Efficiency of mechanical seed harvesting for grassland restoration

Agriculture, Ecosystems and Environment 247 (2017) 195–204

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Agriculture, Ecosystems and Environment journal homepage: www.elsevier.com/locate/agee

Efficiency of mechanical seed harvesting for grassland restoration a,⁎

Michele Scotton , Magdalena Ševčíková a b

MARK

b

Department of Agronomy, Food Natural Resources, Animals and Environment, University of Padova, Italy OSEVA Development and Research Ltd., Zubří, Czech Republic

A R T I C L E I N F O

A B S T R A C T

Keywords: Combining Harvesting efficiency Green hay Hay-making Seed harvesting Seed stripping Standing seed yield Semi-natural grasslands

Maintaining and re-creating species-rich semi-natural grasslands are important issues in current agricultural policy in Europe. The seed that is required for their establishment can be obtained through direct harvesting from semi-natural herbaceous vegetation. To test the efficiency of mechanical seed harvesting on donor Arrhenatherion elatioris grasslands, experiments were performed in northern Italy and eastern Czech Republic. Trials were organized with a randomized block design and involved harvesting as green hay (grass mowing and immediate collection), dry hay (grass mowing and collection after drying on the field), direct combining (grass cutting and threshing at the same combine passage) and seed stripping with pull-type equipment (seed removal without grass cutting with a brush harvester pulled by a tractor). Harvesting was carried out at the time of maximum ripe standing seed yield (SSY) in the first and second regrowth. The harvested materials were analysed for seed number and weight and compared with the SSY. The species composition and phenology were also surveyed. In all methods, the seed mixture obtained contained the species present as seed at harvest time and was correlated with SSY. However, with regard to the seed number collected, the harvesting efficiency changed in relation to species group (grasses or forbs), individual species, seed maturation and regrowth. The most efficient method was harvesting as green hay (efficiency of approximately 71% of SSY and seed mixture composition that was very highly correlated with SSY). The least efficient methods were direct combining (30% of SSY and negative selectivity against light or difficult to detach seeds, producing the highest mean 1000-seeds weight) and harvesting as dry hay (39% of SSY and a high sensitivity to seed maturation level). Seed stripping, operated with downward brush rotation at the leading edge, resulted in an intermediate efficiency (60% of SSY and a seed mixture composition highly correlated with SSY). The harvesting efficiency for seed number was higher at the first regrowth than the second one for both species groups and, especially at the second regrowth, higher for forbs than for grasses. Especially forb harvesting presented a problem with regard to the species number collected. The problem was, not due to harvesting inefficiency but rather for phenological reasons, as several forbs did not produce fertile stems at the first or second regrowth and some other early flowering species had already shed the seed at the harvesting time. These results improve our understanding of factors affecting the efficiency of mechanical harvesting and will help in the preparation of efficient harvesting programs.

1. Introduction Semi-natural grasslands play a central role in biodiversity conservation in Europe (EEA, 2004). They represent habitats that have a high natural value, are rich in native species and often contain rare plants, birds and invertebrates (Jefferson, 1999a). In addition to their high natural importance, they contribute to the enhanced value of agricultural products of regional or gastronomic value and to noncommodity outputs, such as agro-tourism and ecosystem functions (Hopkins and Holz, 2006). Semi-natural grasslands were likely widespread in Europe at the beginning of the twentieth century (Van Dijk, 1991), but agricultural

⁎

intensification and abandonment of many marginal areas have strongly reduced their extent (Dietl, 1995; MacDonald et al., 2000). Maintaining and re-creating species-rich semi-natural grasslands has therefore become an important issue of current agricultural policy in Europe. Many types of semi-natural grasslands are included among the habitats to be protected under the Natura 2000 network (EEC, 1992). Additionally, restoring, preserving and enhancing ecosystems related to agriculture have become priorities for EU rural development policy (https://ec. europa.eu/agriculture/rural-development-2014-2020_en). Arrhenatherion elatioris hay meadows are the most important type of European lowland semi-natural grasslands. In the past, they have been extensively mown two to three times a year as a valuable forage source

Corresponding author. E-mail addresses: [email protected] (M. Scotton), [email protected] (M. Ševčíková).

http://dx.doi.org/10.1016/j.agee.2017.06.040 Received 5 March 2017; Received in revised form 25 June 2017; Accepted 26 June 2017 0167-8809/ © 2017 Elsevier B.V. All rights reserved.

Agriculture, Ecosystems and Environment 247 (2017) 195–204

M. Scotton, M. Ševčíková

Ševčíková et al., 2011) and that all of these factors affect restoration success (Kiehl et al., 2010; Münzbergová, 2012; Scotton et al., 2016). Probably, the harvesting year can also affect the composition and amount of the collected seed, as weather is cause of important year-toyear changes of the grassland vegetation (Herbent et al., 2003) and the seed production of herbaceous species (Ryle, 1966). Comparing harvesting methods is then important to understand the main factors that influence their efficiency, which would be useful for optimizing harvesting and increasing the chance of successful restoration. To improve the available knowledge, we therefore compared different methods of mechanical harvesting on Arrhenatherion elatioris grasslands to:

with considerable species richness (Rodwell et al., 2007), but they are currently much less common than before the 1970s and are therefore now regarded as plant communities to be protected and restored (Dietl, 1995). Due to the climatic regions (low altitude) and soil types (middle fertility) where these meadows exist, they could also represent the target vegetation for restoring not only agricultural surfaces but also urbanized areas, e.g., as urban wildflower meadows (Kingsbury, 2004) or anti-erosion covers along roads and railways. Grassland restoration can be accomplished with different methods (Kirmer et al., 2012). Site-specific seed mixtures, seed-enriched chaff from hay-barn floors, hay-flowers from threshing and brush harvesting can be sown manually or with sowing or spreading devices (e.g. mechanical seeder, hydro-seeder). Seed-rich biomass, such as green hay, dry hay or raked material, can also be used to transfer seed and, at the same time, spread a mulch layer to protect soil against erosion and favour seedling establishment. Grassland ca be restored also through spreading plant and seed rich topsoil or transplanting grass turfs. Whatever the method used, a central point in grassland restoration is the need for native seed material. Due to the lack of viable seed in the soil seed bank and limited dispersal of target species seed (Bakker et al., 1996) seed of native species has to be actively introduced if high-nature value grassland is to be achieved within a reasonable time. Seeds from native grassland plants can be obtained through singlespecies seed propagation (Steinauer, 2003) or directly from species-rich grasslands. The second approach is easy when sufficient areas of species-rich grasslands are present in the region in question (Scotton, 2016) and is the only practical field-scale solution for obtaining seeds of known local origin and the full range of species associated with native grasslands, which are often not available from typical commercial sources (Everett, 2003). Several methods are available for harvesting seed from semi-natural grasslands. Hand collection is suitable for obtaining enough seed for restorations of less than a few acres (Steinauer, 2003). Mechanical methods allow for operating on wide surfaces and collecting great quantities of seed from many species that can be used for large restorations (Steinauer, 2003). Among the most frequently used, green or dry hay harvesting (collecting the whole fresh or dried above ground seed-rich biomass), direct combining and seed stripping can be carried out with equipment often available in standard agricultural farms (mowing equipment and wheat threshers) or purchasable at a relatively low price (seed stripper). In the last two decades, many trials have been conducted to test the efficacy of grassland restoration with propagation materials obtained from mechanical harvesting directly from donor sites. With regard to mesic or dry grasslands, green hay harvesting was tested e.g., by Kiehl and Wagner (2006), Donath et al. (2007), Schmiede et al. (2012), and Graiss et al. (2013). Seed from dry hay harvesting was used in the experiments of Wells et al. (1986),and Scotton (2016), seed from direct combining was used in the trials of Schwab et al. (2002), Graiss et al. (2013), Baasch et al. (2016) and Scotton (2016), and seed from seed stripping was used in the experiments of Edwards et al. (2007) and Scotton (2016). However, except for Scotton et al. (2009) peer-reviewed research on native seed harvesting methods is limited, even if many internet sources can supply useful information (Davison, 2004). Compared to cultivated varieties, seed harvesting from semi-natural grasslands is complicated due to the mixed species composition. Native plant species populations mature seed gradually, as they were not selected for phenological uniformity (Burton and Burton, 2003). Further, each one of the many species has its own optimal harvesting method (Mitchell, 2003) and/or settings on the harvesting equipment (Houseal, 2007; Krautzer et al., 2004), which means that finding one harvesting method and equipment setting optimal for all species is not possible. On the other hand, past experience has shown that the harvesting method and regrowth can influence the number of species collected as seed as well as the absolute and relative seed amounts of individual species and species groups in the seed mixture (Scotton et al., 2009;

• quantify the harvesting efficiency of several methods with regard to • • •

species diversity, seed number, seed quality for the whole harvested material and for important grassland species groups (grasses and forbs) and individual species; characterize the variability of the harvesting efficiency as affected by year, regrowth and site; investigate the possible effects of seed traits on harvesting efficiency of the individual methods; identify the stages when the species loss can take place during the process of seed production and harvesting on the donor site.

2. Materials and methods 2.1. Sites, meadows, harvesting trials and field surveys Seed harvesting trials were performed on semi-natural grasslands between 2009 and 2011 in Italy and in the Czech Republic. The Italian trials took place in Pianari (Marostica, 11°37′55′′E, 45°46′38′′N; 435 m a.s.l.) on a 20% sloping, south-facing Arrhenatherion elatioris hay meadow with a 31 cm deep, 7.6-pH soil. Management intensity was low (two to three cuts and 20 kg N fertilization per ha per year) and species richness was high (63 vascular plant species, including 11 grasses and 52 forbs, found within an area of 1800 m2). In March 2009 three randomized blocks, each one with five 100 m2 plots, were marked. On 5 June, at the end of the first regrowth period (the regrowth after winter time) and at the time of maximum ripe standing seed yield (SSY) as estimated through phenological surveys (for methods see later in this section) of the main meadow species (Arrhenatherum elatius and Trisetum flavescens, together percentage cover of 50.5%), four plots in each block were harvested for seed using the following methods:

• green hay harvesting: the grass was cut with a cutterbar mower and immediately collected; • dry hay harvesting: the grass was cut with a cutterbar mower, dried • •

on the field with one manual turning and baled in the afternoon of the day after; direct combining with a wheat thresher (model Laverda 3300); seed stripping with a pull-type stripper (model 410, Prairie Habitats) with downward brush rotation covering the full vegetation height.

In each plot, after weighing the whole harvested material, two samples with weight corresponding to one m2 each were taken. The fifth plot per block was used to survey the standing seed yield (SSY): all fertile stems present on two 1 × 1 m subplots were collected and placed in a paper bag separately for each species. After drying, the samples were analysed in the following months for content of mature seeds, which were extracted, categorized to the species level, counted and weighted. In SSY, green and dry hay harvesting the number of fertile stems of each species was also counted. The harvesting trial was repeated at the second 2009 regrowth and in 2011, where, however, due to less funding available, the treatments considered were less numerous. The methods used were SSY, green hay 196

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the nominal p ≤ 0.05 divided by 3) to avoid increasing Type 1 error (Sokal and Rohlf, 2012). In case of ANOVA rejecting the null hypothesis, the Tukey test was performed on the means. According to Cochran and Cox (1950), as the four Italian trials differed with respect to the number of harvesting methods considered, a one-factor ANOVA was performed for each trial. In contrast, the two Czech trials had the same number of treatments and could be subjected to a single two-way analysis with harvesting method and year as categorical factors. Aside from the total amount of harvested seed, the individual species harvesting efficiency of each method was also analysed. The harvesting efficiency was calculated as the percentage of the number of seeds harvested by each method compared to SSY. Statistical correlations were then calculated among the characteristic and the following possibly influencing parameters retrieved from field observations or the literature. A seed ripeness index was calculated for each species and trial from the phenological surveys performed immediately before harvesting. The values 0–3 were assigned to the phenological stages of flowering or earlier, developing seed or fruit, ripe seed or fruit, and fallen seed or fruit, respectively. The seed ripeness index was then calculated as the mean of the above values weighted by the percentage of flowers recorded in the corresponding stage. From the D3 dispersal database (Hintze et al., 2013) the following diaspore traits were downloaded for each species: morphology (presence of wings, appendages, hooks and mucilaginous surface), size (length and width), terminal velocity, anemochory, epizoochory and hydrochory ranking indices. The plant height of each species was retrieved from Flora alpina (Äschimann et al., 2004). Relations between the harvesting efficiency (response variable) and the explanatory variables (seed ripeness index and variables from the D3 database and Flora alpina) were fitted for grasses and forbs separately according to linear and second-order polynomial regressions and checked for the parametric assumptions of residual normality and homoscedasticity with the QQ-Plot and the Breusch-Pagan test respectively. The effect of the method on the seed amount harvested for each species was analysed based on the seed number collected per fertile stem, calculated from the seed and fertile stem numbers found per m2. In the case of the harvesting techniques where the fertile stem density was not available, the density found in SSY was used for the calculation. After a log transformation needed to homogenize variances, the data obtained for the main species (at least two values available per harvesting treatment and an average fertile stem density of three per m2) were subjected to parametric ANOVA. The similarity of the species composition of seed mixtures harvested with the different methods with the SSY was analysed using standardised major axis estimation after transforming the absolute number of harvested seeds of the individual species into a percentage of the total number of seeds. As data were strongly heteroscedastic, line-fitting was based on log-transformed values. SMA was used instead of linear regression method as the analysis aimed at testing the similarity of compositions and not at predicting one of them (Warton et al., 2006). Slopes and elevations of the calculated lines were tested for differences from 1 and 0, respectively, by checking for correlation between residual and fitted values (slope) and using a one-sample t-test (elevation), as described in Warton et al. (2006). To study the number of species harvested and the stages where species loss can take place during the process of seed production and harvesting on the donor site, species presences recorded in the botanical and phenological surveys and found in the harvested materials were combined separately for each 2009 trial plot. Five types of species presence were considered: presence as plant recorded during the whole growing season, presence as plant at harvesting, presence as fertile stem at harvesting, presence as mature seed in the phenological survey at harvesting and in the harvested material. The presence of donor-site species observed on the plots of the restoration trials in years 2009–2014 (Italian trial) or 2009–2011 (Czech trial) and certainly not originating from the receptor site or its surroundings were also used to

and dry hay in 2009 second regrowth (4 August), to obtain the propagation material necessary for the restoration trial reported in Scotton (2016). To improve the knowledge about dry hay harvesting, which had not been considered in the Czech trials (see later), only this method was further experimented in 2011 first (16 June) and second (2 August) regrowth. In the text the trials are coded with year/regrowth (e.g., trial 2009/1 corresponds to the trial from the first 2009 regrowth). Propagation materials obtained from the 2009 harvests were used to implement a grassland restoration trial, which was surveyed for botanical composition in the years 2009–2014. The trial was located on an ex arable field 11 km apart from the donor site and had an extent of 2400 m2 (Scotton, 2016). In the harvesting trial years, botanical composition was visually estimated (percentage of above ground biomass of each species; Klapp, 1971) in each plot on four dates: at the beginning of the growing season and at the end of the three regrowth periods. Species identification and naming were performed as described by Pignatti (1982). In the month before harvesting the phenology of each species was weekly recorded by visual estimation of the percentage of plants in vegetative and reproductive stages and the percentage of flowers in different development stages. Hess et al. (1997) was used as a reference for phenological stages. The Czech trials took place in the nature reserve Terezské údolí in Central Moravia (Náměšť na Hané, 17°2′24.061′′ E,49°35′48.7′′ N; 248 m a.s.l.) on an alluvial flat Arrhenatherion elatioris hay meadow with a deep (> 80 cm), 6.2-pH soil. The main species were Holcus lanatus, Trifolium campestre and Arrhenatherum elatius. The usual management was two cuts per year and no fertilization, and the botanical composition included 64 species (14 grasses and 50 forbs, found on an area of 2250 m2). In March 2009 three randomized blocks, each with three 250 m2 plots, were marked. On 25 June (at the end of the first regrowth period) the seed was harvested on two plots per block as green hay and with direct combining. In the third plot, SSY was surveyed. The harvesting procedure was the same as in the Italian trials. For each plot, three samples (weight corresponding to one m2 each) were taken. Species composition and phenology were surveyed the day before harvesting with the same method as in the Italy trials. Species identification and naming were performed as described by Kubát et al. (2002). Lab analyses for seed (SSY, green hay and direct combining) and fertile stem (SSY) content were also performed as in the Italian trials. The harvesting trial, botanical and phenological surveys were repeated in 2010 (28 June). Propagation materials from the 2009 trial were used to establish a restoration trial that was surveyed for botanical composition in the years 2009–2011. The trial was located at Bázlerova pískovna on an ex arable field 14.6 km far from the donor site and had an extent of 1500 m2. 2.2. Data analysis The single species values of the numbers of fertile stems and mature seeds, weight of mature seeds and 1000-seeds weight obtained from the laboratory analysis were grouped into grasses, forbs and all species. The number of fertile stems was never found to be affected by harvesting method. Therefore, as the random variability of this characteristic could influence the amount of harvested seed, with possible confounding effects with regard to the efficiency of the harvesting method, the number and weight of harvested seeds of SSY and as green hay and dry hay recorded in the Italian trials were adjusted to the mean fertile stem number of the three methods. After checking for ANOVA assumptions (Bartlett test for variance homoscedasticity and Shapiro-Wilk test for data normality), the values of the four characteristics for each species group were subjected to parametric ANOVA. As the test for each characteristic was performed three times (all species, grasses and forbs) on at least partially the same data, null hypothesis significance thresholds were Bonferroni-corrected (actual p ≤ 0.017 resulting from 197

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integrate the presence in the harvesting material. Indeed, restored plots sometimes contained donor-site species which had not been recorded in the harvesting samples: this could happen especially in harvesting as green or dry hay and for less abundant species, as the samples of harvested material analysed did not necessarily contain mature seed of all species present in the whole harvested material. This analysis was only performed for the 2009 trials, as the 2010 and 2011 trials were not followed by a restoration experiment. After checking for parametric assumptions (variance homoscedasticity and data normality), the recorded number of present species obtained for each trial were subjected to a two-way parametric ANOVA with type of species presence and harvesting method as categorical factors. As the test was performed on the three species groups (all species, grasses and forbs), the significance threshold was again Bonferroni-corrected. In the case of ANOVA giving significant differences among factor levels, the Tukey test was performed on the means. The software used for statistical analyses was R 3.0.0 (Core Team R, 2013) with the package STATS for Bartlett and Shapiro-Wilk tests, ANOVA, Tukey test, linear or polynomial regressions under the Linear Model (LM) approach; the package CAR (Fox and Weisberg, 2011) for validation of parametric assumptions of linear and polynomial regressions; and the package SMATR (Warton et al., 2012) for testing for the difference from 1 and 0 of the slope and elevation of the regressions between species composition of the harvested seed mixtures and of the SSY.

3. Results 3.1. The Italian trials In SSY the fertile stem number was on average 431 and 312 m−2 and the seed number was 8048 and 17,134 at the first and second regrowth, respectively (Table 1). Grasses were highly represented at the first regrowth but much less so at the second growth (respectively 68% and 21% of fertile stems; 82% and 3% of seeds). The fertile stem number collected in green and dry hay harvesting did not differ from SSY in any trial. In all trials except 2009/2, dry hay harvesting, direct combining and seed stripping collected much less seeds than SSY (approximately 35–42%, 30% and 60%, respectively) whereas green hay harvesting (61–81% of seeds harvested) was not significantly different from SSY. Except for harvesting as green hay 2009/1 and as dry hay 2011/1, the efficiency was, especially at the second regrowth, relatively higher for forbs (27–132% of SSY) than for grasses (6–89% of SSY). The percentage of grass seed number in the harvested material was on average

Fig. 1. Relationship between seed ripeness index and harvesting efficiency of individual species in dry hay harvesting (DH: a and b) and seed stripping (SSp: c) from the Italian harvesting trials. Efficiency was calculated as seed number per fertile stem harvested by the method compared to the standing seed yield. Values higher than 100 were set equal to 100.

Table 1 Fertile stems and seeds harvested in the Italian trials. Letters after numbers refer to the Tukey test performed on the means from the same regrowth period and year in the case of ANOVA results showing among-method differences significant at the nominal level p ≤ 0.017 (Bonferroni correction of p ≤ 0.05). Means followed by different letters are significantly different. Regrowth Year

Characteristic

Harvesting method 1) No. fertile stems m−2 2)

No. mature seeds m−2

Weight mature seeds (g m−2)

1000 seeds weight (g)

All species Grasses Forbs All species Grasses Forbs All species Grasses Forbs All species Grasses Forbs

1 2009

1 2009

1 2009

1 2009

1 2009

1 2011

1 2011

2 2009

2 2009

2 2009

2 2011

2 2011

SSY 395 246 149 8364 a 6870 a 1494 10.1 a 6.76 a 3.37 1.22 b 0.98 b 2.26

GH 399 255 144 6800 ab 6112 a 688 7.12 ab 5.49 ab 1.63 1.1 b 0.94 b 2.41

DH 430 265 165 3357 bc 2257 b 1100 3.66 b 1.97 c 1.68 1.07 b 0.89 b 1.42

DC . . . 2495 c 1613 b 882 4.46 b 2.76 c 1.7 1.8 a 1.73 a 1.96

SSp . . . 4995 abc 3028 b 1968 6.2 b 3.13 bc 3.07 1.25 b 1.05 b 1.54

SSY 466 337 129 7732 a 6376 a 1356 8.03 5.2 2.83 1.07 0.84 1.97

DH 511 381 130 3034 b 2669 b 366 3.46 3.08 0.38 1.09 1.10 1.08

SSY 383 84 299 21812 581 a 21231 18.28 a 1.94 a 16.34 a 0.84 3.33 0.77

GH 387 86 302 13411 160 b 13251 14.2 a 0.45 b 13.8 a 1.09 2.84 1.06

DH 308 64 243 9095 108 b 8987 6.9 b 0.28 b 6.61 b 0.76 2.60 0.74

SSY 240 45 195 12457 a 379 12077 a 10.2 a 0.94 9.27 a 0.82 2.32 0.77

DH 309 71 238 4333 b 22 4311 b 3.62 b 0.06 3.56 b 0.84 2.48 0.83

Legend. 1) Harvesting method: SSY standing seed yield; GH green hay; DH dry hay; DC direct combining; SSp seed stripping with pull-type equipment. 2) Fertile stem number not available for DC and SSp.

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Table 2 Number of seeds per fertile stem harvested in the four Italian harvesting trials. ANOVA was used to compare means from each experiment. Letters after numbers refer to the Tukey test performed on the means in the case of ANOVA results showing among-methods differences significant at p ≤ 0.05: means followed by different letters are significantly different.

Year Regrowth Harvesting method 1)

2009 1 SSY

2009 1 GH

2009 1 DH

2009 1 DC

2009 1 SSp

2011 1 SSY

2011 1 DH

2009 2 SSY

2009 2 GH

2009 2 DH

2011 2 SSY

2011 2 DH

Anthoxanthum odoratum Avenula pubescens Brachypodium rupestre Dactylis glomerata Poa pratensis Rhinanthus freynii Salvia pratensis Arrhenatherum elatius Trisetum flavescens Lotus corniculatus Onobrychis viciifolia Trifolium pratense Plantago lanceolata Trifolium repens Clinopodium vulgare Galium album Knautia arvensis

7.6 3.3 a 48 a 54 35 a 31 a 13 8.0 a 69 a 82 a 2.8 12.5 5.9 – – – –

4.4 5.0 a 29 ab 67 36 a 10 b 8 5.5 ab 62 a 51 ab 5.8 5.4 4.2 – – – –

3.6 0.4 b 32 ab 23 29 ab 10 b 7 0.4 c 3b 26 b 2.7 1.8 2.8 – – – –

0.4 1.0 ab 22 bc 23 6b 8b 10 3.2 b 6b 25 b 2.7 4.8 5.7 – – – –

3 1.4 ab 13 c 48 15 ab 32 a 26 3.1 b 29 a 2c 1.1 7.2 2 – – – –

2.3 a 2.2 a 48 65 a 70 a 31 a 13 0.7 a 4a 28 4.5 6.5 a 1.8 – – – –

0.2 b 0.2 b 27 12 b 26 b 3b 7 0.2 b 0b 7 0.3 0.9 b 0.4 – – – –

– – – – – – – 8.6 a 1.2 160 a 10.2 a 29 24 46 37 104 6

– – – – – – – 2.2 b 1.2 114 a 9.5 a 21 9 23 25 58 13

– – – – – – – 1.6 b 0.5 40 b 2.9 b 16 13 32 31 82 5

– – – – – – – 8.2 a 3.5 a 69 a 7.2 a 60 44 63 a 46 a 103 18

– – – – – – – 0.5 b 0.1 b 28 b 0.4 b 39 30 10 b 8b 29 7

Legend. 1) Harvesting method: see legend of Table 1.

76% at the first regrowth and 1% at the second regrowth. The weight of seed harvested varied between 3.5–7.1 g m−2 at the first regrowth and 3.6–14.2 g m−2 at the second regrowth. For the seed number, ANOVA results showed harvesting as green hay collecting similar amounts as SSY and the other methods harvesting clearly lower seed weights. Also in this case, forbs were harvested better than grasses. The ranges of the mean 1000 seed weights were 1.07–1.8 g and 0.82–1.09 g at the first and second regrowths, respectively. The forb values were higher than grass values at the first regrowth but were lower at the second regrowth. Among-methods significant differences were found only for the 2009 first regrowth, where the value of direct combining was particularly high (1.8 g) due to the high weight of grass seed. The grasses harvesting efficiency was found to be negatively correlated with the seed ripeness index in dry hay harvesting (Fig. 1a). In direct combining and seed stripping the relation was also negative but weaker and not significant. In forbs, a negative trend was found for dry hay harvesting (Fig. 1b). The seed ripeness index positively influenced harvesting efficiency in seed stripping (Fig. 1c). The other diaspore or plant traits taken into account were never found to be correlated with the species harvesting efficiency. With reference to the individual species (Table 2), green hay harvesting collected efficiently all species. In direct combining, Arrhenatherum elatius, Salvia pratensis, Plantago lanceolata and Brachypodium rupestre were rather efficiently harvested and Trisetum flavescens and Poa pratensis inefficiently. In dry hay harvesting, Poa pratensis, Salvia pratensis, Plantago lanceolata and Brachypodium rupestre were efficiently collected and Trisetum flavescens inefficiently. In seed stripping, Brachypodium rupestre and Plantago lanceolata were inefficiently harvested. The efficiencies of green and dry hay harvesting recorded at the second regrowth were almost always lower than at the first regrowth for both forbs and (especially) grasses (Table 1). In all trials the relationship between the seed mixture obtained with the harvesting methods and that of SSY yielded highly significant straight lines. In no case did slope and elevation differ significantly from 1 and 0, respectively. However, the percentage of variance explained differed considerably among methods (Table 3). Generally, the R squared values of green hay harvesting were the highest (0.82–0.9). In the 2009/1 trial, direct combining and seed stripping had intermediate R squared values and dry hay harvesting the lowest. However,

Table 3 R-squared values of the SMA lines calculated for the relationship between the percentage botanical composition of the seed mixture in the standing seed yield (X) and in the material harvested with different methods (Y) in the Italian and Czech trials. X and Y data were log10 transformed before analysis. Only R-squared values are shown as lines resulting from the standardised major axis estimation were always significant at p < 0.025 and slope and elevation were never significantly different from 1 and 0, respectively. In the second table part, the mean seed ripeness index is also shown. Trial

Italian

Regrowth Year

1 2009

1 2011

2 2009

2 2011

1 2009

1 2010

GH DH DC SSp

0.90 0.50 0.73 0.86

– 0.85 – –

0.82 0.85 – –

– 0.90 – –

0.49 – 0.19 –

0.72 – 0.28 –

Total Grasses Forbs

2.27 2.27 2.27

2.58 2.68 2.13

1.66 2.03 1.64

1.50 1.84 1.49

1.74 1.95 1.29

1.80 1.80 1.80

Harvesting method

Seed ripeness index

1)

Czech

Legend. 1) Harvesting method: see legend of Table 1.

in the 2009/2 and 2011 trials the values of dry hay harvesting were much higher than in 2009/1. The variable similarity of the dry hay seed mixture to SSY is probably connected to the method’s sensitivity to the seed ripeness. In the 2009/1 trial, the seed ripeness index showed intermediate values (on average 2.3, phenological stage between ripe and fallen seed/fruit) at which both grasses and forbs were prone to shedding (see Fig. 1) and similarity was low (Table 3). In the 2009/2 and 2011/2 trials (almost exclusively forb seed) the mean seed ripeness index was relatively low (1.7 and 1.5 for grasses and forbs, respectively, i.e. between developing and ripen seed/fruit), species were, therefore, less prone to shedding and the similarity to SSY was high. The 2011/1 trial was a special case. The seed ripeness index was relatively high (on average 2.6) so that many species had already shed their seed also in SSY: however, the similarity was high as the main remaining species were less prone to shedding (Poa pratensis) or not yet in advanced seed maturation (Brachypodium rupestre). In the 2009 trials, there were no significant differences among harvesting methods with respect to pattern of species presence on the donor site. Data are therefore shown as an average of the four methods (Fig. 2). At the first regrowth, the total species loss was approximately 19% of all species found on the donor site. This loss was due much more 199

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SSY (−22%). Direct combining was instead significantly less efficient (−65%), with greater seed loss by grasses (−82%) than by forbs (−16%). The seed loss by weight was 17% in green hay harvesting and 43% in direct combining, i.e., considerably less than for seed number. For seed number, direct combining losses compared to SSY were particularly high for grasses (−83%). On average, the number and weight of seed harvested were lower in 2010 (significant Year effect). Further, even if the interaction Year x Method was not significant, green hay harvesting and direct combining were considerably less efficient in 2010 than in 2009 (on average 36 versus 68%). The 1000 seeds weight was similar to SSY in green hay harvesting but significantly higher in direct combining. In this case, the difference was due in large part to forbs (+54% compared to SSY). Numbers of fertile stem and number and weight of harvested seeds were higher in 2009 than in 2010 with significant differences for the last two parameters. No significant relationships were found between the species seed weight or harvesting efficiency and the diaspore or plant traits tested here. Similarly as in the Italian trials, in direct combining Poa pratensis and Trisetum flavescens were badly harvested (Table 5). Plantago lanceolata and Vicia tetrasperma were, instead, well harvested. The species composition of the seed mixtures obtained with green hay harvesting and direct combining were significantly correlated with the SSY seed mixture and in no case did slope and elevation differ from 1 and 0, respectively. The R-squared values were relatively high in green hay harvesting (0.49–0.52) but were low in direct combining (0.19–0.28) (Table 3). There were no significant differences among harvest methods with regard to pattern of species presence on the donor site, so the data were averaged (Fig. 3). The total species loss on the donor site was approximately 46%. The loss was much less for grasses (−26%) than for forbs (−53%) and occurred almost exclusively due to no formation of fertile stems during the harvesting regrowth (−23% and −50% for grasses and forbs, respectively). If all harvesting methods are considered together, 24 out of the 64 meadow species (2 grasses and 22 forbs) were not harvested. The species loss was higher for forbs (44% of the species) than for grasses (14%).

Fig. 2. Pattern of species presence in the donor site and harvested material (mean ± standard error) of the Italian 2009 first and second regrowth harvesting trials. X axis legend: P-WS, presence as Plant recorded during the Whole growing Season; P-H, presence as Plant at Harvesting; FS-H, presence as Fertile Stem at Harvesting; SP-H, presence as mature Seed in the Phenological survey at Harvesting; SH-H, presence as mature Seed in the Harvested material. Letters refer to the Tukey test (nominal level p ≤ 0.017, Bonferroni-corrected p ≤ 0.05) performed on the five means of the same species group.

to forbs (−22%) than to grasses (−8%). The forb loss occurred mainly before harvesting (before SH-H in Fig. 2a). Harvesting methods were able to collect the seed of almost all species present as ripe seed (no significant difference between SP-H and SH-H in Fig. 2a). At the second regrowth, species loss was considerably higher at 31%. In this case, loss was due to both grasses (−53%) and forbs (−25%). In both species groups considerable loss occurred due to no plant growth during the regrowth period (between P-WS and P-H in Fig. 2b). In forbs, loss occurred also due to no fertile stem formation. When taking all harvesting methods into account, the total number of species harvested was 49 and 47 in the 2009/1 and 2009/2 trials, respectively. The species loss was higher for forbs (25 and 21% of the species, respectively, in the two trials) than for grasses (9 and 45% respectively). Only 6 of the 63 meadow species, all forbs, were not harvested in any regrowth. Five of them were not collected because they had no seed at harvest time. Among the 57 harvested species, 39 (5 grasses, 34 forbs) were collected in both regrowths, 10 (5 grasses, 5 forbs) only in the first one and 8 (1 grass, 7 forbs) only in the second one.

4. Discussion 4.1. Efficiency of the harvesting methods From the results obtained, the tested harvesting methods can be subdivided in two groups. The first group includes only green hay harvesting, where in most cases the harvested seed was not significantly different from SSY. The second group includes all other methods, which almost always harvested seed amounts different from SSY. In all trials, green hay harvesting was the most efficient method with regard to the number of seeds (on average, 71 and 72%, respectively, in the Italian and Czech trials) and weight of seeds (74 and 80%, respectively). The characteristics of the harvested seeds were not very different from SSY: the 1000-seeds weight was never significantly different and the botanical composition of the seed mixture was always very similar. In addition, the individual species harvesting efficiency was not affected by the seed maturation level or other seed traits. This method can therefore efficiently harvest seeds of many species and from grasslands in an advanced seed maturation stage. The high efficiency is due to the method implying only a minimum manipulation of plants bearing the seed and an immediate removal of the cut plant material, thus avoiding seed losses as in the other methods when trying to separate the seed from the plant (direct combining and seed striping) or due to spontaneous shattering during drying (dry hay harvesting). Seed harvesting could negatively affect the reproduction of the grassland species, thus modifying the botanical composition of the donor site

3.2. The Czech trials In SSY, the fertile stem number was 261 m−2 in the 2009–2010 average and did not differ among years (Table 4). As in the first-regrowth Italian trial, grasses were more abundant than forbs (60 vs. 40%). The number and weight of seed was 3935 and 3.19 g m−2, respectively. Green hay harvesting did not harvest significantly less seeds than 200

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Table 4 Fertile stems and seeds harvested in the Czech trials. Letters after numbers refer to the Tukey test performed on the means from the same regrowth and year in the case of ANOVA results showing among-methods differences significant at the nominal level p ≤ 0.017 (Bonferroni corrected p ≤ 0.05). Means followed by different letters are significantly different. Regrowth Year

Characteristic

Harvesting method 1) No. fertile stems m−2 2)

No. mature seeds m−2

Weight mature seeds (g m−2)

1000 seeds weight (g)

Total Grasses Forbs Total Grasses Forbs Total Grasses Forbs Total Grasses Forbs

1 2009

1 2009

1 2009

1 2009

1 2010

1 2010

1 2010

1 2010

1 Mean

1 Mean

1 Mean

1 Mean

SSY 320 191 129 5207 3545 1661 3.96 2.99 0.97 0.77 0.89 1.03

GH – – – 4737 2890 1847 4.55 2.61 1.94 0.96 0.90 1.47

DC – – – 2303 872 1431 3.76 0.69 3.07 1.58 0.81 2.05

Mean 320 191 129 4082 a 2436 1647 4.09 a 2.10 2.00 a 1.10 0.87 1.52

SSY 201 126 75 2664 2254 411 2.42 2.03 0.39 1.01 1.01 0.98

GH – – – 1404 1033 371 1.11 0.72 0.39 0.78 0.69 1.02

DC – – – 495 182 314 0.49 0.17 0.32 1.04 1.02 1.06

Mean 201 126 75 1521 b 1156 365 1.34 b 0.97 0.37 b 0.94 0.91 1.02

SSY 261 159 102 3935 a 2899 a 1036 3.19 2.51 a 0.68 0.89 b 0.95 1.00

GH – – – 3071 a 1961 ab 1109 2.83 1.66 ab 1.16 0.87 b 0.79 1.25

DC – – – 1399 b 527 b 872 2.13 0.43 b 1.70 1.31 a 0.92 1.55

Mean 261 159 102 2802 1796 1006 2.72 1.53 1.18 1.02 0.89 1.27

Legend. 1) Harvesting method: see legend of Table 1. 2) Fertile stem number not available for GH and DC.

serious problem in central Europe hay-meadows as they are composed almost exclusively of perennial species and because the hay produced is traditionally removed 1–3 times per year together with the seed not shattering during harvesting. Additionally, due to the uneven seed maturation, at least 40–60% of the seeds produced have shattered before mowing (Scotton et al., 2012). However, Jefferson (1999b) suggested to avoid harvesting every year or to harvest only a proportion of the site in any one year and Natural England (2010) recommended taking green hay from no more than one third (but preferably one fifth) of a site in any one year and to cut a different area each year. In the second method group, direct combining showed the lowest efficiency. The low efficiency was likely due mainly to the fact that the optimal setting of the combine harvester changes among species (Houseal, 2007; Krautzer et al., 2004) while only one setting can be used in each harvesting. Swath combining may have produced higher efficiency. In seed propagation swath combining is suggested for nonshattering species that mature seed unevenly (Stevens et al., 1996), as drying in the swath allows the seed to cure, reduces seed shattering and increases harvesting efficiency (Copeland and Loeffler, 2006). In a mixed grassland, however, the different phenologies of the many species would make it impossible to find a harvesting time that is simultaneously optimal for all of them. Additionally, the 5–10 days needed for grass drying in the swath (Najda et al., 2002) would increase the rain risks and produce high shattering from the more ripe species. The seed loss was more evident for the number of seeds (approximately 70% in the average of all trials) than for weight of seed (approximately 47%). This result was due to the different harvesting efficiency for species having low or high 1000-seeds weight and was responsible for the higher mean 1000-seeds weight found in direct combining. In the Italian trial the relevant species included some grasses: the light seed Poa pratensis and Trisetum flavescens were harvested with low efficiency, while the heavier seed Arrhenatherum elatius, Brachypodium rupestre and Dactylis glomerata, were harvested with high efficiency. In the Czech 2009 trial, the same light seed grasses of the Italian trial were underharvested, and a large amount of the heavy seed Vicia tetrasperma was collected. In general, most combines are designed to harvest heavyseeded agricultural crops. This often makes it difficult to harvest prairie seed that is light and fluffy (Lochner, 1997). A low harvesting efficiency of light seed species, such as Trisetum flavescens and Achillea millefolium, was highlighted by Everett (2003). In the case of combining on a mixed grassland, the different efficiency is likely due to the wide concave setting of the combine that is necessary to avoid damaging larger seeds but is unfavourable for an efficient separation of smaller seeds. However, no relation was found between the harvesting efficiency and 1000-seeds weight. In addition to weight, other seed characteristics are probably important, including morphology, maturity and proneness to

Table 5 Number of seeds per fertile stem harvested in the 2009 and 2010 Czech harvesting trials. ANOVA was used to compare means from the harvesting methods. As ANOVA showed significant difference among years only for Arrhenatherum elatius, the results are shown as 2009–2010 mean. Symbols after numbers refer to the Tukey test performed on the means in the case of ANOVA results showing among-methods differences significant at the p ≤ 0.05): means followed by different letters are significantly different. Harvesting method 1)

SSY

GH

DC

Anthoxanthum odoratum Arrhenatherum elatius Dactylis glomerata Festuca rubra Holcus lanatus Poa pratensis Trisetum flavescens Trifolium campestre Vicia tetrasperma Knautia arvensis Plantago lanceolata Ranunculus acris

5.5 a 7.7 a 46 a 9.2 42 a 15.3 a 14.4 a 24 9 13.5 a 5.2 15.1

4.0 ab 6.4 ab 26 ab 7.2 29 a 3.9 a 7.9 b 12 10 7.6 ab 5.6 9.3

1.6 b 1.2 b 8b 2.9 7b 0.3 b 0c 4 11 1.8 b 4.6 6.1

Legend. 1) Harvesting method: see legend of Table 1.

Fig. 3. Pattern of species presence in the donor site and harvested material (mean ± standard error) of the 2009 Czech harvesting trial. Legend of X axis: see legend of Fig. 2. Letters refer to the Tukey test (nominal level p ≤ 0.017, Bonferroni-corrected p ≤ 0.05) performed on the four means of the same species group.

(Wells et al., 1986). In Minnesota tallgrass prairie, frequent wild-harvesting decreased the frequency of short-lived and non-clonal species and increased the abundance of long-lived and clonal species (Meissen et al., 2015). This problem could be more severe in the case of the highly efficient green hay harvesting. However, this is likely not be a 201

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before harvesting and only one (Scabiosa columbaria) was not harvested but had seeds in the SSY at harvesting. These results confirm those found by Edwards et al. (2007), who also highlighted the relevance of harvesting time with regard to species transfer. Further, they also stress that the main problem of seed harvesting on semi-natural grassland is forb collection, both when harvesting only at the end of the first regrowth (the more frequent situation) and at the end of both regrowths. With regard to number of seeds harvested, the results obtained showed that the main factors affecting harvesting efficiency are the species group, the individual species characteristics, the seed maturity and the harvesting regrowth. Species group was the most important factor. The harvesting efficiency was almost always higher for forbs than for grasses. This was true also in green hay harvesting (only cut plus removing) and in dry hay harvesting (cut and turning, but no further mechanical action for separating the seeds from the plant). This fact suggests that forbs have on average stronger mechanisms to adhere to the stem than grasses: e.g., closed fruit (legume, capsules) in Fabaceae and Plantago spp., involucre in Asteraceae heads, calyx in Lamiaceae. However, as forb harvesting efficiency was higher than grasses in direct combining and seed stripping, this likely means that the mechanical actions exerted by these methods are effective in removing the seed. In the Italian trials, the forbs most efficiently harvested were Salvia pratensis at the first regrowth, and Trifolium pratense, Vicia cracca, Galium spp. and Plantago lanceolata at the second regrowth. In the Czech trials, Trifolium pratense, Vica tetrasperma, Plantago lanceolata and Galium mollugo were particularly well harvested. Seed ripeness was another important factor. However, its effect changed in interaction with the harvesting method and the species group. The negative effect found in dry hay harvesting for grasses was likely due to the seed ripeness favouring the seed shattering during drying, turning and harvesting. Additionally, in harvesting seed from monospecific seed propagation, the crop is cut and swathed on the soil to favour younger seed ripening during drying days (Burton and Burton, 2003) and the following more efficient threshing (Copeland and Loeffler, 2006). As the effect of seed ripening tended to also be negative in direct combining and seed stripping, it is possible that in these two methods some seed is lost at the time of contact of the fertile stems with the cutter bar and that this loss type increases with seed ripening. According to Davison (2004), to reduce this type of loss, the reel pushing the plants being harvested into the cutter bar is often removed when threshing native seed. Also in the case of forbs, a negative effect of seed ripening was found, but only when the ripening level was higher (first regrowth 2009), therefore weakening the mechanisms of the seed adhesion to the plant. The lack of a negative effect of seed ripeness on forb harvesting efficiency of direct combining and seed stripping would confirm that the seeds of these species have stronger seed-plant adhesion mechanisms. Rather, seed ripening positively affected seed stripping efficiency, as it likely favoured the separation of the seed from the plant by the brush. The negative effect of seed ripeness on the efficiency of dry hay harvesting does not exclude the possibility to use this method for seed collecting. According to Coolbear et al. (1997), seed shedding is particularly important after seed ripening (stage 3 of seed development), whereas germinability is attained already during maturing (stage 2 of seed development). If seed ripeness is excluded, no diaspore or plant trait taken into account was found to be correlated with species harvesting efficiency. This corresponds to what was found by Scotton et al. (2009). It is possible that these factors can influence harvesting efficiency but that the effect changes for each species in relation to the harvesting method and seed ripeness. However, in direct combining the harvesting efficiency of Salvia pratensis, Plantago lanceolata and Brachypodium rupestre was probably due to seed of these species being appendage-free and compact, while the inefficient collection of Trisetum flavescens and Poa pratensis was probably connected to the their seed low weight and appendage presence (awn in Trisetum and cotton filaments at the seed

shattering; for example, in Poa pratensis, the separation of the seed from the spikelet is difficult due to the presence of cottony filaments at the base of seeds (Najda et al., 2002). The strong among-species selectivity of direct combining explained why, in both the Italian and Czech trials, the species composition of the seed mixture obtained was the most dissimilar from SSY. The efficiency of dry hay harvesting was approximately 40% at both the first and second regrowth. At the first regrowth the individual species harvesting efficiency was negatively affected by seed maturity in both grasses and forbs. The reason for this is the well-known higher susceptibility to shattering of more ripe seeds (Copeland and Loeffler, 2006). In the case of grasses, this relationship seems to contradict the higher overall efficiency found in the 2011/1 Italian trial (efficiency 42% with mean grasses seed ripeness index = 2.68: Table 3) compared to the 2009/1 trial (33%, seed ripeness index = 2.27). This apparent contradiction was because in 2011 several abundant species had almost completely shattered prior to harvest and Poa pratensis, which does not shatter easily (Najda et al., 2002), and the late, not yet over-ripe, Brachypodium rupestre (seed ripeness index = 2) were abundant. At the second regrowth, no relationship was found between harvesting efficiency and seed ripeness index, either for grasses (only two species with mature seed) or for forbs. For forbs, this difference from the first regrowth was probably due to the harvest taking place in both years with a seed ripeness index much lower than 2, the threshold above which the influence of seed ripeness index becomes evident (Fig. 1b). From comparing the harvesting efficiencies of the individual species found here with those found in Scotton et al. (2009), the results were sometimes similar (Dactylis glomerata, well harvested, and Rhinanthus freynii badly harvested) and sometimes different (Trisetum flavescens, badly harvested here and well harvested in Scotton et al., 2009). In Pianari, the same species could be harvested with different efficiencies in the different trials (e.g. Arrhenatherum elatius and Anthoxanthum odoratum). This only partial agreement of the results is likely due to the high sensitiveness of dry hay harvesting to the seed ripeness. As shown in Section 3.1, this sensitivity to the seed ripeness is probably the main reason for the variable similarity of the dry hay seed mixture to SSY. Seed stripping was the most efficient among the second group methods (approximately 60%). While it is usually regarded as less efficient than combining for harvesting seed from fields of single species seed propagation (Houseal, 2007; Steinauer, 2003), seed stripping is considered to be more efficient for mixed grassland harvesting (Everett, 2003). The high efficiency recorded here was mainly due to the equipment setting used for harvesting. The downward brush rotation on short grasslands allows for harvesting more seed as the stripping action involves the whole vegetation layer and the seed bearing stems are pushed to the horizontal board located under the equipment, which intercepts the seed and conveys it to the hopper (Scotton et al., 2009). When seed stripping was performed on tall meadows where it affected only the upper vegetation layer, the harvesting efficiency was found to be lower (Edwards et al., 2007; Scotton et al., 2009). As a consequence of the relatively high efficiency, the similarity of the harvested seed mixture was high and was only lower than in green hay harvesting. 4.2. Factors affecting harvesting efficiency The species loss observed in the 2009 Italian and Czech trials was higher for forbs than for grasses. This result confirms the findings of Scotton et al. (2009), who, in two first regrowth experiments without integration from restoration trials, found losses amounting to 46 and 65% for forbs and 12 and 18% for grasses. In no trial was the species loss influenced by the harvesting method. The main factor was, instead, the phenology (no seed present at harvesting). In the Italian trials this type of loss was mainly due to the regrowth: the species not flowering or not maturing seed were eight in the first regrowth and ten in the second regrowth. Of the other six lost species (forbs maturing seed only at the first regrowth), five had already set and shattered their seed 202

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low seed loss (e.g., green hay harvesting). To reduce the initial strong grass establishment on re-created grasslands, the seed mixture should contain more propagation material from the second regrowth. The forbs, whose seeds are not mature at the end of the two regrowths, could be collected with seed stripping or manually during the first regrowth before the main harvest.

base in Poa). In dry hay, Poa pratensis, Salvia pratensis and Plantago lanceolata were probably well harvested due to seeds having efficient mechanisms of seed-plant adhesion, such as filaments in Poa, the calyx in Salvia and the closed capsule in Plantago. In seed stripping, the low harvesting efficiency found for Brachypodium rupestre and Plantago lanceolata was probably due to the small contacts between the brush bristles and the narrow spike inflorescence typical of these species. In the Italian trials, the efficiency found at the second regrowth in green and dry hay harvesting was lower than at the first regrowth for both grasses and forbs. This result was more evident for grasses and was not due to more ripe seeds, as the seed ripeness index was lower at the second regrowth. Instead, this was likely due to the higher temperature of the seed development period before harvesting (23.2 and 21.1 °C in the 2009–2011 mean of the three weeks before harvesting, at the second and first regrowth, respectively: data from the ARPAV meteorological station of Lusiana). Higher temperatures likely predisposed plants to an easier seed shattering and loss during harvesting. In several cultivated plants, a higher temperature during fruit development increases fruit shedding. For instance, higher temperature significantly decreased boll retention in cotton (Zhao et al., 2005), and soybean pod shattering accelerated when the plants matured in a period with relatively high temperatures and dry conditions (Zhang and Bellaloui, 2012). Additionally, Simon et al. (1997) found that warmer, dry conditions can increase seed shattering in Lolium perenne. The lower efficiency of green hay harvesting and direct combining recorded in the 2010 Czech trial compared to 2009 was probably also due to the same temperature effect, as in 2010 seed development occurred in a much warmer and drier period (18.5° C and 44.7 mm) than in 2009 (16.4° C and 81.9 mm) (data from the CHMI meteorological station of OlomoucHolice). The decrease in harvesting efficiency from the first to the second regrowth (Italian trial) and from 2009 to 2010 (Czech trial) was lower for forbs than for grasses. This again suggests that, on average, ripe fruits of forbs adhere more strongly to the mother plant.

Acknowledgements This research was funded under project Salvere (EU Interreg programme Central Europe 1CE052P3). The authors are grateful to two anonymous reviewers for their valuable suggestions, which significantly improved the paper. They also thank Antonio Timoni, Claudia Dal Buono, Petra Chalupová, Tomáš E. Vondřejc and Eva Chovančíková for data collection and laboratory analyses, SAGITTARIA (Association for Nature Conservation of Central Moravia) for phenology and phytocenological surveys and harvesting plots, the Forestry Service of the Autonomous Province of Trento for the seed stripper, Matteo Fardo for access to the Italian donor site and ARPAV (Veneto Regional Agency for Environmental Protection) and CHMI (Czech Hydrometeorological Institute) for meteorological data. References Äschimann, D., Lauber, K., Moser, D.M., Theurillat, J.P., 2004. Flora Alpina. Zanichelli, Bologna. Baasch, A., Engst, K., Schmiede, R., Maya, K., Tischew, S., 2016. Enhancing success in grassland restoration by adding regionally propagated target species. Ecol. Eng. 94, 583––591. Bakker, J.P., Poschlod, P., Strykstra, R.J., Bekker, R.M., Thompson, K., 1996. Seed banks and seed dispersal: important topics in restoration ecology. Acta Botanica Neerlandica 45, 461––490. Burton, C.M., Burton, P.J., 2003. A Manual for Growing and Using Seed From Herbaceous Plants Native to the Interior of Northern British Columbia. Symbios Research & Restoration, Smithers, B.C 168 pp. Cochran, W.G., Cox, G.M., 1950. Experimental Designs. New York, John Wiley and Sons, Inc. Chapman and Hall, Limited, London. Coolbear, M.J., Hill, M.J., Win Pe, 1997. Maturation of grass and legume seed. In: Fairey, D.T., Hampton, J.G. (Eds.), Forage Seed Production Volume 1: Temperate Species. CAB International, Oxon, New York, pp. 71––104. Copeland, L.O., Loeffler, T., 2006. Production for sowing. In: Black, M., Bewley, D.J., Halmer, P. (Eds.), The Encyclopedia of Seeds: Science, Technology and Uses. CAB International, Wallingford, Cambridge, pp. 546–551. Core Team R, 2013. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project. org/. Davison, J., 2004. A Field Guide for Collecting Native Seeds in Nevada. University of Nevada Cooperative Extension (135 pp.). Dietl, W., 1995. Wandel der Wiesenvegetation im schweizer Mittelland (Changing of meadows vegetation in the Swiss Plateau). Zeitschrift für Ökologie und Naturschutz 4, 239–249. Donath, T.W., Bissels, S., Hölzel, N., Otte, A., 2007. Large-scale application of diaspore transfer with plant material in restoration practice −impact of seed and microsite limitation. Biol. Conserv. 138, 224–234. European Environment Agency, 2004. EEA Report No 1/2004. High Nature Value Farmland. Characteristics, Trends and Policy Challenges. . Available from: http:// www.eea.europa.eu/publications/report_2004_1 (Accessed 01.02.17). EEC, 1992. Council directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora. Off. J. Eur. Commission No L 206/7. Edwards, A.R., Mortimer, S.R., Lawson, C.S., Westbury, D.B., Harris, S.J., Woodcock, B.A., Brown, V.K., 2007. Hay strewing, brush harvesting of seed and soil disturbance as tools for the enhancement of botanical diversity in grasslands. Biol. Conserv. 134, 372–382. Everett, S., 2003. Seed Collection Methods from Grassland. (Available from:). https:// www.floralocale.org/tiki-print.php?page=page23960. Fox, J., Weisberg, S., 2011. An R Companion to Applied Regression, second edition. Sage, Thousand Oaks CA. Graiss, W., Haslgrübler, P., Blaschka, A., Pötsch, E.M., Krautzer, B., 2013. Establishment of an Arrhenatherion meadow through on-site threshing material and green hay transfer. In: In: Helgadóttir, Á., Hopkins, A. (Eds.), The Role of Grasslands in a Green Future. Threats and Perspectives in Less Favoured Areas. Grassland Science in Europe, vol. 18. pp. 341–343 (Akureyri, Iceland). Herbent, T., Krahulec, F., Hadincová, V., Pecháćková, S., Wildová, R., 2003. Year-to-year variation in plant competition in a mountain grassland. J. Ecol. 91, 103––113. Hess, M., Barralis, G., Bleiholder, H., Buhr, L., Eggers Th Hack, H., Stauss, R., 1997. Use of the extended BBCH-scale −general for the description of the growth stages of monoand dicotyledonous weed species. Weed Res. 37, 433––441. Hintze, C., Heydel, F., Hoppe, C., Cunze, S., König, A., Tackenberg, O., 2013. D3: the dispersal and diaspore database − baseline data and statistics on seed dispersal.

5. Conclusions All of the tested methods collected the species present as seed at harvest time and did not differ with regard to the number of species collected. The species composition of the seed mixture obtained was always correlated with SSY. However, with regard to the number of seeds, the efficiency of the methods changed in relation to the species group, individual species, seed ripeness and regrowth. The most efficient method was green hay harvesting, where the immediate collection of the cut hay allowed for harvesting nearly all seeds in the SSY. The least efficient methods were direct combining and dry hay harvesting. In direct combining, a strong negative selectivity against light or difficult to detach seeds was observed. Dry hay harvesting was found to be very sensitive to the seed ripeness level. For seed stripping, a relatively high efficiency was found, which was related to the downward brush rotation at the leading edge, stripping the whole vegetation layer. Upward rotation would not have the same efficiency. In all methods, the harvesting efficiency for grass seed number was lower than for forbs. However, this is not necessarily a problem, especially at the first regrowth where the high grasses to forbs ratio usually produces herbaceous covers that are strongly unbalanced in favour of grasses (Scotton, 2016). On the other hand, forbs represented a problem with regard to the number of species collected. The problem was not due to the harvesting method but rather due to phenological factors, including no seed production in the harvesting regrowth or seeds already shed at harvest time. A hypothetical optimal (though time-consuming) harvesting programme could be to harvest at both the first and second regrowth. At the first regrowth, the method used should be one that is more efficient in harvesting forbs than grasses (seed stripping, direct combining or dry hay harvesting). At the second regrowth, the method used should have 203

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