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Differential land snail damage to selected species of the lichen genus Peltigera
Biochemical Systematics and Ecology 32 (2004) 127–138 www.elsevier.com/locate/biochemsyseco
Differential land snail damage to selected species of the lichen genus Peltigera Renato Benesperi a, Mauro Tretiach b,∗ a
b
Dipartimento di Biologia Vegetale, Universita` degli Studi di Firenze, Via La Pira 4 - I 50121, Firenze, Italy Dipartimento di Biologia, Universita` degli Studi di Trieste, Via Giorgieri, 10 - I 34127 Trieste, Italy Received 16 December 2002; accepted 27 March 2003
Abstract The intensity of grazing of two land snails, Cantareus aspersa and Limax sp., on three epigaeic species of Peltigera with (P. horizontalis, P. neckeri) and without (P. praetextata) secondary compounds, were studied in the field in three transects of 2 × 50 m along an altitudinal gradient in the surroundings of Pistoia (Tuscany, Italy). The results were confirmed by laboratory experiments and were carried also out on a further species lacking lichen compounds (P. degenii), showing that land snails definitely prefer thalli lacking lichen secondary compounds. Damage intensity is correlated to the thallus size of the lichens. 2003 Elsevier Ltd. All rights reserved. Keywords: Cantareus aspersa; Grazing; Lichens; Limax sp.; Lichen secondary compounds
1. Introduction In natural habitats lichens represent an important food source for many organisms, from reindeers to arthropods and mollusca, and the effects of damage by herbivory on lichens are well documented (Schmid, 1929; Plitt, 1934; Coker, 1967; Peake and James, 1967; Yom-Tow and Galun, 1971; James and Henssen, 1976; Gerson and Seaward, 1977; Holleman and Luick, 1977; Lawrey 1980, 1983 a,b; Seaward, 1988; Baur et al., 1995). The lichens are protected against their predators by a number of mechanisms, the ∗
Corresponding author: Tel.: +39-040-558-3886; fax: +39-040-568-855. E-mail address: [email protected] (M. Tretiach).
0305-1978/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0305-1978(03)00141-8
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most important being the accumulation in large quantities of secondary compounds— the so-called lichen compounds. These substances have several roles, such as UVprotection and allelopathy (for a recent review see Huneck and Yoshimura, 1996; Huneck, 1999), and their anti-herbivorous role has long been suspected (Zukal, 1895; Stahl, 1904, see Hesbacher et al., 1996). Tests for the anti-herbivore properties of lichen compounds have been conducted with whole lichens or crude extracts (e.g. Baur et al., 1992, 1994; Ha¨ tscher et al., 1991; Lawrey, 1980, 1983a,b), and, more recently, with purified substances. Emmerich et al. (1993), for instance, demonstrated acute toxicity and feeding deterrence for frequently occurring lichen compounds such as (+) and (⫺)-usnic acid and vulpinic acid, whereas Slansky (1979) documented the anti-feeding activity of atranorin. The presence of different lichen compounds, together with other features, such as nutrient content and morphology, might account for the differential preference shown by land snails to various species of lichens (Baur et al., 1994; Fro¨ berg et al., 1993). Peltigera species are among the largest and most frequent epigaeic foliose lichens of temperate and boreal forests. They are generally characterised by high growth rates (Webster and Brown, 1997), which are related to their high photosynthetic capacity and nitrogen content, as a result of N2-fixation by the Nostoc cyanobionts (Hahn et al., 1993; Palmqvist and Sundberg, 2000; Schell and Alexander, 1973; Sundberg et al., 2001). Peltigera species may be ecologically important sources of nitrogen in environments with poor nitrogen supply, due to leaching from the thalli during the wet months (Millbank, 1985), or to herbivory by small animals. This last phenomenon has not yet been documented. In this paper we report the results of research aimed at investigating the grazing effects of two land snails on some Peltigera species, which confirms that their palatability is related to the presence of lichen compounds.
2. Data and methods The study is based on two data sets, consisting of field observations and laboratory experiments respectively. In order to estimate the snail grazing of Peltigera species in the field, three transects of 2 × 50 m each were selected in three wooded areas disposed along an altitudinal gradient in the surroundings of Pistoia (Tuscany, Central Italy) (Fig. 1), where different Peltigera species occur sympatrically. Transect 1 was located at 350 m a.s.l. in a mixed Quercus pubescens–Ostrya carpinifolia coppice; transect 2 at 650 m a.s.l. in an aged Castanea sativa coppice, and transect 3 at 1050 m a.s.l. at the border of a young Fagus sylvatica coppice. All thalli encountered in each transect were identified, measured, and the damage caused by snail grazing checked with the aid of an eyepiece (magnification 10× and 20×). The grazing intensity was classified according to a five ordinal scale, as follows: nil=undamaged thalli; low=very small spots of the photobiont layer exposed; medium=large areas of the photobiont layer exposed; high=photobiont layer largely removed; very high=thalli completely destroyed. The frequencies of damaged and undamaged thalli were subdivided
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Fig. 1.
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Location of the three transects (Tr 1–3) in the survey area.
according to their chemistry, organised in a 2 × 2 matrix, and analysed with the c2 test. In the laboratory the same species of Peltigera observed in the transects, as well as P. degenii collected near transect 2, were alternatively offered as food to the snail, Cantareus aspersa O.F. Mu¨ ller and to an undescribed species of slug, Limax (s.str.) sp., which were observed feeding on the thalli and were particularly frequent in the survey area. Fragments of each lichen (measuring ca. 4 cm2) were placed in plastic boxes of 30 × 20 × 30 cm lined with humid paper, covered with a thin mesh, and kept in a cold greenhouse under natural light. Two specimens of C. aspersa or Limax sp., collected with the lichens in the days immediately before the experiments, were introduced into each container without any pre-starvation treatment. To stimulate snail activity the containers were moistened, and the thalli examined daily under a dissecting microscope over a 10 day period to check possible damage. The intensity of final grazing was classified according to the above mentioned scale. Two sets of experiments were carried out. In the first set six fragments of lichen thalli were used; the ratio of thalli containing lichen substances vs thalli without lichen substances was 1:1. In the second set, carried out with a new group of snails, the ratio was 5:1 (Table 1). For identification of the lichens the keys of Vitikainen (1994) and Martı´nez Moreno (1999) were used. Voucher specimens are preserved in the lichen herbarium, Department of Biology, Trieste (TSB): P. degenii 35827; P. horizontalis 35828– 35830; P. neckeri 35831–35833; P. praetextata 35834–35836. Nomenclature of the lichens follows Nimis (2000).
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Table 1 Compounds identified in the four Peltigera species examined in this study Species
Lichen substances
P. degenii
No lichen substances found (Vitikainen, 1981, 1994; White and James, 1987; HoltanHartwig, 1993; Martı´nez Moreno, 1999). Tenuiorin, methyl gyrophorate, gyrophoric acid, zeorin, Phr-1,hopane-7b,22diol (traces), up to seven unidentified terpenoids (White and James, 1987; Holtan-Hartwig, 1993). Two chemotypes: type I with tenuiorin, methyl gyrophorate, gyrophoric acid, zeorin, Phr-1; type II with additional unidentified substances 1-5, 31, 36, 40 (Vitikainen, 1994). Five new terpenoids are added for type I by Miadlikowska and Lutzoni (2000). Tenuiorin, methyl gyrophorate, gyrophoric acid, dolichorrhizin (traces), zeorin, and PnP-1(minor) up to five unidentified triterpenoids (Holtan-Hartwig, 1988, 1993; Vitikainen, 1994) No lichen substances found (White and James, 1987; Ku¨ mmerling, 1991; Holtan-Hartwig, 1993; Vitikainen, 1994; Martı´nez Moreno, 1999; Miadlikowska and Lutzoni, 2000).
P. horizontalis
P. neckeri
P. praetextata
3. Results 3.1. Field observations In the three transects shown in Fig. 1, the lowest number of Peltigera thalli was 10 (transect 2), and the highest 113 (transect 3); a total of 166 thalli were monitored. They belonged to three species: P. praetextata (69% of thalli), present in all transects, P. horizontalis (22%, transects 1 and 3), and P. neckeri (9%, transect 2). Damage caused by snails was observed in 88 thalli (53%). In some cases snails were observed, feeding on the lichens. Young individuals of Limax sp. were observed twice sheltering under the thalli of P. neckeri. Fig. 2 shows the frequency of thalli damaged by snails subdivided in two groups,
Fig. 2. Frequency distribution of damages by slugs and snails observed in the field in two groups of thalli: with lichen compounds (black bars), and without lichen compounds (white bars).
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namely those with and without lichen substances (listed in Table 2). Land snails predominantly fed on thalli without lichen compounds (c 2= 26.7, P ⬍ 0.999): 69% of the thalli of P. praetextata, the most common species, had suffered slight to intense damage notwithstanding the heavy pruina covering its lobes, whereas only 25% of thalli with lichen substances were slightly damaged. In the latter case grazing was often limited to the apothecia. The high frequency of P. praetextata also enabled analysis of the effect of size on grazing intensity. Thalli were subdivided in three arbitrary size classes (small: ⱕ5 cm; medium: 5⬍xⱕ15 cm; large: ⬎15 cm), and the percentage of grazing was calculated for each class (Fig. 3). There was a clear increase in damage intensity in going from small to large thalli, probably because (a) larger thalli are older, and therefore they have been exposed to the feeding activity of snails for a longer time; (b) larger thalli have a higher probability of being found by snails during their erratic exploration of the territory. This observation, however, does not bias the results shown in Fig. 2, because in transects 1 and 3 the species had a similar size distribution (Fig. 4). Only in transect 1 did P. praetextata have a different size distribution (Fig. 4A), but this difference was not considered to be significant due to the low number of thalli (five for each species). In any case, the mean diameter of the two sample groups did not differ significantly (9.0 ± 7.3 against 9.3 ± 7.3 cm). 3.2. Laboratory experiments The first set of experiments showed the clear preference of snails for thalli lacking lichen compounds (Table 2): only the upper cortex of a single fragment of P. neckeri was grazed. When thalli of P. degenii and P. praetextata were placed together in the same containers, the snails did not show any preference: the damage was homogeneously distributed over the thalli of the two species, and reduced in intensity due Table 2 Damages suffered by thalli of Peltigera exposed to the feeding activity of two animals for ten days under controlled conditions. Pd: P. degenii; Ph: P. horizontalis; Pn: P. neckeri; Pp: P. praetextata. Species with lichen substances in italics; (—) not performed Ratio Limax sp.
Cantareus aspersa
Pp/Pn Pp/Ph Pd/Pn Pd/Ph Pp/Pd Ph/Pn Pp/Pn Pp/Ph Pd/Pn Pd/Ph Pp/Pd Ph/Pn
1:1
1:5
High/low High/nil High/nil High/nil Medium/medium Nil/nil Medium/nil Medium/nil Medium/nil Medium/nil Medium/medium Nil/nil
Very high/low Very high/(low) High/(low) Very high/low — — High/(low) High/nil High/nil Very high/nil — —
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Fig. 3. g. 3. Frequency distribution of damages by slugs and snails observed in the field in thalli of Peltigera praetextata of different size class: small (A) maximum diameter = x ⱕ 5 cm; medium (B) 5 ⬍ x ⱕ 15 cm; large (C) x ⬎ 15 cm. The number of thalli is reported in square parenthesis.
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Fig. 4. Size distribution of two groups of thalli (with and without lichen compounds, respectively on the left and the right column) in the three transects of Fig. 1.
to the increase in the surface area at disposal for grazing. These results are congruent with those of the second set of experiments. Although in this case the ratio between thalli with and without lichen compounds was 5:1 (the probability that the animals encountered thalli with lichen compounds being thus considerably higher), only thalli of P. degenii and P. praetextata were heavily grazed: in some cases their thalli were completely destroyed (Table 1). When thalli of P. neckeri and P. horizontalis were placed together in the same containers, no signs of damage were evident even after 10 days.
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4. Discussion Our data show that Cantareus aspersa and the Limax sp. preferred the two Peltigera species lacking lichen compounds, in good accordance with the avoidance hypothesis of Lawrey (1983a). Damage was heavier in the first group of species, both in the field and in the laboratory, whereas in the second group they were generally limited to the upper cortical layer (Fig. 2 and Table 1). The two animals grazed the thalli with different intensity: snails were generally less active than slugs (Table 2), but we ignored whether this was due to their preference for Peltigera species as food, or to their hunger, because the animals used in the experiments had not been pre-starved. Which lichen substance(s) gave protection against the two herbivores? Peltigera neckeri and P. horizontalis have a rather complex chemistry (Table 1), which comprises two main classes of compounds: gyrophoric acid and its derivatives (methylgyrophorate, tenuiorin) on the one hand, and terpenoids, with zeorin the most abundant, on the other. The harmful effects of zeorin to herbivores has been suggested by Mattsson (1987), but the antifeedant role of this and others lichen terpenoids has not been tested (Baur et al., 1994; Lawrey, 1980, 1983a,b; Emmerich et al., 1993). However, in the case of gyrophoric acid some further facts can be mentioned. This molecule, a tridepside, occurs in a wide range of lichens (Culberson et al., 1977), and is generally deposited in the upper cortex (Kurokawa and Takahashi, 1970) or in the medulla, where it can be easily detected by a selective histofluorescent method (Modenesi and Lajolo, 1992). Interestingly, a recent survey of the damage caused by the polyphagous herbivorous beetle Lasioderma serricorne to specimens of Physcia and Parmelia s.lat. kept in the TSB herbarium, confirmed that this substance has antifeedant activity (N. Skert & P.L. Nimis, pers. comm.), which is however less intense than that of other phenolic compounds (Lawrey, 1983a). The deterrence by gyrophoric acid might be due to the antiproliferative and cytotoxic activity of this substance, recently demonstrated in human keratinocyte (Kumar and Mu¨ ller, 1999), and is probably related to cytological aberrations induced during the mitotic phase (Reddy and Rao, 1978). According to Lawrey (1984, p. 254), protection against grazing is expected to be greater in reproductive tissues rather than in vegetative tissue, and, according to Fro¨ berg et al. (1993, p. 93), at least some lichens contain substances in the hymenium of the fruiting bodies that prevent grazing. In our Peltigera species, however, the hymenium was the more frequently eaten tissue in the two species containing lichen compounds. TLC analyses carried out by standard techniques (Culberson and Ammann, 1979) showed that these structures contain the same compounds as the vegetative thallus. Therefore it must be argued that their concentration in the apothecia is lower, or that the palatability of these reproductive structures is higher. The latter hypothesis appears to contradict the results of Lawrey (1983a). In order to test the so-called ‘preference hypothesis’, the latter author analysed lichens for their concentration of twelve elements, and demonstrated that slugs avoided species containing higher quantities of N, P and Ca, species which were also protected by secondary products. Palatability, however, is probably more related to the caloric content
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rather than to the elemental composition of the tissue: the fruiting bodies of Peltigera are usually rich in lipids, organic compounds with the highest caloric content per unit of dry weight, and this probably explains the selective grazing observed in our lichens. It is noteworthy that in the field the most heavily damaged species, P. praetextata, was also the most common, in term of both absolute and relative frequency. Does this imply that snail predation increases the dispersal efficiency of P. praetextata or is its abundance simply due to the wider ecological tolerance of the species (Vitikainen, 1994; Martı´nez Moreno, 1999)? McCarthy and Healy (1978) demonstrated the viability of both lichen spores and photobiont cells from faecal pellets of the slug Limax flavus, whereas Fro¨ berg et al. (2001) showed that faeces of the land snail Helicigonia lapicida contain, among the lichen remains, a few seemingly undamaged algae that withstand the enzymes and mechanical damage of digestion. According to these authors, these viable cells might have the potential to reconstitute a lichen symbiosis. Although endozoochory thus seems to be a potential mechanism for lichen dispersal, a better alternative is offered by symbiotic propagules. Along lobe margins and thallus cracks, many species of Peltigera form the so-called phyllidia, small dorsiventral lobules constricted at the base which are easily detached (Bu¨ del and Scheidegger, 1996), giving origin to independent thalli under laboratory conditions (Kershaw and Millbank, 1970). The formation of these propagules is mediated by lectins, and interestingly, they are often induced by wounding (Lallemant and Savoye, 1985). Unlike soredia, which are lighter and smaller and easily transported by small organisms such as ants (Lorentsson and Mattsson, 1999), neuroptera (Slocum and Lawrey, 1976), mites (Stubbs, 1995), or by fluids such as air and water (Armstrong, 1987, 1994; Harmata and Olech, 1991; Marshall, 1996), phyllidia and isidia (other common vegetative propagules typical of the lichen symbiosis) are larger and relatively heavy, and therefore their dispersal is generally restricted to small distances (Awasthi, 1983; Bjelland, 2001), although this has been questioned by several authors (e.g. Jahns, 1984; Ka¨ rnefelt, 1990). Slugs and snails are however sufficiently large to act as active, efficient carriers of these structures: when visiting the Peltigera thalli for food (or refuge: see Peake and James, 1967; Baur and Baur, 1997, and our obs.), phyllidia can easily adhere to their sticky bodies, detaching off later, when the protecting mucus is left behind the animal due to the body movements, forming the characteristic slime track. Interestingly, in P. praetextata phyllidia are very common, in P. degenii they are present occasionally (but are less luxuriant than in the former species), while in P. neckeri and P. horizontalis they are very rare or absent (Martı´nez Moreno, 1999; Vitikainen, 1994, and our obs.). Apart from some observations on the occurrence of lichen propagules on the shell surfaces of Clausilia bidentata (Peake and James, 1967), there is no accurate experimental information on the extent and importance of the role living snails and slugs might play in the dispersal of lichens.
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Acknowledgements We are grateful to Dr O. Vitikainen (Helsinki) for identification of problematic specimens of Peltigera, to Prof. Dr Folco Giusti (Siena) for identification of snails, and to Dr L. Baruffo (Trieste) for TLC analyses. Dr P. Crisafulli (Trieste) and Mr. Elia Menicagli (Florence) are acknowledged for technical assistance, and Dr M. Codogno (Trieste) for helpful suggestions. References Armstrong, R.A., 1987. Dispersal in a population of the lichen Hypogymnia physodes. Environ. Exp. Bot. 27, 357–363. Armstrong, R.A., 1994. Dispersal of soredia from individual soralia of the lichen Hypogymnia physodes (L.) Nyl. in a simple wind tunnel. Environ. Exp. Bot. 34, 39–45. Awasthi, D.D., 1983. Reproduction in lichens. Phytomorphology 33, 26–30. Baur, B., Baur, A., 1997. Xanthoria parietina as a food resource and shelter for the land snail Balea perversa. Lichenologist 29, 99–102. Baur, A., Baur, B., Fro¨ berg, L., 1992. The effect of lichen diet on growth rate in the rock-dwelling land snails Chondrina clienta (Westerlund) and Balea perversa (Linnaeus). J. Moll. Stud. 58, 245–247. Baur, A., Baur, B., Fro¨ berg, L., 1994. Herbivory on calcicolous lichens: different food preferences and growth rates in two co-existing land snails. Oecologia 98, 313–319. Baur, B., Fro¨ berg, L., Baur, A., 1995. Species diversity and grazing damage in a calcicolous lichen ¨ land, Sweden. Ann. Bot. Fenn. 32, 239–250. community on top of stone walls in O Bjelland, T., 2001. Comparative studies of the distribution and ecology of some oceanic species in the genus Leptogium (Lecanorales, Ascomycotina) in Norway. Nova Hedwigia 72, 1–44. Bu¨ del, B., Scheidegger, C., 1996. Thallus morphology and anatomy. In: Nash III, T.H. (Ed.), Lichen biology. Cambridge University Press, Cambridge, pp. 37–64. Coker, P.D., 1967. Damage to lichen by gastropods. Lichenologist 3, 428–429. Culberson, C.F., Ammann, K., 1979. Standard-methode zur Du¨ nnschicht-chromatographie von Flechtensubstanzen. Herzogia 5, 1–24. Culberson, C.F., Culberson, W.L., Johnson, A., 1977. Second supplement to Chemical and Botanical Guide to Lichen Products. American Bryological and Lichenological Society, Missouri Botanical Garden, St Louis. Emmerich, R., Giez, I., Lange, O.L., Proksch, P., 1993. Toxicity and antifeedant activity of lichen compounds against the polyphagous herbivourous insect Spodoptera littoralis. Phytochemistry 33, 1389–1394. Fro¨ berg, L., Baur, A., Baur, B., 1993. Differential herbivore damage to calcicolous lichens by snails. Lichenologist 25, 83–95. Fro¨ berg, L., Bjo¨ rn, L.O., Baur, A., Baur, B., 2001. Viability of lichen photobionts after passing through the digestive tract of a land snail. Lichenologist 33, 543–545. Gerson, U., Seaward, M.R.D., 1977. Lichen–invertebrate association. In: Seaward, M.R.D. (Ed.), Lichen Ecology. Academic Press, London, pp. 69–119. Hahn, S.C., Tenhunen, J.D., Popp, P.W., Meyer, A., Lange, O.L., 1993. Upland tundra in the foothills of the Brooks Range, Alaska: diurnal CO2 exchange patterns of characteristic lichen species. Flora 188, 125–143. Harmata, K., Olech, M., 1991. Transect for aerobiological studies from Antarctica to Poland. Grana 30, 458–463. Ha¨ tscher, I., Veit, M., Proksch, P., Lange, O.L., Zellner, H., 1991. Feeding deterrency and growth retarding activity of lichen substances against Spodoptera littoralis. Planta Medica A 57 (Suppl. 2), 26. Hesbacher, S., Fro¨ berg, L., Baur, A., Baur, B., Proksch, P., 1996. Chemical variation within and between individuals of the lichenized ascomycete Tephromela atra. Biochem. Syst. Ecol. 24, 603–609.
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Schell, D.M., Alexander, V., 1973. Nitrogen fixation in arctic coastal tundra in relation to vegetation and micro-relief. Arctic 26, 130–137. Schmid, G., 1929. Endolithische Kalkflechten und Schneckenfrass. Biol. Zentralbl. 49, 28–35. Seaward, M.R.D., 1988. Contribution of lichen ecosystems. In: Galun, M. (Ed.), Handbook of Lichenology, II. CRC Press, Boca Raton, pp. 107–129. Slansky, F. Jr, 1979. Effect of the lichen chemicals atranorin and vulpinic acid upon feeding and growth of larvae of the yellow-striped armyworm, Spodoptera ornithogalli. Environ. Poll. 8, 865–868. Slocum, R.D., Lawrey, J.D., 1976. Viability of the epizoic lichen flora carried and dispersed by green lacewing (Nodita pavida) larvae. Can. J. Bot. 54, 1827–1831. Stahl, E., 1904. Die Schutzmittel der Flechten gegen Tierfrass. In: Festschrift zum sienzigsten Geburtstage von Ernst Haeckel. Fischer, Jena, pp. 355–376. Sundberg, B., Na¨ sholm, T., Palmqvist, K., 2001. The effect of nitrogen on growth and key thallus components in the two tripartite lichens, Nephroma arcticum and Peltigera aphthosa. Plant Cell Environ. 24, 517–527. Stubbs, C.S., 1995. Dispersal of soredia by the oribatid mite, Humerobates arborea. Mycologia 87, 454–458. Vitikainen, O., 1981. Peltigera Willd nom. cons. Biblioth. Lichenol. 16, 236–242. Vitikainen, O., 1994. Taxonomic revision of Peltigera ( lichenized Ascomycotina) in Europe. Acta Bot. Fenn. 152, 1–96. Webster, M., Brown, D.H., 1997. Preliminary observations on the growth of transplanted Peltigera canina under semi-natural conditions. Lichenologist 29, 91–96. White, F.J., James, P.W., 1987. A chemical check-list of British lichens. 2. Bull. British Lichen Society 60, 42–47. Yom-Tow, Y., Galun, M., 1971. Note on feeding habits of the desert snail Sphincterochila boisseri Carpentier and Trochoidea (Xerocrassa) seetzeni Carpentier. Veliger 14, 86–88. Zukal, H., 1895. Morphologische und biologische Untersuchungen u¨ ber die Flechten I, II. Sitzb. Kais. Akad. Wissen. Math.-Nat. Classe. Abt. 1, 104, 529–574, 1303–1395.
Differential land snail damage to selected species of the lichen genus Peltigera Renato Benesperi a, Mauro Tretiach b,∗ a
b
Dipartimento di Biologia Vegetale, Universita` degli Studi di Firenze, Via La Pira 4 - I 50121, Firenze, Italy Dipartimento di Biologia, Universita` degli Studi di Trieste, Via Giorgieri, 10 - I 34127 Trieste, Italy Received 16 December 2002; accepted 27 March 2003
Abstract The intensity of grazing of two land snails, Cantareus aspersa and Limax sp., on three epigaeic species of Peltigera with (P. horizontalis, P. neckeri) and without (P. praetextata) secondary compounds, were studied in the field in three transects of 2 × 50 m along an altitudinal gradient in the surroundings of Pistoia (Tuscany, Italy). The results were confirmed by laboratory experiments and were carried also out on a further species lacking lichen compounds (P. degenii), showing that land snails definitely prefer thalli lacking lichen secondary compounds. Damage intensity is correlated to the thallus size of the lichens. 2003 Elsevier Ltd. All rights reserved. Keywords: Cantareus aspersa; Grazing; Lichens; Limax sp.; Lichen secondary compounds
1. Introduction In natural habitats lichens represent an important food source for many organisms, from reindeers to arthropods and mollusca, and the effects of damage by herbivory on lichens are well documented (Schmid, 1929; Plitt, 1934; Coker, 1967; Peake and James, 1967; Yom-Tow and Galun, 1971; James and Henssen, 1976; Gerson and Seaward, 1977; Holleman and Luick, 1977; Lawrey 1980, 1983 a,b; Seaward, 1988; Baur et al., 1995). The lichens are protected against their predators by a number of mechanisms, the ∗
Corresponding author: Tel.: +39-040-558-3886; fax: +39-040-568-855. E-mail address: [email protected] (M. Tretiach).
0305-1978/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0305-1978(03)00141-8
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most important being the accumulation in large quantities of secondary compounds— the so-called lichen compounds. These substances have several roles, such as UVprotection and allelopathy (for a recent review see Huneck and Yoshimura, 1996; Huneck, 1999), and their anti-herbivorous role has long been suspected (Zukal, 1895; Stahl, 1904, see Hesbacher et al., 1996). Tests for the anti-herbivore properties of lichen compounds have been conducted with whole lichens or crude extracts (e.g. Baur et al., 1992, 1994; Ha¨ tscher et al., 1991; Lawrey, 1980, 1983a,b), and, more recently, with purified substances. Emmerich et al. (1993), for instance, demonstrated acute toxicity and feeding deterrence for frequently occurring lichen compounds such as (+) and (⫺)-usnic acid and vulpinic acid, whereas Slansky (1979) documented the anti-feeding activity of atranorin. The presence of different lichen compounds, together with other features, such as nutrient content and morphology, might account for the differential preference shown by land snails to various species of lichens (Baur et al., 1994; Fro¨ berg et al., 1993). Peltigera species are among the largest and most frequent epigaeic foliose lichens of temperate and boreal forests. They are generally characterised by high growth rates (Webster and Brown, 1997), which are related to their high photosynthetic capacity and nitrogen content, as a result of N2-fixation by the Nostoc cyanobionts (Hahn et al., 1993; Palmqvist and Sundberg, 2000; Schell and Alexander, 1973; Sundberg et al., 2001). Peltigera species may be ecologically important sources of nitrogen in environments with poor nitrogen supply, due to leaching from the thalli during the wet months (Millbank, 1985), or to herbivory by small animals. This last phenomenon has not yet been documented. In this paper we report the results of research aimed at investigating the grazing effects of two land snails on some Peltigera species, which confirms that their palatability is related to the presence of lichen compounds.
2. Data and methods The study is based on two data sets, consisting of field observations and laboratory experiments respectively. In order to estimate the snail grazing of Peltigera species in the field, three transects of 2 × 50 m each were selected in three wooded areas disposed along an altitudinal gradient in the surroundings of Pistoia (Tuscany, Central Italy) (Fig. 1), where different Peltigera species occur sympatrically. Transect 1 was located at 350 m a.s.l. in a mixed Quercus pubescens–Ostrya carpinifolia coppice; transect 2 at 650 m a.s.l. in an aged Castanea sativa coppice, and transect 3 at 1050 m a.s.l. at the border of a young Fagus sylvatica coppice. All thalli encountered in each transect were identified, measured, and the damage caused by snail grazing checked with the aid of an eyepiece (magnification 10× and 20×). The grazing intensity was classified according to a five ordinal scale, as follows: nil=undamaged thalli; low=very small spots of the photobiont layer exposed; medium=large areas of the photobiont layer exposed; high=photobiont layer largely removed; very high=thalli completely destroyed. The frequencies of damaged and undamaged thalli were subdivided
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Fig. 1.
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Location of the three transects (Tr 1–3) in the survey area.
according to their chemistry, organised in a 2 × 2 matrix, and analysed with the c2 test. In the laboratory the same species of Peltigera observed in the transects, as well as P. degenii collected near transect 2, were alternatively offered as food to the snail, Cantareus aspersa O.F. Mu¨ ller and to an undescribed species of slug, Limax (s.str.) sp., which were observed feeding on the thalli and were particularly frequent in the survey area. Fragments of each lichen (measuring ca. 4 cm2) were placed in plastic boxes of 30 × 20 × 30 cm lined with humid paper, covered with a thin mesh, and kept in a cold greenhouse under natural light. Two specimens of C. aspersa or Limax sp., collected with the lichens in the days immediately before the experiments, were introduced into each container without any pre-starvation treatment. To stimulate snail activity the containers were moistened, and the thalli examined daily under a dissecting microscope over a 10 day period to check possible damage. The intensity of final grazing was classified according to the above mentioned scale. Two sets of experiments were carried out. In the first set six fragments of lichen thalli were used; the ratio of thalli containing lichen substances vs thalli without lichen substances was 1:1. In the second set, carried out with a new group of snails, the ratio was 5:1 (Table 1). For identification of the lichens the keys of Vitikainen (1994) and Martı´nez Moreno (1999) were used. Voucher specimens are preserved in the lichen herbarium, Department of Biology, Trieste (TSB): P. degenii 35827; P. horizontalis 35828– 35830; P. neckeri 35831–35833; P. praetextata 35834–35836. Nomenclature of the lichens follows Nimis (2000).
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Table 1 Compounds identified in the four Peltigera species examined in this study Species
Lichen substances
P. degenii
No lichen substances found (Vitikainen, 1981, 1994; White and James, 1987; HoltanHartwig, 1993; Martı´nez Moreno, 1999). Tenuiorin, methyl gyrophorate, gyrophoric acid, zeorin, Phr-1,hopane-7b,22diol (traces), up to seven unidentified terpenoids (White and James, 1987; Holtan-Hartwig, 1993). Two chemotypes: type I with tenuiorin, methyl gyrophorate, gyrophoric acid, zeorin, Phr-1; type II with additional unidentified substances 1-5, 31, 36, 40 (Vitikainen, 1994). Five new terpenoids are added for type I by Miadlikowska and Lutzoni (2000). Tenuiorin, methyl gyrophorate, gyrophoric acid, dolichorrhizin (traces), zeorin, and PnP-1(minor) up to five unidentified triterpenoids (Holtan-Hartwig, 1988, 1993; Vitikainen, 1994) No lichen substances found (White and James, 1987; Ku¨ mmerling, 1991; Holtan-Hartwig, 1993; Vitikainen, 1994; Martı´nez Moreno, 1999; Miadlikowska and Lutzoni, 2000).
P. horizontalis
P. neckeri
P. praetextata
3. Results 3.1. Field observations In the three transects shown in Fig. 1, the lowest number of Peltigera thalli was 10 (transect 2), and the highest 113 (transect 3); a total of 166 thalli were monitored. They belonged to three species: P. praetextata (69% of thalli), present in all transects, P. horizontalis (22%, transects 1 and 3), and P. neckeri (9%, transect 2). Damage caused by snails was observed in 88 thalli (53%). In some cases snails were observed, feeding on the lichens. Young individuals of Limax sp. were observed twice sheltering under the thalli of P. neckeri. Fig. 2 shows the frequency of thalli damaged by snails subdivided in two groups,
Fig. 2. Frequency distribution of damages by slugs and snails observed in the field in two groups of thalli: with lichen compounds (black bars), and without lichen compounds (white bars).
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namely those with and without lichen substances (listed in Table 2). Land snails predominantly fed on thalli without lichen compounds (c 2= 26.7, P ⬍ 0.999): 69% of the thalli of P. praetextata, the most common species, had suffered slight to intense damage notwithstanding the heavy pruina covering its lobes, whereas only 25% of thalli with lichen substances were slightly damaged. In the latter case grazing was often limited to the apothecia. The high frequency of P. praetextata also enabled analysis of the effect of size on grazing intensity. Thalli were subdivided in three arbitrary size classes (small: ⱕ5 cm; medium: 5⬍xⱕ15 cm; large: ⬎15 cm), and the percentage of grazing was calculated for each class (Fig. 3). There was a clear increase in damage intensity in going from small to large thalli, probably because (a) larger thalli are older, and therefore they have been exposed to the feeding activity of snails for a longer time; (b) larger thalli have a higher probability of being found by snails during their erratic exploration of the territory. This observation, however, does not bias the results shown in Fig. 2, because in transects 1 and 3 the species had a similar size distribution (Fig. 4). Only in transect 1 did P. praetextata have a different size distribution (Fig. 4A), but this difference was not considered to be significant due to the low number of thalli (five for each species). In any case, the mean diameter of the two sample groups did not differ significantly (9.0 ± 7.3 against 9.3 ± 7.3 cm). 3.2. Laboratory experiments The first set of experiments showed the clear preference of snails for thalli lacking lichen compounds (Table 2): only the upper cortex of a single fragment of P. neckeri was grazed. When thalli of P. degenii and P. praetextata were placed together in the same containers, the snails did not show any preference: the damage was homogeneously distributed over the thalli of the two species, and reduced in intensity due Table 2 Damages suffered by thalli of Peltigera exposed to the feeding activity of two animals for ten days under controlled conditions. Pd: P. degenii; Ph: P. horizontalis; Pn: P. neckeri; Pp: P. praetextata. Species with lichen substances in italics; (—) not performed Ratio Limax sp.
Cantareus aspersa
Pp/Pn Pp/Ph Pd/Pn Pd/Ph Pp/Pd Ph/Pn Pp/Pn Pp/Ph Pd/Pn Pd/Ph Pp/Pd Ph/Pn
1:1
1:5
High/low High/nil High/nil High/nil Medium/medium Nil/nil Medium/nil Medium/nil Medium/nil Medium/nil Medium/medium Nil/nil
Very high/low Very high/(low) High/(low) Very high/low — — High/(low) High/nil High/nil Very high/nil — —
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Fig. 3. g. 3. Frequency distribution of damages by slugs and snails observed in the field in thalli of Peltigera praetextata of different size class: small (A) maximum diameter = x ⱕ 5 cm; medium (B) 5 ⬍ x ⱕ 15 cm; large (C) x ⬎ 15 cm. The number of thalli is reported in square parenthesis.
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Fig. 4. Size distribution of two groups of thalli (with and without lichen compounds, respectively on the left and the right column) in the three transects of Fig. 1.
to the increase in the surface area at disposal for grazing. These results are congruent with those of the second set of experiments. Although in this case the ratio between thalli with and without lichen compounds was 5:1 (the probability that the animals encountered thalli with lichen compounds being thus considerably higher), only thalli of P. degenii and P. praetextata were heavily grazed: in some cases their thalli were completely destroyed (Table 1). When thalli of P. neckeri and P. horizontalis were placed together in the same containers, no signs of damage were evident even after 10 days.
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4. Discussion Our data show that Cantareus aspersa and the Limax sp. preferred the two Peltigera species lacking lichen compounds, in good accordance with the avoidance hypothesis of Lawrey (1983a). Damage was heavier in the first group of species, both in the field and in the laboratory, whereas in the second group they were generally limited to the upper cortical layer (Fig. 2 and Table 1). The two animals grazed the thalli with different intensity: snails were generally less active than slugs (Table 2), but we ignored whether this was due to their preference for Peltigera species as food, or to their hunger, because the animals used in the experiments had not been pre-starved. Which lichen substance(s) gave protection against the two herbivores? Peltigera neckeri and P. horizontalis have a rather complex chemistry (Table 1), which comprises two main classes of compounds: gyrophoric acid and its derivatives (methylgyrophorate, tenuiorin) on the one hand, and terpenoids, with zeorin the most abundant, on the other. The harmful effects of zeorin to herbivores has been suggested by Mattsson (1987), but the antifeedant role of this and others lichen terpenoids has not been tested (Baur et al., 1994; Lawrey, 1980, 1983a,b; Emmerich et al., 1993). However, in the case of gyrophoric acid some further facts can be mentioned. This molecule, a tridepside, occurs in a wide range of lichens (Culberson et al., 1977), and is generally deposited in the upper cortex (Kurokawa and Takahashi, 1970) or in the medulla, where it can be easily detected by a selective histofluorescent method (Modenesi and Lajolo, 1992). Interestingly, a recent survey of the damage caused by the polyphagous herbivorous beetle Lasioderma serricorne to specimens of Physcia and Parmelia s.lat. kept in the TSB herbarium, confirmed that this substance has antifeedant activity (N. Skert & P.L. Nimis, pers. comm.), which is however less intense than that of other phenolic compounds (Lawrey, 1983a). The deterrence by gyrophoric acid might be due to the antiproliferative and cytotoxic activity of this substance, recently demonstrated in human keratinocyte (Kumar and Mu¨ ller, 1999), and is probably related to cytological aberrations induced during the mitotic phase (Reddy and Rao, 1978). According to Lawrey (1984, p. 254), protection against grazing is expected to be greater in reproductive tissues rather than in vegetative tissue, and, according to Fro¨ berg et al. (1993, p. 93), at least some lichens contain substances in the hymenium of the fruiting bodies that prevent grazing. In our Peltigera species, however, the hymenium was the more frequently eaten tissue in the two species containing lichen compounds. TLC analyses carried out by standard techniques (Culberson and Ammann, 1979) showed that these structures contain the same compounds as the vegetative thallus. Therefore it must be argued that their concentration in the apothecia is lower, or that the palatability of these reproductive structures is higher. The latter hypothesis appears to contradict the results of Lawrey (1983a). In order to test the so-called ‘preference hypothesis’, the latter author analysed lichens for their concentration of twelve elements, and demonstrated that slugs avoided species containing higher quantities of N, P and Ca, species which were also protected by secondary products. Palatability, however, is probably more related to the caloric content
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rather than to the elemental composition of the tissue: the fruiting bodies of Peltigera are usually rich in lipids, organic compounds with the highest caloric content per unit of dry weight, and this probably explains the selective grazing observed in our lichens. It is noteworthy that in the field the most heavily damaged species, P. praetextata, was also the most common, in term of both absolute and relative frequency. Does this imply that snail predation increases the dispersal efficiency of P. praetextata or is its abundance simply due to the wider ecological tolerance of the species (Vitikainen, 1994; Martı´nez Moreno, 1999)? McCarthy and Healy (1978) demonstrated the viability of both lichen spores and photobiont cells from faecal pellets of the slug Limax flavus, whereas Fro¨ berg et al. (2001) showed that faeces of the land snail Helicigonia lapicida contain, among the lichen remains, a few seemingly undamaged algae that withstand the enzymes and mechanical damage of digestion. According to these authors, these viable cells might have the potential to reconstitute a lichen symbiosis. Although endozoochory thus seems to be a potential mechanism for lichen dispersal, a better alternative is offered by symbiotic propagules. Along lobe margins and thallus cracks, many species of Peltigera form the so-called phyllidia, small dorsiventral lobules constricted at the base which are easily detached (Bu¨ del and Scheidegger, 1996), giving origin to independent thalli under laboratory conditions (Kershaw and Millbank, 1970). The formation of these propagules is mediated by lectins, and interestingly, they are often induced by wounding (Lallemant and Savoye, 1985). Unlike soredia, which are lighter and smaller and easily transported by small organisms such as ants (Lorentsson and Mattsson, 1999), neuroptera (Slocum and Lawrey, 1976), mites (Stubbs, 1995), or by fluids such as air and water (Armstrong, 1987, 1994; Harmata and Olech, 1991; Marshall, 1996), phyllidia and isidia (other common vegetative propagules typical of the lichen symbiosis) are larger and relatively heavy, and therefore their dispersal is generally restricted to small distances (Awasthi, 1983; Bjelland, 2001), although this has been questioned by several authors (e.g. Jahns, 1984; Ka¨ rnefelt, 1990). Slugs and snails are however sufficiently large to act as active, efficient carriers of these structures: when visiting the Peltigera thalli for food (or refuge: see Peake and James, 1967; Baur and Baur, 1997, and our obs.), phyllidia can easily adhere to their sticky bodies, detaching off later, when the protecting mucus is left behind the animal due to the body movements, forming the characteristic slime track. Interestingly, in P. praetextata phyllidia are very common, in P. degenii they are present occasionally (but are less luxuriant than in the former species), while in P. neckeri and P. horizontalis they are very rare or absent (Martı´nez Moreno, 1999; Vitikainen, 1994, and our obs.). Apart from some observations on the occurrence of lichen propagules on the shell surfaces of Clausilia bidentata (Peake and James, 1967), there is no accurate experimental information on the extent and importance of the role living snails and slugs might play in the dispersal of lichens.
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Acknowledgements We are grateful to Dr O. Vitikainen (Helsinki) for identification of problematic specimens of Peltigera, to Prof. Dr Folco Giusti (Siena) for identification of snails, and to Dr L. Baruffo (Trieste) for TLC analyses. Dr P. Crisafulli (Trieste) and Mr. Elia Menicagli (Florence) are acknowledged for technical assistance, and Dr M. Codogno (Trieste) for helpful suggestions. References Armstrong, R.A., 1987. Dispersal in a population of the lichen Hypogymnia physodes. Environ. Exp. Bot. 27, 357–363. Armstrong, R.A., 1994. Dispersal of soredia from individual soralia of the lichen Hypogymnia physodes (L.) Nyl. in a simple wind tunnel. Environ. Exp. Bot. 34, 39–45. Awasthi, D.D., 1983. Reproduction in lichens. Phytomorphology 33, 26–30. Baur, B., Baur, A., 1997. Xanthoria parietina as a food resource and shelter for the land snail Balea perversa. Lichenologist 29, 99–102. Baur, A., Baur, B., Fro¨ berg, L., 1992. The effect of lichen diet on growth rate in the rock-dwelling land snails Chondrina clienta (Westerlund) and Balea perversa (Linnaeus). J. Moll. Stud. 58, 245–247. Baur, A., Baur, B., Fro¨ berg, L., 1994. Herbivory on calcicolous lichens: different food preferences and growth rates in two co-existing land snails. Oecologia 98, 313–319. Baur, B., Fro¨ berg, L., Baur, A., 1995. Species diversity and grazing damage in a calcicolous lichen ¨ land, Sweden. Ann. Bot. Fenn. 32, 239–250. community on top of stone walls in O Bjelland, T., 2001. Comparative studies of the distribution and ecology of some oceanic species in the genus Leptogium (Lecanorales, Ascomycotina) in Norway. Nova Hedwigia 72, 1–44. Bu¨ del, B., Scheidegger, C., 1996. Thallus morphology and anatomy. In: Nash III, T.H. (Ed.), Lichen biology. Cambridge University Press, Cambridge, pp. 37–64. Coker, P.D., 1967. Damage to lichen by gastropods. Lichenologist 3, 428–429. Culberson, C.F., Ammann, K., 1979. Standard-methode zur Du¨ nnschicht-chromatographie von Flechtensubstanzen. Herzogia 5, 1–24. Culberson, C.F., Culberson, W.L., Johnson, A., 1977. Second supplement to Chemical and Botanical Guide to Lichen Products. American Bryological and Lichenological Society, Missouri Botanical Garden, St Louis. Emmerich, R., Giez, I., Lange, O.L., Proksch, P., 1993. Toxicity and antifeedant activity of lichen compounds against the polyphagous herbivourous insect Spodoptera littoralis. Phytochemistry 33, 1389–1394. Fro¨ berg, L., Baur, A., Baur, B., 1993. Differential herbivore damage to calcicolous lichens by snails. Lichenologist 25, 83–95. Fro¨ berg, L., Bjo¨ rn, L.O., Baur, A., Baur, B., 2001. Viability of lichen photobionts after passing through the digestive tract of a land snail. Lichenologist 33, 543–545. Gerson, U., Seaward, M.R.D., 1977. Lichen–invertebrate association. In: Seaward, M.R.D. (Ed.), Lichen Ecology. Academic Press, London, pp. 69–119. Hahn, S.C., Tenhunen, J.D., Popp, P.W., Meyer, A., Lange, O.L., 1993. Upland tundra in the foothills of the Brooks Range, Alaska: diurnal CO2 exchange patterns of characteristic lichen species. Flora 188, 125–143. Harmata, K., Olech, M., 1991. Transect for aerobiological studies from Antarctica to Poland. Grana 30, 458–463. Ha¨ tscher, I., Veit, M., Proksch, P., Lange, O.L., Zellner, H., 1991. Feeding deterrency and growth retarding activity of lichen substances against Spodoptera littoralis. Planta Medica A 57 (Suppl. 2), 26. Hesbacher, S., Fro¨ berg, L., Baur, A., Baur, B., Proksch, P., 1996. Chemical variation within and between individuals of the lichenized ascomycete Tephromela atra. Biochem. Syst. Ecol. 24, 603–609.
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