided clear, reproducible bands which were sufficiently polymorphic to show variation within and among populations. Concerning the laboratory protocols, we followed the recommendations published in Oberprieler et al. (2011). The samples were sent to LGC (Cologne/Germany) for fragment visualization. The data matrix was established using GENOGRAPHER 2.1.4 (Benham et al. 1999). Each AFLP fragment was scored using the “thumbnail” option, which allows for the comparison of signals per locus over all samples. If possible, peaks of low intensity were additionally scored by eye and included into the analysis. Standard lanes, carrying identical samples on each plate were added as quality check. For five samples, the AFLP analysis for each primer combination was repeated twice per 96 well plate and the phenotypic error rate was calculated as number of phenotypic differences related to the total number of phenotypic comparisons and subsequently averaged over the three combinations (Bonin et al. 2004, Pompanon et al. 2005). We carefully analysed the data twice and the AFLP gel images were very good and thorough, the final overall error rate was under the 5% level (2.09%) (Hansen et al. 1999, Jones et al. 1997). Genetic diversity estimates were analysed with AFLPsurv 1.0 (Vekemans 2002) which calculates percentage of polymorphic loci (PLP) and genetic diversity (He) after Lynch and Milligan (1994). For allele frequency estimation the default option in AFLPsurv was used: Bayesian method with non-uniform prior distribution of allele frequencies (Vekemans 2002, Zhivotovsky 1999). HeØ after rarefaction was calculated due to the unequal sample sizes of the different populations by multiple recalculations of He with the smallest population size. Significance tests between He values were done in R (R Core Team 2013) with a posthoc test.
Genetic variation within and among populations was evaluated using Analysis of Molecular Variance (AMOVA) in ARLEQUIN 3.5.1.2 (Excoffier and Lischer 2010), with 10,000 permutations for significance tests. Patterns of genetic population structure were visualized with a principal coordinates analysis (PCoA) using the R package ADEGENET v1.4-2 (Jombart 2008, R Core Team 2013). Pairwise FST was calculated with GenAlEx (Peakall and Smouse 2012). To further explore the genetic affiliation of individuals to genetic clusters, STRUCTURE 2.3.3 (Pritchard et al. 2000) was applied, by using the admixture model with 100,000 Markov Chain Monte Carlo replicates, a burn-in period of 50,000, and ten repeats per run for each chosen cluster number (i.e. K = 1-14), PLOIDY = 2 and RECESSIVEALLELES = 1. For all other settings, the default options were used. To identify the most likely modal K distribution, ΔK (Evanno et al. 2005) was determined using STRUCTURE HARVESTER (Earl and vonHoldt 2012). To verify the most probable cluster membership coefficient among the ten runs of STRUCTURE and STRUCTURE HARVESTER we used CLUMPP vs. 1.1.2. (Jakobsson and Rosenberg 2007). Corresponding graphs were constructed with DISTRUCT (Rosenberg 2004).
RESULTS
The three analysed individuals of the 14 analysed L. vulgare agg. populations, respectively, revealed on average 1C values of 19.86 pg (Table 1). However, the sampled individuals of two indigenous populations (I04 and I05) resulted in mean genome values of 11.5 pg +/–0.2 pg and thus differed significantly from all others with on average mean genome values of 21.36 pg +/–0.4 pg.
The L. vulgare agg. data set, derived from three AFLP primer combinations with 173 successfully analysed individuals, resulted in a total of 263 unambiguously scorable loci (Table 1). The percentage of polymorphic loci (PLP) across all taxa showed a mean value of 84.0% (73.8–90.1%, Table 1). The average PLP of the indigenous populations (84.6%) was not significantly different from that of the restored populations (83.4%, none significant paired T-test). The arithmetic mean genetic diversity of all investigated L. vulgare agg. populations was He = 0.271 with genetic diversities non-significantly varying between He = 0.220 and 0.298. The mean genetic diversity of the indigenous populations He = 0.281 was slightly but non-significantly higher (p = 0.32) than the one of the restored populations He = 0.261 (Table 1). Two of the 14 L. vulgare populations had fewer individuals, but even after a rarefaction, the values hardly changed (Table 1).
L. vulgare agg. featured very low population genetic differentiation between the indigenous and restored provenances. The AMOVA revealed, that only 1.56% of genetic variation explained the provenance-specific divergence (Table 2), while 9% of the genetic variation was explained by among population deviation. The percentage of within population variation was 89.34% with a moderate FST = 0.107 (p < 0.001). If only I-sites were considered, the FST values were slightly higher (FSTI = 0.089), compared to the restored one (FSTR = 0.077). The overall range of pairwise FST values varied between 0.013–0.176 with the populations from the R-sites contributing most to this range. If only the I-site populations were considered, the range varied between 0.013–0.166. However, if the two populations where we detected the lower genome sizes were excluded, the maximum divergence was reduced to 0.083 (Table 3).
Analysis of molecular variance (AMOVA) for populations of Leucanthemum vulgare agg. with two different groupings (restored and indigenous). Statistics include degrees of freedom (Df), AMOVA sums of squares (SS), variance components (VC), percentage of variation, differentiation values (FCT, FSC, FST) and their high significance level (p < 0.001)
| Sites | Source of variation | Df | SS | VC | Percentage variation | F-statistics (p < 0.001) | |
|---|---|---|---|---|---|---|---|
| all | among groups (I, R) | 1 | 154.716 | 0.725 | 1.565 | FCT | 0.016 |
| among populations | 12 | 1101.920 | 4.207 | 9.087 | FSC | 0.092 | |
| within populations | 159 | 6401.998 | 41.361 | 89.347 | FST | 0.107 | |
| total | 172 | 7658.634 | 46.292 | 100 | |||
| indigenous | among populations | 6 | 603.017 | 4.501 | 8.890 | ||
| within populations | 78 | 3597.736 | 46.125 | 91.110 | FST | 0.089 | |
| total | 84 | 4200.753 | 50.625 | 100 | |||
| restored | among populations | 6 | 534.990 | 3.621 | 7.658 | ||
| within populations | 81 | 3536.829 | 43.665 | 92.342 | FST | 0.077 | |
| total | 87 | 4071.818 | 47.286 | 100 | |||
Pairwise FST of the different provenances of Leucanthemum vulgare agg.
| Species | Overall FST | Range of pairwise FST values |
|---|---|---|
| Leucanthemum vulgare | indigenous (all) | 0.013–0.166 |
| indigenous (without I04, I05) | 0.013–0.083 | |
| restored | 0.003–0.176 | |
| all | 0.003–0.176 | |
| all (without I04, I05) | 0.003–0.176 |
The AMOVA results were substantiated by the PCoA (Fig. 2), which depicted one undifferentiated cloud for all analysed L. vulgare agg. samples with no obvious provenance or population specific genotypic differentiations. For L. vulgare agg. the STRUCTURE analysis resulted in a distinct modal maximum of ΔK = 2 (Evanno et al. 2005) with genotypic clusters being neither indicative for provenance nor population affiliation (Fig. 3A). The next higher peak of the STRUCTURE analysis shows K = 5 (Fig. 3B) here we can see a slight difference between I and R. I has more of the yellow part in some populations (LI05, LI06) and R has more of the green part in some populations (LR071, LR02). Nevertheless, as well as in Figure 3A the results shows no genotypic clusters according to provenance or population affiliation.
Principal coordinates analyses (PCoA) summarizing the AFLP marker results of Leucanthemum vulgare agg. individuals. Each circle represents one individual. Individuals from restored sites (R) are depicted in red, and from indigenous sites (I) are depicted in blue. Both axes explain 2.82% und 2.68% of the data, respectively
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.8
AFLP results evaluated in a STRUCTURE analysis for the surveyed populations of Leucanthemum vulgare agg. A) ΔK = 2; according to Evanno et al. 2005 and B) K = 5 the next higher peak for K
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.8
DISCUSSION
This research was carried out to analyse whether the earlier use of nonlocal seeds in restoration projects is still detectable today and differs from the population genetic pattern of the ox-eye daisy in the region. Genome size measurements of most of the populations studied, irrespective of their indigenous or restored origin suggest, that the most common ox-eye daisies in the region have similar genome sizes and most likely belong to the tetraploid L. ircutianum (2n = 36). We are aware that genome sizes do not automatically reflect the degree of ploidy, but the only two indigenous L. vulgare agg. populations from the most northwestern part of the study area in the Hainich (I04, I05), Thuringia, had lower 1C-values, which might possibly indicate the diploid L. vulgare (2n = 18). Both species are known to frequently occur in this region of Central Germany (Oberprieler et al. 2011, Scholz and Uhlemann 2001 a, b).
Our analysis support earlier findings by Oberprieler et al. (2011) who already noted that most of the AFLP markers studied are shared among the different species within the L. vulgare agg. with only a few markers being unique to the respective species. While the genetic diversity of one of the potential L. vulgare populations within the L. vulgare agg. (HeI04 = 0.282) is similar to the average genetic diversity of all indigenous populations (ØHeI = 0.281) mainly belonging to L. ircutianum, the L. vulgare population I05 is genetically depleted (HeI05 = 0.249, Table 1). However, a reduced genetic diversity was also found at a restored site with potentially tetraploid L. ircutianum individuals (HeIR01 = 0.220). The population is on a compensatory site surrounded by intensively farmed agricultural land on very productive soils. These most likely form an isolation barrier that prevents gene flow from the environment, which could explain the genetic impoverishment of the population (Grime et al. 2007, Vil-lard 1970, Wagenitz et al. 1987). On average genetic diversities in populations on I-sites (ØHeI = 0.281) were slightly higher than the ones on R-sites (ØHeR = 0.261), which even became more prominent after rarefaction. Nevertheless, the AFLP-data are suitable to cover 90% of the population genetic diversity (Leipold et al. 2020, McGlaughlin et al. 2015). According to Leipold et al. (2020), an average of 14 samples and 120 loci is required for insect-pollinated species, which we come close to with an average of 13 samples per population, but with 263 loci we have more than twice as many loci analysed (Reisch and Bernhardt-Römermann 2014). However, no information about the initial genetic diversity in the region was available and we are unaware if the genetic diversity in the non-local seed mixtures was significantly different from the ones of the indigenous populations. Other studies on the same sites on Pimpinella saxifraga (Gemeinholzer et al. 2020) have shown that there is a genetic difference which might indicate the previous use of non-local seed mixtures. Morphological studies on Daucus carota (Reiker et al. 2020) have also shown a difference in generative characters that significantly differed between indigenous and restored sites in the same system.
Throughout the investigation area, there were no distinct pattern differences between L. vulgare agg. populations of indigenous and formerly restored sites (Figs 2–3). This supports earlier findings in Daucus carota (Reiker et al. 2015), another common insect pollinated plant species frequently present in seed mixtures for Central European restoration projects. Reiker et al. (2015) also conducted population genetic analyses in the same investigation area of Hesse, Thuringia and NW-Bavaria with the same study design. In L. vulgare agg. (FST = 0.106, p < 0.001) as well as in D. carota (FST = 0.034, p < 0.01, Reiker et al. 2015) moderate to negligible patterns of genetic differentiation between populations of indigenous and restored sites were retrieved. Even though L. vulgare agg. is known to be self-compatible (e.g. Andersson 2008, Knuth et al. 1898, 1906, Villard 1970) showed that cross-pollinated flower heads had a much higher fruit set (Ø 57%) than heads subject to self-pollination (Ø 2.5%). This may explain why the here found outcrossing rates are high, similar to the ones found in D. carota (Reiker et al. 2015). However, the genetic structuring for L. vulgare agg. in the investigation area is distinctly lower than typical for common perennial outcrossing plant species (FST = 0.22, Nybom et al. 2004), FST = 0.20–0.17, (Reisch and Bernhardt-Römermann 2014). The lower result might be due to the regional focus of our investigation (Reisch and Bern-hardt-Römermann 2014) but could also be the result of the human induced introduction of non-local seed sources in the region. In L. vulgare agg. most of the molecular variance (Ø 89.3%, 73.8–90.1) was found within the 14 populations, irrespective of whether only indigenous populations or sites restored with non-local seed material were analysed. The values are high compared to other AFLP based population genetic investigations in central European perennial Asteraceae, e.g. in Senecio vulgaris L. Ø 81.9 % (Haldimann et al. 2003) or Lactuca serriola L. 78.0% (Lebeda et al. 2009), and higher than the ones found by Oberprieler et al. (2011) who screened the central European Leucanthemum species from the northwestern Alps and the Franconian Jura (78.0%). The lower within population divergence retrieved by Oberprieler et al. (2011) might be due to the larger investigation area of their analysis and the fact that they included the hexaploid alpine L. adustum into their investigation, which was not present in our analysis. We performed the AMOVA with several alternative groupings (L. vulgare agg., L. vulgare, L. ircutianum of indigenous and/or restored sites, Table 4) but the genetic differentiations of the L. vulgare populations did not distinctly differ from the overall pattern.
Based on these results, similar to D. carota (Reiker et al. 2015), the conclusion is obvious to use non-local seed material for the current restoration practice for L. vulgare agg. Nevertheless, studies on phenotypic variability in D. carota (Durka et al. 2019, Reiker et al. 2020) showed that there are generative traits in seed maturation towards the end of the season that give the local seeds an advantage. P. saxifraga (Gemeinholzer et al. 2020), Centaurea cyanus L. and Lychnis flos-cuculi L. (Bucharova et al. 2017) were also found to have an average 1.3–1.4-times higher reproductive output compared to non-native plants.
Finally, the genetic patterns did not reflect the generative traits and fitness parameter (Durka et al. 2019, Reiker et al. 2015). Thus solely based on the genetic outcome in L. vulgare agg. we are not aware of local adaption in a small region like the investigation area in Hesse, Thuringia, and NW-Bavaria within a range of 40,000 km2.
In other population genetic analyses on seven common grassland species in Germany (Arrhenatherum elatius (L.) P. Beauv. ex J. Presl et C. Presl , Centaurea jacea L. , Daucus carota, Galium album Mill. , Hypochaeris radicata L. , Knautia arvensis (L.) Coult. and Lychnis flos-cuculi; Durka et al. 2019) and on Pimpinella saxifraga (Reiker et al. 2015) no or low genetic differentiation was found but both analyses could reveal generative trait differences between indigenous and restored populations. Durka et al. (2019) were able to show that local seed usage is the better solution by also considering mix-or-match (Lesica and Allendorf 1999), thus, we endorse this recommendation. In the case that there are not enough local seeds available, renaturation should be replenished with the nearest of the 22 regions of origin, to fulfil ecosystem function. Potential genetic benefits associated with population admixture are increased genetic variation and the formation of novel trait combinations due to segregation and recombination, which could result in increased individual fitness and population growth rates (e.g. Hufford and Mazer 2003, Verhoeven et al. 2004, 2011).
CONSEQUENCES FOR RESTORATION PRACTICES
In the German Nature Projection and Landscape Conservation act (Schumacher and Werk 2010, §40 Abs. 4 BNatSchG 2010) seed transfer zones were defined, based on climate, geological substrate and soil types (Meyen 1959–1962). Within each seed transfer zone, the mixing of seeds from several source populations is recommended to increase genetic variation (Durka et al. 2017, ErMiV 2011, Prasse et al. 2010). Durka et al. (2017) screened the population genetic differentiation of seven common grassland species in eight regions of the 22 seed transfer zones throughout Germany and revealed significant differentiation in most of the investigated species. As a consequence they postulated genetic structuring across Germany in common grassland plants to be more common than formerly assumed (Durka et al. 2017). However, their data only allows for a recommendation in support of the eight seed transfer zones while in our analysis on smaller scales within the investigation area in Hesse, Thuringia, and NW-Bavaria the indigenous populations featured negligible population genetic differentiation within a range of 40,000 km2. The investigation area represents on average 2.5 times the size of a seed transfer zone in Germany (357,376 km2 in total and divided into 22 seed transfer zones) and therefore, we would recommend to use the envisaged 22 seed transfer zones and support the demand for a population genetic monitoring of these zones. However, this would require a much more in-depth analysis across species, as already proposed by Durka et al. (2017). They argued that ideally a population genetic monitoring of a large number of the 150 grassland species used for restoration purposes in Germany on a grid-based scheme would be necessary to put the whole seed provenancing system on a solid empirical basis. However, this requires careful experimental considerations as proposed by Bucharova et al. (2017).
CONCLUSIONS
Without trait information, we cannot make any conclusive recommendations for seed use of L. vulgare agg. for restoration practices. Based on the data of other species studied (e.g. D. carota; Reiker et al. 2015, 2020 and P. saxifraga; Gemeinholzer et al. 2020) and comparable research (Bucharova et al. 2017, 2019, Durka et al. 2017), it can only be recommended that it is best to use seeds that are local and similar to the site intended for sowing, and otherwise to use seeds from nearby/similar origins.
*
Acknowledgements –
We are grateful for the financial support from the Deutsche Bundesstiftung Umwelt (DBU, 20011/122), and the Heidehofstiftung (57190.01.1/4.11). Sampling was conducted with support from the Association of German Wild Seeds and Wild Plants Producer Association (Markus Wieden), as well as the road authorities Frankfurt (Gerd Ledergerber), Schotten (Alexander Greb), and Herleshausen (Helmut Byczysko). For permits to collect plants in nature conservation areas we thank Thomas Keller (NSG “Schwarze Berge”), Norbert Mitter (NSG “Schwarze Berge”), Jürgen Busse (NSG “Oberes Verstal”, Kirchvers), and Manfred Grossman (Hainich National Park). For support in the molecular laboratory, we thank the bio-technical assistants (Sabine Mutz and Helene Krufczik) and the students (Tatjana Lapin and Hannah Nebelung). We would also like to thank Benjamin Schulz and the members of the Systematic Botany group in Gießen. The manuscript developed during the EU funded COST action CA18201: An integrated approach to conservation of threatened plants for the 21st Century.
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