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Simulated Nitrogen Deposition Influences Gastropod Grazing in LichensAuthor(s): Johan Asplund, Otilia Johansson, Line Nybakken, Kristin Palmqvist, Yngvar GauslaaSource: Ecoscience, 17(1):83-89. 2010.Published By: Centre d'études nordiques, Université LavalDOI: http://dx.doi.org/10.2980/17-1-3331URL: http://www.bioone.org/doi/full/10.2980/17-1-3331

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17 (1): 83-89 (2010)

The deposition of anthropogenic nitrogen (N) from the atmosphere onto plants and soils currently causes global-scale changes to terrestrial ecosystems (as reviewed by Matson, Lohse & Hall, 2002). Altered N availability may

affect plant–herbivore interactions in various ways. Nitrogen content is considered a good predictor of food quality for herbivores (Mattson, 1980). In addition, increased N supply may decrease the concentration of carbon-based second-ary compounds (CBSCs) in plants (Koricheva et al., 1998; Hamilton et al., 2001) and hence reduce the plants' herbi-vore defence. Studies on insect–plant interactions show that N-deposition in most cases increases insect perform-ance (as reviewed by Kytö, Niemelä & Larsson, 1996; Throop & Lerdau, 2004). However, there is a discrepancy between reviewed studies. Some experimental studies have

Simulated nitrogen deposition influences gastropod grazing in lichens1

Johan ASPLUND2, Department of Ecology and Natural Resource Management, Norwegian University

of Life Sciences, PO Box 5003, NO-1432 Ås, Norway, e-mail: johan.asplund@umb.no

Otilia JOHANSSON, Department of Ecology and Environmental Science, Umeå University,

901 87 Umeå, Sweden.

Line NYBAKKEN3, Department of Ecology and Natural Resource Management,

Norwegian University of Life Sciences, PO Box 5003, NO-1432 Ås, Norway.

Kristin PALMQVIST, Department of Ecology and Environmental Science,

Umeå University, 901 87 Umeå, Sweden.

Yngvar GAUSLAA, Department of Ecology and Natural Resource

Management, Norwegian University of Life Sciences, P. O. Box 5003,

NO-1432 Ås, Norway.

Abstract: Lichens are often important photosynthetic organisms in oligotrophic environments where high-quality fodder plants are rare. A strong herbivore defence and/or low nutritional quality allows the accumulation of a high lichen biomass in such areas. However, it is not known how N deposition influences lichen palatability. This study analyzes possible changes in gastropod grazing preference after 3 months simulated N deposition on 3 foliose (Lobaria scrobiculata, Platismatia glauca, and Xanthoria aureola) and 1 pendulous lichen species (Alectoria sarmentosa). Lichens were daily irrigated in the field with rainwater containing 1.625 mM NH4NO3 from June to September, equivalent to a deposition of 50 kg N·ha–1·y–1. Irrigations applied at night, morning, or noon simulated different C-gain regimes. Afterwards in the lab, we offered 2 common lichen-feeding gastropods the choice between N-fertilized thalli and control thalli irrigated with artificial rainwater. The gastropods clearly preferred the unfertilized thalli of the 3 foliose species. For the pendulous A. sarmentosa, the gastropods preferred N-enriched thalli (irrigated at night) to controls. In conclusion, N-enrichment changes the palatability of lichens in species-specific ways. Keywords: Alectoria sarmentosa, herbivory, Lobaria scrobiculata, mollusc, Platismatia glauca, Xanthoria aureola.

Résumé : Les lichens sont souvent importants comme organismes photosynthétiques dans les environnements oligotrophiques où les plantes fourragères de haute qualité sont rares. Une forte défense contre les herbivores et/ou une faible qualité nutritionnelle permet une grande accumulation de biomasse de lichens dans de tels secteurs. Cependant, on ne sait pas comment la déposition d’azote (N) influence le goût des lichens. Cette étude analyse les possibles changements dans la préférence de broutement de gastéropodes après 3 mois de déposition de N simulée pour 3 espèces de lichens foliacés (Lobaria scrobiculata, Platismatia glauca et Xanthoria aureola) et une espèce de lichen pendant (Alectoria sarmentosa). Sur le terrain, les lichens ont été arrosés quotidiennement entre juin et septembre avec l’eau de pluie contenant 1,625 mM NH4NO3, ce qui équivalait à une déposition de 50 kg N·ha–1·an–1. Les irrigations appliquées la nuit, le matin ou à midi simulaient des régimes différents d’accumulation de C. Au laboratoire, nous avons offert à 2 gastéropodes communs se nourrissant de lichens, le choix entre des thalles fertilisés au N et des thalles contrôles irrigués avec l’eau de pluie artificielle. Les gastéropodes ont clairement préféré les thalles non fertilisés des 3 espèces foliacées. Quant à l’espèce de lichen pendant, A. sarmentosa, les gastéropodes ont préféré les thalles enrichis en N (irrigués la nuit) aux contrôles. En conclusion, l’enrichissement en N modifie le goût des lichens de façon spécifique selon l’espèce.Mots-clés : Alectoria sarmentosa, herbivorie, Lobaria scrobiculata, mollusque, Platismatia glauca, Xanthoria aureola.

Nomenclature: Lid et al., 2005; Gilbert, James & Wolseley, 2009.

Introduction

1Rec. 2009-11-24; acc. 2010-01-27. Associate Editor: Håkan Rydin.2Author for correspondence. 3Present address: Natural Product Research Laboratory, Department of Biology, University of Eastern Finland, PO Box 111, FIN-80101 Joensuu, Finland.

DOI 10.2980/17-1-3331

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addressed effects of increased N deposition on gastro-pods' plant feeding behaviour. For example, Deroceras agreste preferred fertilized lettuce with higher N contents (Pakarinen, Niemelä & Tuomi, 1990), and Arion subfuscusconsumed more Salix eriocephala seedlings fertilized with medium to high levels of nitrogen, phosphorus, and potassium than those receiving low additions (Albrectsen et al., 2004). However, increased N deposition did not influ-ence overall gastropod consumption rates in a grassland community (Cleland et al., 2006). Generalist herbivores, such as some gastropod species, often show food prefer-ences when offered a variety of options. They optimize their diet by balancing the intake of essential nutrients and energy with toxic secondary compounds (Speiser, 2001). In gen-eral, lichen palatability is determined by surface toughness, energy content, nutrient composition, and concentration of defensive compounds.

Gastropods grazing on lichens can be frequent in habitats such as calcareous stone walls (Fröberg, Baur & Baur, 1993), trunks of broadleaved deciduous trees with high bark pH (Asplund & Gauslaa, 2008), and some deciduous tree trunks in the northern boreal zone (J. Asplund, pers. observ.). A number of acidic and nutri-ent-limited ecosystems are dominated by lichens that are rarely grazed. Most lichens are susceptible to N depos-ition (van Herk, Mathijssen-Spiekman & de Zwart, 2003). However, we do not yet know how N deposition influ-ences grazing by lichen-feeding gastropods. A number of CBSCs in lichens deter grazing (Lawrey, 1983; Gauslaa, 2005). Recently, factors regulating the concentration of herbivore defensive lichen compounds have been stud-ied (Hyvärinen, Walter & Koopmann, 2002; Asplund & Gauslaa, 2007; Nybakken et al., 2007; Asplund, Solhaug & Gauslaa, 2009). The concentration of CBSCs was shown to be robust against increased N deposition in 3 of 4 of the lichen species shared by Nybakken, Johansson, and Palmqvist (2009) and this study, which is consistent with a constitutive type of defence.

Our aim was to study the effect of simulated N depos-ition on invertebrate–lichen interactions in northern Sweden with low anthropogenic N deposition. To do so, we used the facilities of an artificial N-deposition study with or without added N (50 kg N·ha–1·y–1) applied at 3 different times (night, early morning, and day) each day. We shared the experimental design and sample pool of study species with Nybakken, Johansson, and Palmqvist (2009) and ran the 2 transplantation studies simultan-eously. In this way, the quantification of CBSCs done by Nybakken, Johansson, and Palmqvist (2009) was repre-sentative also for our lichen samples. After exposure to the N-deposition regimes, our lichens were offered to 2 species of lichen-feeding gastropods to quantify grazing preference. As increased N-availability either reduced the CBSCs (Platismatia glauca) or did not influence the con-centration of CBSCs (Lobaria scrobiculata, Xanthoria aureola, Alectoria sarmentosa) according to Nybakken, Johansson, and Palmqvist (2009), we hypothesized that gastropods would favour lichens exposed to simulated N-deposition because of higher nitrogen content. We also hypothesized that the hydration regime might play a role

in the grazing pattern. As lichens are poikilohydric organ-isms, their carbon balance should depend on the duration of the wet and active state and the level of irradiance during hydration events. Hydration during night is likely to increase respirational losses and lower the C gain. Hydration at noon is likely to be followed by rapid desic-cation (cf. Jonsson, Moen & Palmqvist, 2008) also leading to low C gain. The highest C gain can be expected when hydration occurs in the early morning, because of slow desiccation. The long photosynthetically active period in the morning (Nybakken, Johansson & Palmqvist, 2009) presumably causes higher concentration of carbohydrates. If high-energy fodder is the important resource for gastropods when they graze on lichens, one may expect a higher prefer-ence for the morning treatment.

Methods

This study focused on A. sarmentosa, L. scrobicu-lata, P. glauca, and X. aureola, which exhibit contrasting environmental preferences. The N-fixing cyanobacterial L. scrobiculata grows mainly in old forests, and has strongly declined in areas with anthropogenic N depos-ition (Hallingbäck, 1989). It was collected from trunks of Alnus incana in a ravine forest in Verdalen, Nord-Trøndelag, Norway (64° 25' N, 11° 54' E) and from Salix caprea in Trondheim, Sør-Trøndelag, Norway (63° 28' N, 10° 42' E). The green algal P. glauca dominates a number of nutrient-poor habitats, such as acidic tree bark and rocks. Nevertheless, it is tolerant to additional N supply (Dahlman et al., 2003). P. glauca was collected from a spruce forest in Omagaliden, Västerbotten, Sweden (64° 9' N, 19° 50' E). X. aureola is a green-algal nitrophytic lichen growing on sea cliffs fertilized by bird droppings. It was collected from sea cliffs at Strömstad, Västra Götaland, Sweden (58° 52' N, 11° 7' E). These 3 genera are all foliose lichens and inhabit substrates that are often visited by gastropods. Foliose lichens offer shelter to gastropods and are easily accessible due to their flat growth form. The last species, A. sarmen-tosa, is a pendulous green-algal old forest lichen inhabiting thin canopy branches of conifer forests. A. sarmentosa was collected from Picea abies twigs in boreal spruce forests in Omagaliden (64° 8' N, 19° 48' E) and Gunnarn (64° 55' N, 17° 20' E), both sites in Västerbotten, Sweden. This long, pendulous lichen on canopy twigs is mainly accessible to gastropods as litter on the forest floor. All lichens were col-lected in May 2006, air-dried in the laboratory, and rinsed to remove debris. During storage prior to and after transplanta-tion, they were stored in a desiccated state at –18 °C.

NITROGEN TREATMENTS OF LICHENS

The N-deposition simulations were run at the Unit for Field-based Forest Research, Swedish University of Agricultural Sciences (Vindeln, Sweden), as described by Nybakken, Johansson, and Palmqvist (2009). Our lichens were sampled and treated together with those used by Nybakken, Johansson, and Palmqvist (2009). They were all transplanted in an open, old growth spruce- (P. abies) dominated forest at Kulbäcksliden (64° 12' N, 19° 33' E). Here, the lichens received 6 irrigation treatments: rainwater

at 0000 (Night), rainwater at 0640 (Morning), rainwater at 1210 (Noon), and rainwater + N at these 3 occasions (Night+N, Morning+N, and Noon+N). We used 12 thalli from each of the foliose species and 16 thalli of A. sarmen-tosa for each spraying regime.

Our lichen thalli were randomly located among the thalli analyzed for N and CBSC. The foliose thalli were sewed to wooden sticks and placed across each treatment plot. We sewed the pendulous A. sarmentosa to vertical plastic sticks mounted onto the wooden sticks. Six sprink-lers irrigated the sample plots. In order to ensure homogen-eous irrigation, the irrigation tube was placed 1 m above the ground with evenly distributed sprinklers. Open-top chambers of transparent PVC film (0.4 mm) minimized cross-contamination between treatments but did not exclude natural rainfall. We used artificial rainwater, 8.8 mg·L–1 K2CO3, 4.6 mg·L–1 Na2CO3, 5 mg·L–1 CaCO3, 4.4 mg·L–1

NaH2PO4, 0.25 mg·L–1 Fe2SO47H2O, and 0.6 mg·L–1

MnSO4H2O (Tamm, 1953) with or without 1.625 mM NH4NO3. Nitrogen equivalent to the background deposition (0.04 mM NH4NO3) represented the control treatment. Each irrigation treatment consisted of 2 min spraying equivalent to 1.2 mm rainfall. Sunrise occurred at 0100 at the start of the experiment and at 0600 at the end. The transplantation was done 14 June 2006 with the exception of X. aureola (21 June). The irrigation started 16 June and continued until 19 September 2006, thus including 95 irrigation–fertil-ization days (89 for X. aureola). The samples were har-vested October 3–5. Natural precipitation was 211 mm during the experiment (June 20–October 3); the irrigation added 110 mm. Irrigation amounted to an N deposition of 50 kg·ha–1·y–1 in fertilization treatments. The natural back-ground deposition of N was 2 kg N·ha–1·y–1 (Forsum et al., 2006). Climatic data are presented by Nybakken, Johansson, and Palmqvist (2009).

COLLECTION AND PRE-TREATMENT OF GASTROPODS

The snail Cepaea hortensis and the slug Arion fuscus were used. These gastropods are generalist herbivores. C. hortensis is common and widespread in various habi-tats (Cameron, 2003). A. fuscus is common in forests and feeds primarily on fungi (including lichens), but also on fresh leaves and dead animals (Andersson et al., 1980). Both species frequently climb trees to feed on epiphytic lichens, and they share habitats with the studied lichen species. However, A. sarmentosa is mostly inaccessible to gastropods until it falls to the ground as litter. Cepaea hor-tensis has been used in controlled experiments to test the defensive role of CBSCs in lichens (Gauslaa, 2005). Adult specimens of C. hortensis were collected from Ås, Akershus and Oslo, both in southeastern Norway. They were kept in glass containers with a perforated lid and fed a mixed diet of Lactuca sativa, Taraxacum F. H. Wigg spp., and lichens, including the study species prior to feeding experiments. Thus, the lichens were not novel to the snails. The glass containers were cleaned and fresh food was supplied every day. The snails were starved for 24 h before the start of each feeding experiment. The slugs, A. fuscus, were collected from Ås on early rainy mornings and kept without food in

plastic containers with the bottom lined with a humid filter paper for 24 h before the start of the feeding experiments.

FEEDING EXPERIMENTS

The ability to detect preferences depends on the num-ber of concurrent choices (Raffa, Havill & Nordheim, 2002). Hence, we performed a number of two-choice experiments rather than multiple-choice experiments. In the experiments with the 3 foliose species we used C. hortensis. In each experiment the snail was given the choice between 1 N-fertilized thallus and 1 unfertilized thallus given the same irrigation regime. In the experiments with the fruticose A. sarmentosa we used the slug A. fuscus since the snail C. hortensis tends to push and mix the 2 thallus portions with its shell. In addition, we repeated the feeding experi-ments using A. fuscus on L. scrobiculata and P. glauca irrig-ated at night and at noon. Only a limited number of lichen thalli were then available, and the results from the 2 time treatments were thus merged to increase the sample size. For the only foliose species that showed no significant dif-ferences between +N/–N treatment at any of the 3 hydration regimes (P. glauca), we added a 3-choice feeding experi-ment to quantify the effects of hydration treatment within each of the 2 N treatments. Prior to the feeding experi-ments the lichens had been rinsed with water to remove any remaining NH4NO3 on the thallus surface.

All thalli were weighed air-dry and photographed wet. Thalli in each treatment were ranked according to dry weight. The N-fertilized and the irrigated thalli of the same rank were placed together, minimizing the within-box varia-tion in thallus weight. The initial weights of the 2 thallus parts in each box were not significantly (paired t-test) dif-ferent except for X. aureola (t = 4.3, df = 35, P < 0.001, paired t-test). Immediately after the weighing procedure, each pair was placed in 1 plastic box (A. sarmentosa: 12 × 9 cm; foliose species: 9 × 7 cm) and randomly picked gastropods were placed in between the thalli. Two snails, or 3–5 slugs, per box were used in order to reduce individual feeding variability (Hanley, Bulling & Fenner, 2003). The boxes with lichens and gastropods were sprayed with water and closed with a perforated lid, allowing air circulation and preventing snails from escaping. Thalli were sprayed repeat-edly, compensating for evaporation. We used a fixed time stopping rule (Lockwood, 1998) by removing all snails after 24 h regardless of the amount consumed. The 2 snails from each box were weighed together on an analytical balance. The weight of the slugs was not recorded.

NITROGEN CONCENTRATION OF STUDIED SPECIES

The nitrogen concentration of separate thalli that were transplanted together with the thalli used in this experiment was quantified as described by Nybakken, Johansson, and Palmqvist (2009).

STATISTICAL ANALYSIS

Relative consumption was used to quantify the prefer-ence. This was calculated by dividing the amount consumed (DMend – DMstart) by total box consumption. Difference in preference was tested with a modified Hotelling’s T 2 test followed by a pair-wise comparison as described by Lockwood (1998). With 2 choices, the test is reduced to a

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traditional two-sided t-test (Lockwood, 1998). Differences in initial weights of paired N-fertilized and rainwater-treated thalli were analyzed with paired t-test.

Results

All green-algal lichens exposed to the N treatment had higher N concentration than those exposed to rainwater only (Table I). The 2 species from oligotrophic habitats, A. sarmentosa and P. glauca, more than doubled their N concentration, whereas the nitrophytic X. aureola exhibited a lower relative enrichment in N. The N-fixing cyano-bacterial L. scrobiculata did not increase its N concentra-tion regardless of N-deposition regime. Thus, the studied species differed in N concentration. L. scrobiculata con-trols had 5 times higher N concentration than A. sarmen-tosa controls.

In all 3 foliose lichen species, C. hortensis (Figure 1) and A. fuscus (Figure 2a,b) consistently preferred rainwater-treated thalli rather than N-enriched thalli. These species-specific patterns were similar despite the large contrasts in N concentration (Table I). The only exception occurred when C. hortensis did not discriminate between +/–N treat-ments sprayed in the morning for P. glauca (Figure 1b). For all foliose lichens, preferences were less clear in the morning treatment compared to night and day treatments (Figure 1). Nitrogen concentration was not affected by time of irrigation (data not shown).

As the gastropods did not discriminate between the +N/–N treatments given in the morning for P. glauca (Figure 1b), there was a need to check for possible effects of timing of the treatment for this species. When given the choice between thalli irrigated at different times, the snail preferred P. glauca irrigated in the morning. However, this preference was only significant for the control thalli (Figure 3; +N: T 2 = 5.1, df = 10, ns; –N: T 2 = 16.1, df = 10, P < 0.05, Hotelling’s T 2).

The grazing response for the +N/–N treatments on the pendulous lichen A. sarmentosa, given to A. fuscus only, contrasted with those on foliose lichens (Figure 2c). For this lichen species the slug strongly preferred fertilized thalli when treatment was given at night, but there were no sig-nificant differences in preference for +/– N treatments given in the morning and at noon.

Both gastropods grazed all layers of the thin P. glauca thalli simultaneously. Neither did C. hortensis show any preferences for particular parts of the X. aureola thalli. Presumably because of high thallus thickness in X. aureola, grazing seldom comprised the entire vertical section in this

TABLE I. Concentrations (mg·g–1 DW ± SE) of nitrogen in 4 lichen species subjected to irrigation with rainwater with or without nitro-gen every day. Sample sizes are in parentheses. Level of significance is denoted by *P < 0.05, and ***P < 0.001. One-way ANOVA.

Rainwater Rainwater + N MS F

Alectoria sarmentosa 6.1 ± 0.4 (10) 13.2 ± 0.6 (15) 364.2 112.0***Lobaria scrobiculata 30.3 ± 0.3 (3) 31.0 ± 0.8 (6) 0.82 0.3Platismatia glauca 6.4 ± 0.2 (19) 17.5 ± 0.8 (19) 1530.4 260.3***Xanthoria aureola 22.6 ± 2.6 (3) 28.8 ± 0.9 (6) 78.4 8.3*

FIGURE 1. Preference (+ SE) of the snail Cepaea hortensis for 3 lichen species subjected to irrigation with rainwater every day with or without nitro-gen at 3 different times (Night, Morning, Noon). Difference in preference was tested with a two-tailed t-test. However, for Platismatia glauca a Wilcoxon rank-sum test was performed. Data for X. aureola irrigated in the morning and at night were arcsine transformed. Numbers in boxes represent total amount DW consumed in mg ± SE, n = 12 (number of boxes). Filled bars = nitrogen, open bars = control. Differences in preference were tested with a two-tailed t-test. P-values are denoted with * P < 0.05, or *** P < 0.001.

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species. The 2 gastropods made species-specif ic graz-ing marks on L. scrobiculata. Cepaea hortensis grazed L. scrobiculata from the lower side, preferably on the naked, tomentum-free patches. The snails avoided the cyanobacterial layer, although they perforated the entire thallus in a few cases. In addition, C. hortensis grazed on free-living epi-phytic green algae that had grown outside the upper cortex of some L. scrobiculata thalli. In contrast, A. fuscus grazed exclusively from the upper side through both the upper cor-tex and the cyanobacterial layer, but left the white medulla behind. This is a common type of grazing mark in nature, although grazing from the lower side sometimes occurs. The total grazing of L. scrobiculata was much lower with A. fuscus than with C. hortensis.

Discussion

N additions caused a significant reduction in palat-ability of all 3 foliose lichens (Figure 1; 2a,b). However, the palatability of the pendulous A. sarmentosa was unchanged or increased (night irrigation) when treated with nitrogen (Figure 2c). The observed responses cannot be explained by the CBSCs that often function as herbivore deterrent compounds in lichens (Gauslaa, 2005), as these were hardly affected by the nitrogen treatment (Nybakken, Johansson & Palmqvist, 2009). Even in P. glauca, where CBSC con-centration was significantly lower in N-treated thalli com-pared with those that only got rainwater, the N-treated thalli were avoided. Apart from CBSCs, other factors such as nutrient composition and energy also control lichen palatability. But contrary to our expectations, increase in nitrogen (Table I) did not increase the palatability of the foliose lichens. Likewise, Lawrey (1983) found that slugs frequently avoided N-rich lichen species. However, these lichens were better defended in terms of CBSCs than our study species. The changed energy status of the lichens

FIGURE 2. Preference (+SE) of the slug Arion fuscus for a) Lobaria scrob-iculata (n = 12) and b) Platismatia glauca (n = 14) irrigated every day with or without nitrogen at 2 different times (Night and Noon merged). c) Alectoria sarmentosa subjected to irrigation with rainwater every day with or with-out nitrogen at 3 different times (Night, Morning, Noon). n = 16, data are arcsine transformed. Numbers in boxes represent total amount DW con-sumed in mg ± SE, n = 12 (number of boxes). Filled bars = nitrogen, open bars = control. Differences in preference were tested with a two-tailed t-test. P-values are denoted with * P < 0.05, or *** P < 0.001.

FIGURE 3. Preference (+SE) of the snail Cepaea hortensis for Platismatia glauca subjected to irrigation at night, morning, or noon. Irrigations were in the form of rainwater or rainwater + N. Differences in preference were tested with Hotelling’s T2 test followed by a pair-wise comparison. Bars not sharing the same letter are significantly (P < 0.05) different.

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may explain the preferences. For lichen-feeding reindeers, a lichen diet provides sufficient energy but is too low in N to sustain their muscle mass during a long winter (Gaare & Staaland, 1994). Lichens are apparently eaten as an energy source rather than a protein source (Nieminen & Heiskari, 1989; Dubay, Hayward & Martínez del Rio, 2008). Content, composition, and solubility of the fibre fraction have been considered important factors determining the nutritive value of lichens for reindeer (Svihus & Holand, 2000). During the treatment period, most of our lichen transplants showed substantial growth, particularly those receiving additional N (Nybakken, Johansson & Palmqvist, 2009). Gaio-Oliveira et al. (2005) found a trade-off between mannitol accumu-lation and N uptake in Xanthoria parietina, suggesting that excess assimilates can be stored as soluble polyols when lack of N limits cell division and growth. However, studies on the relationship between N deposition and the concentration of polyols are ambiguous (Dahlman et al., 2003; Palmqvist & Dahlman, 2006). Furthermore, lichenan (lichen starch) and other polysaccharides may also act as feeding stimulants for gastropods. Senseman (1977) showed that concentration of starch in agar pellets increased the feeding period of the slug Ariolimax californicus. Lichenan is broken down to sugars by the snail lichenase (Llano, 1948) and may thus represent a good source of energy. Surface toughness is an important factor for lichen palat-ability, but we have no reason to believe that this would be affected by the nitrogen treatment.

The smallest difference in gastropod preference between +N/–N treatments occurred when treatment was applied in the morning, which was the period associ-ated with the highest mean irradiance during hydration and thus high photosynthetic activity (Jonsson, Moen & Palmqvist, 2008; Nybakken, Johansson & Palmqvist, 2009). This hydration timing resulted in the highest preference (Figure 3) and likely favoured the accumulation of storage compounds rich in energy.

Another hypothesis is that the 3 foliose species may produce an N-based defence when N is added in excess. An N-based herbivore defence in lichens has not been experi-mentally documented in the literature as far as we know. However, carbohydrate-binding proteins known as lectins occur in a number of lichens species, including X. parietina (Legaz et al., 2004). Lectins from both lichens and non-lichenized fungi have recently been shown to be insecticidal (Hamshou, Van Damme & Smagghe, 2010; Silva et al., 2009). The role of lectins in plant defence is well docu-mented (Peumans & Van Damme, 1995). Nitrogen fertil-ization increases lectin concentration in Japanese chestnut (Castanea crenata; Nomura et al., 2008). Furthermore, alkaloids has been detected in lichens (Collema spp.: Temina, Levitsky & Dembitsky, 2010; X. parietina: Solberg, 1971), but it has not yet been shown to deter grazing. Nevertheless, moth larvae had lower pupal weights and longer development times when reared on an artificial diet supplemented with allantoin (Wilson & Stinner, 1984). Endophytic fungi of the genus Neotyphodium produce alka-loids that have been shown to protect the host grasses from slug grazing (Barker, 2008). Alkaloids in plants have a well

documented influence on gastropod herbivory (Speiser & Rowell-Rahier, 1991; Speiser, Harmatha & Rowell-Rahier, 1992). Nitrogen-based defences, such as alkaloids or cyano-genic glycosides, increase in concentration when nitrogen availability to plants increases (Bernays, 1983).

In conclusion, increased N deposition changes palat-ability in different ways for different species. The under-lying mechanism is not yet known, and factors controlling gastropods' lichen preferences, as well as possible N-based defence compounds, need to be identified in future studies. Still, we have shown that grazing preferences for cer-tain lichen species will decrease at higher N deposition, while other lichens will face more grazing. Consequently, changed N-deposition rates will alter the competition between lichen species and likely impact the structure of lichen-dominated habitats.

Acknowledgements

We would like to thank K. Asbjørn Solhaug for discussions and for collecting X. aureola and C. hortensis, O. Wiggo Røstad for collecting C. hortensis, and A. Sehlstedt for lab and field assistance.

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