Desirable site conditions for introduction sites for a locally rare and threatened fern species Asplenium septentrionale (L.) Hoffm

Journal for Nature Conservation 22 (2014) 272–278

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Desirable site conditions for introduction sites for a locally rare and threatened fern species Asplenium septentrionale (L.) Hoffm Kai Rünk ∗ , Karin Pihkva, Kristjan Zobel University of Tartu, Institute of Ecology and Earth Sciences, 40 Lai Street, 51005 Tartu, Estonia

a r t i c l e

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Article history: Received 12 April 2013 Received in revised form 21 November 2013 Accepted 11 February 2014 Keywords: Asplenium septentrionale Light availability Reintroduction Soil biota Soil pH

a b s t r a c t Forked spleenwort, Asplenium septentrionale (L.) Hoffm., is mainly a petrophilous fern species of European mountains and very rare in Estonia, a country with flat topography and few rocky habitats. The single extant population is very small, occupies a restricted area, and is threatened seasonally by adverse human activity. An introduction project of the species was prepared according to the A. septentrionale Management Plan. The goal of the project is to introduce new populations of the species in new protected sites from ex situ propagated young sporophytes. The aim of this study was to obtain detailed data on the species ecology and provide information for selecting suitable introduction sites. We carried out a factorial pot experiment on light availability, soil pH and soil biota gradient in order to ascertain optimal growth and development conditions for A. septentrionale. Soil and light conditions must be considered the most important factors affecting species growth and reproduction, particularly under conditions of largely fluctuating water regime. Experimental plants grown in abiotic conditions closely emulating the natural habitat were the most successful and may, therefore, represent the optimal establishment potential. The survival of plants was greater, and plants grew larger with a higher number of longer leaves in acidic soil (pH 4.9) than in neutral soil. Plants performed best under high, approximately 75% total available illumination. Deep shade (90%) and well as full daylight had strong negative effect on the plants. No positive effect of “home-soil” biota was detected. © 2014 Elsevier GmbH. All rights reserved.

Introduction In situ conservation of species is considered the most successful and practical strategy to maintain biodiversity (Hamilton & Hamilton 2006; Kull 1999; Primack & Drayton 2011; United Nations Organization 1992). However, limiting protection of rare species in natural sites may be insufficient when small populations of rare species inhibit areas of detrimental human activity. In such cases, the introduction of new populations in protected public land is an additional and effective conservation method (Godefroid et al. 2011; Guerrant & Kaye 2007; Primack 2010). The main goal of (re)introduction projects is to establish resilient, self-sustaining populations to decrease the likelihood of species extinction (Guerrant & Kaye 2007; Guerrant 1996; Pavlik 1996). The planning of species (re)introduction demands consideration of many factors, such as species biology, ecology, socio-economic and legal requirements (IUCN 1998). Factors associated with the selection of suitable new habitats are as important

∗ Corresponding author. Tel.: +372 7376382; fax: +372 7376380. E-mail address: [email protected] (K. Rünk). http://dx.doi.org/10.1016/j.jnc.2014.02.001 1617-1381/© 2014 Elsevier GmbH. All rights reserved.

as the selection of suitable plant material. Ecological habitat suitability is the foremost criterion when evaluating the suitability of a site for introduction, because the habitat needs to meet all the life history requirements of the species – for establishment, growth, reproduction, dispersal, germination (Falk et al. 1996). High ecological similarity between an introduction site and natural habitats is essential since local genetic adaptation of populations may result in highly specialised genotypes (Hufford & Mazer 2003; Lawrence & Kaye 2011; Pavlik et al. 1993), even in comparatively small geographic scales in the case of isolated populations of fragmented habitats (Raabová et al. 2007). The abundance or rarity of plant species can be determined by a multitude of different factors, operating at different spatial and temporary scales. It is evident that in situ conservation of endangered populations demands a good understanding of the organisation of genetic diversity as a key to the long-term survival of species (Berry 1971; Schemske et al. 1994). On the other hand, successful introduction of new populations in previously unoccupied habitats can only be based on solid autecological research on the basic habitat requirements of species (Brussard 1991; Simberloff 1988). Several case studies have shown the usefulness of ecological research for planning effective protection measures

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of rare and/or endangered species (Buchele et al. 1991; Burmeier & Jensen 2009; Fowler et al. 2012; Gawler et al. 1987; Jusaitis et al. 2004; Vivian 1967), including ferns (Chau & Reyes 2013; Rünk & Zobel 2007; Rünk et al. 2010). Light and edaphic conditions of the habitat are crucial environmental criteria to be considered for plants (e.g. Björkman 1981; Fiedler & Laven 1996; Lambers et al. 2008; Rünk et al. 2010), particularly in sites never previously occupied by the species. Undoubtedly, one of the most efficient ways to obtain reliable information about habitat requirements is to design autecological experiments based on a priori knowledge of habitat conditions in extant populations (Falk et al. 1996; Guerrant & Kaye 2007; Lawrence & Kaye 2011; Rünk & Zobel 2007), using the same plant material that will be used in the introduction project. In locally rare species ecological information comes from a small number of isolated localities and may be insufficient for elaborating proper introduction criteria. Also, it can be very difficult, if not impossible, to judge the interactive effects of different environmental factors on species performance on the basis of in situ measurements only (Rünk et al. 2010). In such cases factorial experimental tests of basic environmental requirements can easily provide necessary practical information, including this on complex interactive effects, and also make a contribution to species autecology and (re)introduction theory (Guerrant & Kaye 2007). The populations closest to the only Estonian station of Asplenium septentrionale (L.) Hoffm. can be found across the Baltic Sea, in southern Finland and eastern Scandinavia (Jalas & Suominen 1972). There the species occurs in rather specific habitat conditions, characterised by full exposure to sunlight and acidic soil (Øvstedal & Tigerschiöld 2000). A. septentrionale is very rare in Estonia with three historically known localities, only one of which is extant. This small population, consisting of 190 individuals in 2009 is located on an island1 in the Gulf of Finland, at the south-western edge of the Fennoscandian distribution subarea (Jalas & Suominen 1972). This population is restricted to an area of about 140 m2 , in a habitat seasonally subjected to detrimental human impact, mainly physical disturbance. Plants are rooted in soil among granite stones (diameter 20–80 cm) in a man-made wall surrounding raised ground. Most of the plants are located on the southern side of the wall. This Estonian Red List of Threatened Species 2008 classifies A. septentrionale as critically endangered, CR (eBiodiversity 2008); the Government of the Republic of Estonia (2004) places the species in the first (strictest) category of protected species. This study represents the initial stage of an ongoing A. septentrionale introduction project in Estonia (Rünk 2009). The main aim was to obtain detailed data on the basic ecological requirements of the species and to provide information for selecting suitable introduction sites for the local Estonian population. We use the term “introduction” to refer to planting of A. septentrionale ex situ propagated sporophytes within its normal range but into previously unoccupied areas (sites) (Allen 1994). We carried out a factorially designed common garden pot experiment with manipulated light availability, soil pH and soil biota in order to assess the optimal growth and development conditions for the siblings of plants in the single Estonian population of A. septentrionale. We estimated the ecological performance of the species through plant growth, assuming that larger plants are expected to be more vigorous (Harper 1977), experience lower mortality (Bloom et al. 2001), and hence have greater establishment potential (Guerrant 1996). Given the environmental conditions prevailing in

1 According to the Nature Conservation Act (Estonian: Looduskaitseseadus) §53 (1) the public disclosure of the exact localities of species belonging to the I category of protected species in Estonia is prohibited (Riigi Teataja (State Gazette) I 2004, 38, 258. https://www.riigiteataja.ee/akt/12808270?leiaKehtiv).

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the only natural site in Estonia, we hypothesised that A. septentrionale plants would perform better and attain greater biomass (i) when light availability exceeds 40% of full sunlight, (ii) in acidic soil, (iii) in the presence of “home” soil biota from the natural habitat. Material and methods Study species Forked spleenwort A. septentrionale (Aspleniaceae) is a low to short-growing rhizomatous herbaceous perennial tetraploid (2n = 144, Crabbe et al. 1993) fern with linear simple or dichotomously 1–3 times forked lamina (Crabbe et al. 1993; Wagner et al. 1993). The short rhizome branches densely and forms compact, grass-like clumps of wintergreen leaves. The species is distributed in Europe, Asia and North America (Hultén & Fries 1986). In Europe, A. septentrionale is more common in Scandinavia, Western Europe and in the mountains of Southern Europe (Jalas & Suominen 1972). The species is less common in Eastern Europe. In Asia, A. septentrionale is distributed mostly in central areas of the continent, in Western Siberia and in the mountains of Central Asia. In Caucasus and Asia Minor, a diploid (2n = 72) cytotype (referred as A. septentrionale subsp. caucasicum Fraser-Jenkins&Lovis) with narrower lamina segments and smaller spores has been found (Davis et al. 1988). Gametophytes and sporophytes of A. septentrionale are considered non-mycorrhizal (Harley & Harley 1987; Wang & Qiu 2006). Plant species nomenclature follows Crabbe et al. (1993) and Frey (2009). Preliminary study: soil and light conditions of the habitat Soil samples were collected on June 21, 2006 and hemispherical digital images were taken on 1 August 2006 at the location of the single natural population. The soil samples were collected from the rhizosphere of A. septentrionale plants, avoiding their disturbance. The samples were pooled to three replicates to attain the necessary weight for the analysis. The samples were analysed for soil pH in the Laboratory of Plant Biochemistry of the Estonian University of Life Sciences. Light conditions of the habitat were assessed from hemispherical digital images using WinSCANOPY software (Regent Instruments Inc., Canada), assuming Standard Overcast Sky model. Seventeen hemispherical images were taken throughout the habitat as close to the nearest plant individual as possible with a Nikon Coolpix 4500 digital camera equipped with a Nikon Fisheye Converter FC-E8. The camera was mounted on a tripod, kept horizontal and orientated according to azimuth. The penetration of indirect and direct radiation through tree canopy – indirect site factor (ISF) and direct site factor (DSF) defined as the proportion of indirect and direct radiation, respectively, reaching a plant individual under the tree canopy during the growth period relative to that in the open sky (Anderson 1964) – were calculated for each plant. Experimental design Experiment 1 Effects of light availability, soil pH and presence of “home” soil biota on A. septentrionale growth (biomass) and morphology were assessed in a factorial pot experiment in a common garden during 2004–2007. A. septentrionale spores were collected from seven individuals from the single Estonian population in 2004, and mixed before germination. The substrate used for spore germination and young sporophytes transplantations consisted of three parts horticultural peat and one part fine-grade sand and was sterilised. Spores were sown in Petri dishes on September 2, 2004. The Petri

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dishes (later plastic boxes) were kept in a greenhouse at 22 ± 2 ◦ C with photoperiod of 12:12 h (fluorescent light: daylight tubes, photon flux density 40 ␮mol m−2 s−1 ) and watered as required to keep the soil moist. Sporophytes emerged in March 2005. Sporophytes were later transplanted into larger plastic boxes (15–50 individuals evenly spaced per box). The plants were placed outdoors in the experimental garden from September 26, 2005 to April, 30 2006. On May 1, 2006 the plants were returned to the greenhouse and planted individually in plastic pots (10 cm diameter, 8 cm deep) on July 20–21. Ninetysix plants were planted in an acidic soil (pHKCl = 4.9) consisting of three parts horticultural peat and one part medium-grade sand. The remaining 192 plants were planted in a neutral soil (pHKCl = 6.9), i.e. in the same substrate with 137 g limestone flour per 10 l of soil added (“Nordkalk” pH+ Natural, Nordkalk AS Rakke lubjatehas, Estonia). The larger number of plants in neutral soil arose from our original plan of planting one third of the plants in alkaline soil. Earlier tests of the effect of limestone flour on soil acidity revealed that pH 6.9 was attained at 73 g of limestone flour per 10 l; additional limestone flour did not increase the pH of the soil solution. Therefore, we increased the amount of limestone flour in the pots (137 g per 10 l) and treated these as one treatment variant. Half of the soil was inoculated with local, so-called “home” soil biota, for both soil treatments. “Home” soil was collected between plants from the Estonian population and mixed carefully with planting soil (23 ml per 10 l soil). On August 1 the plants were relocated to a shading tent (50% shade, spectrum neutral) in the experimental garden, located in Tartu (N 58.36230 ◦ N, E 026.67964 ◦ E; 75 m.s.l.), in south-eastern Estonia, where the average annual temperature is 5.0 ◦ C and the average annual rainfall is 550 mm (Jaagus 1999). On August 10 the pots were distributed randomly among four shading treatments: 100%, 50%, 25% and 10% of full daylight, with 12 (soil pHKCl = 4.9) or 24 (soil pHKCl = 6.9) replicate plants per treatment, and grown for 87 days in 2006 and 80 days in 2007. Shade was increased using aluminium-coated shade cloths (spectrum neutral; Ludvig Svensson, Kinna, Sveden). Plants were watered as required to keep the soil moist and randomised weekly to prevent an edge effect. From November 2006 to April 2007 the plants wintered in an unheated greenhouse, covered with white peat to simulate fallen leaves and their decayed remnants.

watered as required to keep the soil moist and randomised weekly to minimise edge effects. In both experiments the number of leaves, and in Experiment 2, the number of leaves with sori were counted once, immediately before harvest on July 15, 2007 and September 30, 2008. After harvest, plants were separated into leaves, rhizomes and roots and dried at 75 ◦ C for 48 h. All biomass fractions were weighed separately. Leaf length (in 2008 only the length of the longest leaf) was measured to the nearest millimetre. Statistical analysis Differences in mortality among plants grown in different soil treatments were analysed using 2 test. The effects of light availability (three levels – 10%, 25%, 50% of full daylight), soil pH (two levels – acidic and neutral) and “home” soil biota (two levels – with and without) on biomass and morphological traits of A. septentrionale in 2006–2007 (Experiment 1) was analysed using three-way ANOVA. In the second experiment (2008) the effect of light availability (three levels – 50%, 75%, and 95% of full daylight) on biomass and morphological traits was analysed using one-way ANOVA (using Statistica data analysis software system version 7.0; StatSoft Inc., 2004). All dependent variables were log-transformed before performing ANOVA. Differences in relative biomass allocation in 2006–2007 were tested using one-way ANOVA after arcsine square root transformation of the data. Tukey HSD multiple-comparison test with 0.05 significance level was used to detect significant differences between individual experimental treatment combinations (Sokal & Rohlf 1995). Results Preliminary study: soil and light conditions in the habitat Soil pHKCl of A. septentrionale natural habitat ranged from 4.06 to 4.40 (n = 3, mean = 4.18, SE 0.11). The mean proportion of total radiation (indirect + direct radiation reaching the plants under the tree canopy during the growing period relative to the open sky, Anderson 1964) ranged from 24.0% to 86.0% (n = 17, mean = 64.8%, SE 0.04). Experiment 1

Experiment 2 Due to the loss of all plants in the 100% daylight treatment in Experiment 1 (destruction by birds, see Results), and in order to evaluate more exactly the effect of light availability on A. septentrionale biomass and morphology at high radiation intensity, an additional factorial experiment was carried out in 2008. For this experiment was performed on two-year old plants. The spores were collected from the single Estonian population on August 12, 2006, sown on August 25, 2006 and plants grown as in Experiment 1. On May 17, 2008, 60 young sporophytes were planted individually in plastic pots (10 cm diameter, 8 cm deep) containing acidic soil (pHKCl = 5.12), consisting of three parts horticultural peat and one part medium-grade sand. On May 20, plants were relocated to an unheated plastic greenhouse in the experimental garden and covered with neutral shade cloth (50% shade). After ten days the pots were distributed randomly among three shading treatments: 50%, 75% and 95% of full daylight. The 50% and 75% shade treatments were conducted using tents made of aluminiumcoated shade cloths (spectrum neutral; Ludvig Svensson, Kinna, Sveden) and 95% treatment with an aluminium wire mesh (mesh size 3 cm × 3 cm). The 95% treatment was the closest to full light treatment we could get and still protect against birds. Plants were

In July 2007 plants growing in full sunlight were attacked and completely damaged by crows. As a result, data from the fulllight treatment was unavailable for analysis. Subsequently, only the plants from the three shaded treatments of the first experiment are considered. Ninety-nine percent of the plants survived the experiment in acidic soil. Mortality of plants growing in neutral soil in 10%, 25% and 50% light treatments was considerable: 81% (n = 39), 50% (n = 24) and 54% (n = 26), respectively. This was significantly greater than in acidic soil (2 test for 10% light availability: Df = 1, p < 0.0001; for 25% light availability: Df = 1, p < 0.0001, for 50% light availability: Df = 1, p < 0.0001). A negative effect of “home” soil biota was evident only in neutral soil in 50% light availability: mortality of plants grown in soil with “home” biota was significantly greater (79%; n = 19) than in soil without “home” biota (29%, n = 7; 2 test: Df = 1, p = 0.0005). The presence of “home” soil biota had a significant negative effect on total biomass, root mass, belowground mass and leaf length of A. septentrionale plants (Table 1). The effect of soil pH on A. septentrionale plants was highly significant (p < 0.0001, Table 1). Plants grown in acidic soil were larger and had with a greater number of longer leaves.

2.454 1.316 0.949 1.954 1.410 2.458 1.255 0.853

0.091 0.273 0.390 0.147 0.249 0.091 0.290 0.430

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2.516 0.483 8.403 1.779 7.812 0.357 2.384 0.193 0.702 0.480 0.956 0.280 0.891 0.059 0.063 0.823

Fig. 1. Mean ± SE of the belowground biomass of Asplenium septentrionale plants grown in 2006–2007 under 10, 25, and 50% light availability in neutral and acidic soil. Bars with the same letter are not significantly different (p < 0.05, Tukey test).

All studied morphological characteristics responded significantly to light availability. Plants were larger and had a greater number of shorter leaves in 50% daylight than in the 25% and 10% daylight (Table 1). The one significant interactive effect of the experimental treatments was soil pH x light availability on belowground growth (Table 1). The response pattern was quite clear (Fig. 1); root mass and belowground mass were greater in plants grown in acidic soil than in plants grown in neutral soil only in better illumination conditions (25% and 50% light availability). In deep shade (10% light availability), the difference was not significant. Comparison of relative biomass allocations (Fig. 2) to leaves, rhizome and roots in different light availability and soil reaction treatments revealed two significant differences. First, the allocation of biomass into rhizomes in 50% light was significantly less and allocation into leaves notably more in acidic soil than in neutral soil. Second, plants grown in acidic soil allocated significantly less into roots in more shaded (10% and 25% of full daylight) conditions (Fig. 2).

Total mass Leaf mass Root mass Rhizome mass Below-ground mass Length of longest leaf Mean leaf length Number of leaves

4.275 3.181 4.576 0.460 4.073 1.452 4.051 0.632

0.041 0.078 0.035 0.499 0.046 0.231 0.047 0.429

54.727 59.009 109.536 26.060 103.700 26.451 35.640 46.455

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

4.151 3.218 7.146 7.761 2.227 3.806 10.107 4.939

0.019 0.044 0.001 0.001 <0.001 0.026 <0.001 0.009

0.228 0.269 0.159 0.366 0.012 0.170 3.316 0.394

0.634 0.605 0.691 0.546 0.911 0.681 0.072 0.532

0.355 0.740 0.045 1.288 0.115 2.915 2.844 0.195

0.086 0.618 <0.001 0.174 0.001 0.701 0.098 0.825

F F p F p F

p

F

p

F

p

F

p

p

Soil biota × soil pH × light availability Soil pH × light availability Soil biota × light availability Soil biota × soil pH Light availability Soil pH Soil biota Source of variation

Table 1 Results of three-way ANOVA: effects of soil biota, soil pH and light availability and their interaction on parameters of Asplenium septentrionale grown under different light availability in 2006–2007 (10, 25, and 50% full daylight). F = F-ratio and p = probability value. Significant values are in bold.

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Fig. 2. Relative biomass allocation in Asplenium septentrionale plants grown in 2006–2007 under 10, 25, and 50% of light availability in neutral (N) and acidic (A) soil. Proportions with the same letter are not significantly different (p < 0.05, Tukey test).

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Table 2 Results of one-way ANOVA: effect of light availability on parameters of Asplenium septentrionale grown under different light availability in 2008 (50, 75, and 95% full daylight). F = F-ratio and p = probability value. Source of variation

Total mass Leaf mass Root mass Rhizome mass Length of longest leaf Number of leaves Number of leaves with sori

Light availability F

p

15.849 17.018 11.477 10.538 13.752 4.082 16.727

<0.001 <0.001 <0.001 <0.001 <0.001 0.028 <0.00

Experiment 2 The additional experiment, carried out to explore the morphology and growth of A. septentrionale at well illuminated conditions (50–95% of full daylight), showed a highly significant effect of light availability on all studied characteristics (Table 2) and revealed the possible optimum for light availability for the species; total biomass and all biomass fractions were greatest at 75% light availability (Fig. 3). Discussion Generally, the results from the two garden experiments confirmed that the earlier conjecture on the abiotic ecological preferences of A. septentrionale, based on observational data are correct. Furthermore, the results provided us numerical data, allowing an improvement in the habitat selection criteria for the species. Firstly, the better ecological performance of A. septentrionale in acidic soil than in neutral soil was not surprising. The species is known generally in Europe as a habitant of acidic siliceous rocks (Crabbe et al. 1993; Øvstedal & Tigerschiöld 2000; Page 1997; Reichstein 1984). According to the Ellenberg ecological indicator values for soil reaction, indicating the synecological optimum of the species on pH gradient (using ordinal scores ranging from 1 to 9) A. septentrionale inhabits acidic soils (Ellenberg’s score = 2; Ellenberg et al. 1991). Likewise, soil in the only Estonian habitat is acidic (pHKCl = 4.18). High mortality and slow growth of the experimental plants in neutral soil could account for the sparse and ephemeral distribution of the species in Estonia throughout history (Vaga

Fig. 3. Mean ± SE of the total biomass of Asplenium septentrionale plants grown in 2008 under 50, 75, and 95% light availability. Bars with the same letter are not significantly different (p < 0.05, Tukey test.

1960) and may be the reason behind the very restricted distribution of the species on basic rocks in Europe (Øllgaard & Tind 1993; Øvstedal & Tigerschiöld 2000). Secondly, experimental results on the performance of A. septentrionale on the light availability gradient largely confirmed the conclusions drawn from observational data. The species is known in Europe to prefer exposed, sunny habitats (Øvstedal & Tigerschiöld 2000; Page 1997; Reichstein 1984) or, especially in Central Europe, slight shade (Hill et al. 2004). It has rarely been found in habitats where average illumination level is less than 40% full daylight (Ellenberg’s score for light = 8; Ellenberg et al. 1991). Moderate shade tolerance of A. septentrionale has also been observed in Scandinavia (Øvstedal & Tigerschiöld 2000). For the single Estonian population the average transmission of total photosynthetically active radiation has been measured to be around 65% (n = 17, min = 24.0%, max = 86.0%). This study shows that strong shade (10% of full daylight) had a strong negative effect on A. septentrionale growth. The second experiment demonstrated that under 75% full daylight A. septentrionale plants were almost twice as large as under 95%. Such a sharp decrease of growth in near-full light could be attributed to stress caused by intense radiation and hence greater evaportranspiration (Larcher 2003). The same phenomenon is observable in natural habitats; only in microhabitats such as rock fissures, where roots are shaded and protected from desiccation, are plants able to survive long enough to result in large individuals. Although plants in both acidic and neutral soil had similarly small belowground biomass under low light, plants grown in neutral soil also exhibited significantly higher mortality (81.25%) than plants grown in acidic soil (4.2%). Plant biomass increased significantly under better illumination, but only in acidic soil. The positive effect of increased light in neutral soil was diminished by the high soil reaction. In neutral soil, biomass remained low and mortality high (50–54.17%) regardless of the illumination regime. This shows that not only the main effect of basic environmental factors but also their interactive effects may considerably affect the success of a (re)introduction effort. Diminished growth of plants in the presence of “home” soil biota has been reported in several earlier studies and attributed to species-specific pathogenic microorganisms in “home” soil (Callaway et al. 2004; Klironomos 2002; Van Grunsven et al. 2009). This indicates that introduction of A. septentrionale into new habitats requires no soil inoculation of microbes from the established population. Fern rhizome has several functions – it supports fronds, contains conducting tissue (White & Turner 1995) and serves as a reproductive structure. Rhizome functions also as a storage and perennating organ (Grime 2001). We registered notably higher biomass allocation to rhizome in 50% full daylight, on account of leaves and roots biomass, in plants grown in neutral soil. One can speculate that in unfavourable soil conditions, plants adopt a more parsimonious strategy to store existing resources rather than allocating resources to the development of resource acquiring organs. The diminished growth of roots in deep shade and in acidic soil is likely due to an expected active plastic response to very low light resources. According to the results there is no clear evidence of a local adaption in the Estonian A. septentrionale population, as far as we can judge using published data. Still, it cannot be excluded, given our experiment was not specifically designed to ascertain the existence of local adaption. In conclusion, the following criteria should be considered when selecting new sites for the introduction of new A. septentrionale populations, in order to enhance the likelihood of successful establishment: 1. Introduction sites with acidic soil (pHKCl = 4–5) should be chosen. Negative effect of neutral soil should be avoided.

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