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Trophic ecology of the supralittoral rocky shore (Roscoff, France): A dual stable isotope (δ13C, δ15N) and experimental approach
Journal of Sea Research 56 (2006) 27 – 36 www.elsevier.com/locate/seares
Trophic ecology of the supralittoral rocky shore (Roscoff, France): A dual stable isotope (δ 13 C, δ 15 N) and experimental approach Sandrine Laurand, Pascal Riera ⁎ Station Biologique de Roscoff, Université Pierre & Marie Curie Paris VI-CNRS-INSUE, Place Georges-Teissier, BP 74, 29682 Roscoff cedex, France Received 30 March 2005; accepted 2 March 2006 Available online 13 March 2006
Abstract The present study investigates the trophic transfers on the upper littoral rocky shore (i.e. the supralittoral zone together with the upper midlittoral and adlittoral) of northern Brittany. The population mainly consists of four invertebrate species: the littorinids Littorina saxatilis and Melarhaphe neritoides, the isopod Ligia oceanica and the insect Petrobius maritimus. The utilisation of food sources available to these grazers was examined in a laboratory microcosm feeding experiment and a field study using stable isotopes (δ13C, δ15N). The results indicated that although Ligia oceanica preferentially occurs in the supralittoral zone, its trophic subsidies originate mostly from the adlittoral and lower intertidal zones. The stable isotope data also suggested that adlittoral terrestrial organic material may be the major food source of Petrobius maritimus. δ15N of Littorina saxatilis indicated a highly variable diet consisting of supralittoral lichens, midlittoral macroalgae and other food sources (e.g. microalgae). Both feeding experiments and stable isotope data show that only Melarhaphe neritoides has a clearly identifiable diet based on a mixture of lichens, mostly Verrucaria maura and Caloplaca marina, as estimated by an isotopic mixing model. Hence, the food web of this intertidal zone appears largely based on trophic subsidies from other habitats (i.e. upper and lower intertidal zones). © 2006 Elsevier B.V. All rights reserved. Keywords: δ13C; δ15N; Food web; Rocky shore; Supralittoral
1. Introduction Due to the diversity and abundance of benthic communities of the rocky sea shore, it is necessary to study food preferences of primary consumers to better understand the trophic functioning of these ecosystems. So far few attempts have been made to define the trophic spectrum of primary consumers inhabiting the supralittoral zone of the North–East Atlantic coasts. The supralittoral zone is rarely submerged and typically ⁎ Corresponding author. E-mail address: [email protected] (P. Riera). 1385-1101/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2006.03.002
covered by a vegetation of lichens and a fauna comprising littorinid gastropods, isopods, barnacles and insects (Russel, 1991). Along the coast of Brittany, the main invertebrates inhabiting the supralittoral rocky shore are the littorinids Littorina saxatilis and Melarhaphe neritoides, together with the isopod Ligia oceanica and the insect apterygote Petrobius maritimus (Castric-Fey et al., 1997). Littorinids are the main grazers inhabiting supralittoral and upper midlittoral rocky shores (Hawkins and Hartnoll, 1983). Most of the diet studies on littorinids have focused on feeding preferences for macroalgae (Norton et al., 1990). However, by using their taenioglosse
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radula, littorinids are able to feed on a variety of algae in several habitats (Kim and De Wreede, 1996). In Vancouver Island, Voltolina and Sacchi (1990) also found cyanobacteria and lichens in the gut contents of Littorina scutulata inhabiting the supralittoral rocky shore. Littorina saxatilis is generally thought to be omnivorous because it feeds on fresh macroalgae from its proximal environment (Sacchi et al., 1981; Hawkins and Hartnoll, 1983), but also on detritic algae (Daguzan, 1976; Norton et al., 1990) and on benthic diatoms in environments without macroalgal cover (Sacchi et al., 1977). Although Littorina saxatilis largely occupies the Verrucaria maura zone (Hawkins and Hartnoll, 1983), there have been few attempts to compare the role of lichens in its diet with that of other food sources. Melarhaphe neritoides has been reported as lichenophagous because it lives too far up to feed on algae (Colman, 1939; Daguzan, 1976). These dietary patterns were mostly based on field observations or assumptions. In addition, a potential feeding selectivity among co-occurring lichens may characterise the diet of Melarhaphe neritoides. Previous investigations on feeding preferences of Ligia spp. in different areas have yielded contrasting results. In a gut content study, Carefoot (1973) found that the main food sources of Ligia pallasii were encrusting diatoms, insect larvae, a variety of green and red seaweeds and occasional specimens of Ligia pallasii. Carefoot (1973) also found a feeding preference of Ligia pallasii for the green algae Ulva sp. rather than for brown algae. In contrast, Fucus vesiculosus was reported as the main food source of Ligia oceanica by Nicholls (1931). In experimental conditions, Pennings et al. (2000) reported a feeding preference for detrital macroalgae. However, the question of the role of the supralittoral area as a trophic habitat for Ligia oceanica has not been clearly solved. To our knowledge, the feeding ecology of the common supralittoral insect Petrobius maritimus is still unknown. Stable isotopes allow the identification of different types of organic matter as food sources (Fry and Sherr, 1984; Deegan et al., 1990), and are valuable in feeding studies when food items in the gut contents are difficult to identify due to digestion and trituration. Food items may also be digested with different efficiencies, distorting their real dietary importance (Pinn et al., 1998), and the actual ingestion of food items is difficult to observe permanently in field studies. In addition, multiple isotope methods are also powerful tools in ecological studies (Peterson et al., 1985). A recent study has indicated significant differences in δ13C and δ15N between the co-occurring lichens of the rocky coast of
Brittany (Riera, 2005). These data provided the opportunity to investigate lichen-based food chains and to demonstrate feeding selectivity among lichens. The present study investigates the trophic subsidies of the most common invertebrate species inhabiting the upper zone of an intertidal rocky shore: Littorina saxatilis, Melarhaphe neritoides, Ligia oceanica and Petrobius maritimus. Particular attention was given to the feeding utilisation of supralittoral lichens which were examined in laboratory microcosm experiments and a field study using δ13Cand δ15N. 2. Methods 2.1. Sampling and preparation The sampling station is situated in the supralittoral zone of a rocky islet facing the Perharidy head at Roscoff (France) on the northern coast of Brittany (48°43′N, 4°2′W). At this station, the upper midlittoral, supralittoral and adlittoral zones exhibit characteristic fauna and flora of rocky shores along the coasts of the North Atlantic and the Channel (Cabioc'h et al., 1992; Little and Kitching, 1996). For the stable isotopes study, sampling was carried out in March 2001. Lichens were collected by gently scraping the surface of the rocky substratum, and thalli that were free of extraneous material were selected for analysis. The lichens included five phycobiont associations: Xanthoria parietina, Caloplaca marina, Verrucaria maura, Ramalina siliquosa, Tephromela atra inhabiting the supralittoral, and the cyanobiont-alone lichen Lichina pygmaea (Raven et al., 1990) inhabiting the upper midlittoral rocky shore. The macroalgae Pelvetia canaliculata, Fucus spiralis and Enteromorpha sp. inhabiting the upper midlittoral zone, and terrestrial Gramineae present in the adlittoral zone were also collected as potential food sources. In the supralittoral zone, four common invertebrates were collected by hand: two littorinids gastropods Littorina saxatilis and Melarhaphe neritoides, the isopod Ligia oceanica and the apterygote Petrobius maritimus. The macroalgae, lichens and Gramineae were cleared of epibionts, quickly acidified with 10% HCl and rinsed with distilled water. These samples were dried (60 °C) for 48 h and ground to a fine powder using a mortar and a pestle. The invertebrates were kept alive for 24 h in the laboratory to allow evacuation of gut contents and then killed by freezing (−20 °C). The gastropods were extracted from their shell, acidified with 10% HCl to remove any residual carbonates, rinsed with distilled water, dried (60 °C, 48 h) and ground to a fine powder. Each individual isopod and insect was acidified (10%
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HCl) to remove any residual carbonates from the cuticles, rinsed with distilled water and dried (60 °C). Then, cuticles were removed from each specimen, which was ground to a fine powder using a mortar and a pestle. All samples were kept frozen (− 32 °C) until analysis. Individual analyses were performed for each consumer species. 2.2. Stable isotope measurements Carbon and nitrogen isotope ratios were determined using a Fisons CN analyser coupled on line, via a Finnigan Con-Flo 3 interface, with a Finnigan Delta S mass-spectrometer. Data are expressed in the standard δ unit notation where dX ¼ ½ðRsample =Rreference Þ 1 103; with R = 13C/12C for carbon and 15N/14N for nitrogen, and reported relative to the Vienna Pee Dee Belemnite standard (PDB) for carbon and to air N2 for nitrogen. A laboratory working standard (Peptone) was run for every ten samples. Average reproducibilities based on replicate measurements, using the Peptone standard, for δ13C and δ15N were 0.1 and 0.13‰, respectively. 2.3. Feeding experiments Laboratory feeding experiments were conducted to complete the stable isotope results. These experiments aimed to determine the food sources not or rarely used by a consumer (1) to eliminate these sources as primary food components in natural conditions, (2) to exclude these sources in the application of the mixing equations. To make the different lichens available to grazers in the same way as in natural conditions we had to keep the lichens fixed on their rocky substrates during the experiments. Consequently, any quantification of the grazing on lichens could not be performed. To obtain uniformity of response in feeding experiments and minimise dietary pre-conditioning, the consumers were starved for 6 d before the feeding experiments started. The lichens (Xanthoria parietina, Caloplaca marina, Verrucaria maura, Ramalina siliquosa, Tephromela atra and Lichina pygmaea) and macroalgae (Fucus spiralis, Pelvetia canaliculata and Enteromorpha sp.) from the sampling site were used for the feeding experiments. Lichens were taken out together with their rocky substrates using a chisel and a hammer. Rocky fragments were then hewed precisely to obtain a small piece supporting only the lichen
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species desired. Macroalgae were collected a few hours before the experiment started, immediately cleared of epibionts and examined to ensure they did not present any previous grazing marks. For each consumer species, 10 replicate experiments were performed. Each experiment involved 2, 3 and 1 individuals for Littorina saxatilis, Melarhaphe neritoides and Ligia oceanica, respectively. Preliminary investigations indicated (1) that the feeding activity of littorinids became rapidly reduced when tested individually, and (2) two or three individuals tested together produced sufficient faecal pellets to accurately evaluate feeding activity. The duration of each consumer-source exposure was 72 h for littorinids (determined in preliminary investigations), and 36 h for Ligia oceanica (as indicated by Carefoot, 1973). After each consumersource exposure, the organisms were starved until a total excretion of faeces occurred, which took 3 to 6 d for gastropods and 2 d for Ligia oceanica (determined in preliminary investigations). Then the specimens were submitted to another food source. During experiments, the macroalgae were immersed twice a day in filtered seawater, while microcosms with lichens were slightly raised twice a day. In the macroalga microcosms, the seawater was renewed every day taking care to conserve faecal pellets. Because Ligia oceanica do not support permanent immersion, the macroalgae were not immersed but vaporised twice a day to maintain humidity. The estimation of the feeding activity on the different food sources was based on the production of faeces in the microcosms. The number of faecal pellets excreted by littorinids was considered to be a measure of the feeding activity on a source due to easy identification. As littorinids produced size-constant faecal pellets (about 0.8 mm length for Littorina saxatilis and 0.4 mm for Melarhaphe neritoides), a comparison of the number of pellets produced and the different food sources can be used to estimate the preferentially ingested food. However, in contrast to the gastropods, Ligia oceanica did not produce uniform and individually identifiable faecal pellets (observed in preliminary investigations). Therefore, the food sources of Ligia oceanica were not estimated quantitatively but through the production of faeces. 3. Results 3.1. Isotopic characterisation of sources and primary consumers δ13C vs δ15N values of the different sources of organic matter and the intertidal benthic invertebrates
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Table 1 δ13C and δ15N (range of values) of lichens, algae and invertebrates from the supralittoral zone of a rocky shore δ13C (‰)
δ15N (‰)
Lichens Caloplaca sp. Tephromela atra Lichina pygmaea Ramalina silicosa Verrucaria maura Xanthoria parietina
− 21.9 to − 21.6 − 20.9 to − 20.7 − 11.4 to − 10.5 − 19.6 to − 19.2 − 17.5 to − 16.7 − 19.1 to − 19.0
− 3.4 to − 2.6 0.4 to 1.2 2.1 to 2.3 − 0.2 to 0.7 − 0.4 to 1.7 − 1.9 to 0.3
Macroalgae Enteromorpha sp. Fucus spiralis Pelvetia canaliculata Detrital Gramineae
− 10.0 to − 9.7 − 20.5 to − 19.3 − 18.6 to − 18.3 − 29.8 to − 25.9
3.9 to 4.6 2.9 to 4.2 3.6 to 4.2 − 1.3 to 1.2
3 3 3 11
Invertebrates Melarhaphe neritoides Ligia oceanica Littorina saxatilis Petrobius maritimus
− 23.8 to − 18.2 − 23.2 to − 17.9 − 23.0 to − 21.1 − 30.9 to − 22.7
0.1 to 3.1 3.9 to 7.9 0.2 to 7.8 − 2.2 to 3.0
7 8 6 11
n 3 3 3 3 5 3
n: number of individuals.
are presented in Table 1. δ13C and δ15N values for living Enteromorpha sp. (from −10.0 to −9.7‰ and from 3.9 to 4.6‰) were more 13C-enriched and 15N-enriched than stranded Enteromorpha sp. previously measured in this area by Adin and Riera (2003). Significant interspecific differences were observed for δ13C and δ15N among the lichens (Kruskall-Wallis test, p = 0.02 and
p = 0.01 for δ13C and δ15N, respectively). The lichens exhibited an isotopic pattern distribution from lowest δ13C and δ15N values for Caloplaca marina to highest values for Verrucaria maura and Lichina pygmaea. δ13C and δ15N also discriminate these lichens from the co-occurring macroalgae of the intertidal rocky substrate, particularly due to lower δ15N (Fig. 1). Among the four consumers considered, Petrobius maritimus was much more depleted in 13C than other invertebrates. Littorina saxatilis, Melarhaphe neritoides and Ligia oceanica had similar δ13C values but differed in δ15N (Kruskall-Wallis test, p = 0.125 and p = 0.003 for δ13C and δ15N, respectively) showing increasing mean δ15N from Melarhaphe neritoides to Ligia oceanica and high standard deviation (Fig. 1). 3.2. Utilisation of the different food sources For each consumer species, the number of times that an effective ingestion of a food source occurred throughout the ten replicate feeding experiments can be defined as the ingestion frequency. This measure was used to compare the utilisation of the different food sources by Littorina saxatilis, Melarhaphe neritoides and Ligia oceanica (Fig. 2). The results show that the lichens Xanthoria parietina, Tephromela atra and Ramalina siliquosa were much less frequently consumed by Littorina saxatilis than were the other food sources (Fig. 2A). These three lichens were also slightly less used by Melarhaphe neritoides, but it used all food
Fig. 1. δ13C vs δ15N (mean ± SD) for the organic matter sources and the invertebrates of the upper littoral rocky shore in March 2001. (●): Average δ13C and δ15N values corresponding to the theoretical food source of invertebrates taking into account the mean trophic enrichment of 1‰ and 3.4‰ for δ13C and δ15N, respectively. (- - -): Trophic enrichment of carbon and nitrogen.
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Fig. 2. Frequencies of ingestion of the different food sources throughout the ten replicate feeding experiments for Littorina saxatilis (A), Melarhaphe neritoides (B) and Ligia oceanica (C), defined as the number of times that an effective ingestion was observed during each feeding experiment.
sources (Fig. 2B). Verrucaria maura, Lichina pygmaea, Caloplaca marina and the macroalgae were consumed by both gastropods in almost all replicate experiments (Fig. 2A, B). Ligia oceanica fed mainly on Fucus spiralis but also consistently used Enteromorpha sp. and Pelvetia canaliculata (Fig. 2C). Except for the midlittoral Lichina pygmaea, no lichens were consumed by Ligia oceanica.
In addition to the frequencies of ingestion, the number of faecal pellets produced by the littorinids can give a more precise measure of feeding preferences (Fig. 3A, B). On this basis, the consumption intensity of the various sources was significantly different for the two gastropods (Kruskall-Wallis test, p < 0.0001 and p < 0.0001 for Littorina saxatilis and Melarhaphe neritoides, respectively). These measurements indicate
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Fig. 3. Number of faecal pellets (mean ± SD, n = 10) produced by Littorina saxatilis (A), Melarhaphe neritoides (B) throughout the ten replicate feeding experiments corresponding to each food source.
a preferential utilisation of Verrucaria maura and Lichina pygmaea by Littorina saxatilis (means 438 ± 146 and 624 ± 267 faecal pellets produced, respectively) and Melarhaphe neritoides (means 488 ± 244 and 1146 ± 739 faecal pellets produced, respectively). The other lichen species were much less intensively consumed by both gastropods, as also observed for the macroalgae (Fig. 3). 4. Discussion 4.1. Determination of the food sources most utilised by the supralittoral invertebrates The different organic matter sources had distinct δ13C vs δ15N values, which allowed their use to infer food sources for the consumers considered. The mean isotopic composition of the diet of the invertebrates can be estimated by considering a mean trophic fractionation in δ13C of 1‰ (De Niro and Epstein, 1978; Rau et
al., 1983) and a mean trophic enrichment in δ15N of 3.4‰ (De Niro and Epstein, 1981; Minagawa and Wada, 1984; Post, 2002) as a result of the assimilation of food. For each invertebrate species, the trophic enrichments in 13C and 15N are shown in Fig. 1 (dashed line) starting from the mean δ13C vs δ15N of the consumer. The closer the theoretical δ13C vs δ15N value of a consumer's food is to the one of a pure food source, the higher must be the proportion of that source in the diet of the consumer, as compared to other sources. However, the isotopic composition of these invertebrates is perhaps best explained by a mixed diet. In fact, the δ13C vs δ15N for the theoretical food could result not from a preferential utilisation of a particular source, but from a mixed diet of the main food sources potentially available. Consequently, the ability of consumers to use the different sources, as determined through feeding experiments, should help in eliminating the sources not or rarely used by the different consumers. Estimating quantitatively the contribution of the main sources to the
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diet of the consumers is possible through the utilisation of the mixing model Isosource (Phillips and Gregg, 2003). Before the Isosource analysis, the mean trophic fractionation coefficients of 1‰ for δ13C and of 3.4‰ for δ15N were subtracted from the consumers' δ13C and δ15N values, respectively. 4.2. The insect Petrobius maritimus According to this procedure, the expected δ13C and δ15N of the preferentially exploited food source by Petrobius maritimus would be ca. −27.3‰ and ca. −3.1‰ for δ13C and δ15N, respectively. These δ13C and δ15N values are clearly different from the corresponding values obtained for the lichens and macroalgae. In addition, due to the absence of food sources with more positive δ13C values, no feasible mixing solution could be modelled for this species, which is consistent with the fact that lichens and macroalgae can be excluded as main contributors to the diet of Petrobius maritimus. In contrast, the mean δ13C is close to that of C3 plants (Fry and Sherr, 1984) and to the −27‰ obtained for detrital graminae of the adlittoral zone (Fig. 1). Hence, although Petrobius maritimus largely occurs in the supralittoral zone, its diet is most likely based on terrestrial organic matter from the adlittoral zone. Consequently, because these results indicated that supralittoral lichens and macroalgae did not contribute to the diet of Petrobius maritimus, no feeding experiments including these sources were performed with this insect. 4.3. The littorinids Littorina saxatilis and Melarhaphe neritoides The isotopic differences observed between the supralittoral species Littorina saxatilis and Melarhaphe neritoides suggest differences in the utilisation of the trophic resources available in the upper rocky shore. The theoretical food source of Littorina saxatilis has mean δ13C and δ15N values of −23.1‰ and 1.4‰, respectively, which sets it apart from the area defined by supralittoral lichens (Fig. 1). The δ15N and the results of the microcosm experiment suggest that the diet of Littorina saxatilis can include a mixture of macroalgae (Pelvetia canaliculata and Fucus spiralis) and several supralittoral lichens. The experimental study excludes Xanthoria parietina, Tephromela atra and Ramalina siliquosa as major contributors to the diet of Littorina saxatilis (Figs. 2A and 3A). In contrast, Verrucaria maura and Caloplaca marina (> 400 faecal pellets produced) were consistently used by Littorina saxatilis
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(Fig. 3A). Thus, among the potential food sources considered, the diet of Littorina saxatilis is likely to include mainly Verrucaria maura and Caloplaca marina in addition to the upper midlittoral Lichina pygmaea, Enteromorpha sp., Pelvetia canaliculata and Fucus spiralis. However, based on these sources, no feasible mixing solution could be modelled for this species due the absence of a lower δ13C component. This result suggests that other less 13C-enriched sources might also be consumed, which may explain the difference observed between the δ13C of the theoretical food source of Littorina saxatilis and the inferred sources. In addition, the variation in the mean δ15N value of Littorina saxatilis indicates a high variability in its diet. This variability is consistent with the littorinid’s distribution between the lower part of the supralittoral fringe and the high midlittoral, where Pelvetia canaliculata and Fucus spiralis occur. Other sources consistently used by Littorina saxatilis could include epiphytic microalgae inhabiting mid-littoral macroalgae and/or epilithic microalgae from crevices and cracks (Sacchi et al., 1977; Voltolina and Sacchi, 1990). However, according to Sacchi et al. (1977) microphagy in this species is a necessity imposed by the trophic conditions of its habitat rather than an effective choice. Unfortunately, due to methodological constraints, these microalgae have not been extracted during this study. The theoretical mean δ13C and δ15N of the food of Melarhaphe neritoides are estimated at −21‰ and −1.9‰, respectively (Fig. 1). These δ13C and δ15N values are similar to average values of the supra-littoral lichens, in particular when considering the 15 Ndepletion of these lichens as compared to other sources. In addition, although the upper midlittoral Enteromorpha sp. and the lichen Lichina pygmaea can be used by Melarhaphe neritoides (Figs. 2B and 3B), δ13C and δ 15 N results suggest that these sources did not contribute to its diet due to much higher 13 Cenrichment, suggesting, in turn, an exclusive contribution of the supra-littoral lichens in its diet. This is consistent with the vertical distribution of Melarhaphe neritoides on the rocky shore and with previous observations of Colman (1939) and Daguzan (1976). This gastropod is generally located in rock crevices and cracks of the supralittoral zone, which act as a shelter from predators, wave impact and desiccation (Bosch and Moreno, 1986). In addition, Melarhaphe neritoides is considered not to be very mobile, covering maximum distances of about 20 cm (Daguzan, 1976). This restricts it to feeding on supralittoral lichens. As the feeding experiments indicated that the different supralittoral lichens were consumed by Melarhaphe
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neritoides (Fig. 3B), the five lichen species were considered in the application of the mixing equations. Outcomes shown in Fig. 4 indicate that Caloplaca marina played a major role in the diet of Melarhaphe neritoides (mean 62.3%) followed by lower contributions of the other lichens, from 11.2% (mean value) for Xanthoria parietina to 5.8% for Verrucaria maura. Hence, Melarhaphe neritoides can be clearly defined as lichenophagous. The variation in the utilisation of the different lichens species by Littorina saxatilis and Melarhaphe neritoides could be linked with toxic lichenic substances. For example, such substances produced by Xanthoria parietina could inhibit growth and development of the insect Spodoptera littoralis (Huneck, 1999). Lichen physionomy may also play an inhibiting role due to the encrusting thallus of Tephromela atra, or the dry thallus
of Ramalina siliquosa, which gastropods probably cannot digest. 4.4. The isopod Ligia oceanica The microcosm experiments have indicated the absence of lichens in the diet of Ligia oceanica (Fig. 2C), whereas the upper midlittoral macroalgea and Lichina pygmaea were consistently used. Consequently, these sources were considered in the application of mixing equations together with adlittoral Gramineae because Ligia oceanica can occur in this zone (Riera, pers. obs.). Mixing model results showed a decreasing contribution of Fucus spiralis and Gramineae (37.2 and 32.4%, respectively) to Pelvetia canaliculata (23%) while Enteromorpha sp. and Lichina pygmaea contributed a minor part to the diet of Ligia oceanica (Fig. 4).
Fig. 4. Mean contributions (% together with ranges in brackets) of the main organic matter sources in the diet of Melarhaphe neritoides and Ligia oceanica, calculated by a mixing model run according to Phillips and Gregg (2003) using an increment of 1% and a tolerance of 0.1 per mil.
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However, Ligia oceanica was also reported to have tendencies towards scavenging and cannibalism (Nicholls, 1931; Carefoot, 1973), which suggest a higher trophic level than primary consumers. This higher trophic level is consistent with the mean δ15N value observed for Ligia oceanica, higher than for other invertebrates inhabiting the supralittoral zone (Fig. 1). Therefore, the present results support the statute of ‘opportunistic omnivorous’ attributed to Ligia oceanica in the literature (Carefoot, 1973), its diet depending partly on small invertebrates and various fresh or detritic macroalgae. The high standard deviation for δ15N and δ13C values of Ligia oceanica confirms the variability of its diet. A similar observation was previously reported for another opportunistic crustacean, viz. Carcinus meanas (Riera et al., 1999). Hence, these results indicate that although Ligia oceanica mainly occurs in the supralittoral zone, it obtains its trophic subsidies from upper and lower intertidal zones. 5. Conclusions This study showed that the combination of stable isotope measurements based on field sampling and laboratory feeding studies can provide significant insight into food preferences of primary consumers. Our results show that a large proportion of the food web in the supralittoral zone of rocky shores is based on trophic subsidies from other habitats. Although Ligia oceanica and Petrobius maritimus occur mainly in this supralittoral, their feeding habitat is not this zone. In addition, the results underline the importance of supralittoral lichens as the major food source for Melarhaphe neritoides, while lichens were used together with macroalgae by Littorina saxatilis. Feeding preferences by these gastropods among the lichens were also observed. The dual methodological approach indicated that several sources which were easily and consistently used when individually offered to a consumer did not contribute a major part of its diet when available together with other sources in natural conditions. In particular, Lichina pygmaea and the macroalgae can be largely consumed by Melarhaphe neritoides, but they do not actually contribute to its diet, because they are not directly available in natural conditions. Among the four consumers most frequently occurring in this supralittoral zone, Melarhaphe neritoides is the only species characterised by a lichenophagous feeding mode, using the supralittoral zone as its feeding habitat. Hence, the food web of the supralittoral zone appears largely based on trophic subsidies from other habitats.
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References Adin, R., Riera, P., 2003. Preferential food source utilisation among stranded macroalgae by Talitrus saltator (Amphipod, Talitridae): a stable isotopes study in the northern coast of Brittany (France). Estuar. Coast. Shelf Sci. 56, 91–98. Bosch, M., Moreno, I., 1986. Spatial distribution of Littorina neritoides (L. 1758) (Mollusca, Gastropoda) in the supralittoral zone in the Balearic Islands. Cah. Biol. Mar. 27, 53–62. Cabioc'h, J., Floc'h, J.Y., Le Toquin, A., Boudouresque, C.F., Meinesz, A., Verlaque, M., 1992. La végétation marine Atlantique. In: Perret, D. (Ed.), Guide des Algues des Mers d'Europe. Delachaux & Niestlé, Neuchâtel, pp. 30–42. Carefoot, T.C., 1973. Feeding, food preference, and the uptake of food energy by the supralittoral isopod Ligia pallasii. Mar. Biol. 18, 228–236. Castric-Fey, A., Girard-Descatoire, A., Gentil, F., Davoult, D., Dewarumez, J.M., 1997. Macrobenthos des substrats durs intertidaux et subtidaux. In: Dauvin, J.C. (Ed.), Les Biocénoses marines et littorales françaises des côtes atlantiques, Manche et Mer du Nord: Synthèse, menaces et perspectives. Institut d'Ecologie et de gestion de la Biodiversité, Coll. Patrimoines Naturels, vol. 28. MNHN, pp. 83–95. Colman, J., 1939. On the faunas inhabiting intertidal seaweeds. J. Mar. Biol. Assoc. UK 24, 129–183. Daguzan, J., 1976. Contribution à l'écologie des Littorinidae (Mollusques Gastéropodes Prosobranches): I. Littorina neritoïdes (L.) et Littorina saxatilis (Olivi). Cah. Biol. Mar. 17, 213–236. Deegan, L.A., Peterson, B.J., Portier, R., 1990. Stable isotopes and cellulase activity as evidence for detritus as a food source for juvenile Gulf menhaden. Estuaries 13, 14–19. De Niro, M.J., Epstein, S., 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochim. Cosmochim. Acta 42, 495–506. De Niro, M.J., Epstein, S., 1981. Influence of diet on the distribution of nitrogen isotopes in animals. Geochim. Cosmochim. Acta 45, 341–351. Fry, B., Sherr, E.B., 1984. δ13C Measurements as indicators of carbon flow in marine and freshwater ecosystems. Contrib. Mar. Sci. 27, 13–47. Hawkins, S.J., Hartnoll, R.G., 1983. Grazing on intertidal algae by marine invertebrates. Oceanogr. Mar. Biol. Annu. Rev. 21, 195–282. Huneck, S., 1999. The significance of lichens and their metabolites. Naturwissenschaften 86, 559–570. Kim, J.H., De Wreede, R.E., 1996. Distribution and feeding preference of a high intertidal littorinid. Bot. Mar. 39, 561–569. Little, C., Kitching, J.A., 1996. The Biology of Rocky Shores. Oxford University Press, Oxford. Minagawa, M., Wada, E., 1984. Stepwise enrichment of 15N along food chains: further evidence and the relation between δ15N and animal age. Geochim. Cosmochim. Acta 48, 1135–1140. Nicholls, A.G., 1931. Studies on Ligia oceanica: Part II. The processes of feeding, digestion and absorption, with a description of the structure of the foregut. J. Mar. Biol. Assoc. UK 17, 675–707. Norton, T.A., Hawkins, S.J., Manley, N.L., 1990. Scraping a living: a review of littorinid grazing. Hydrobiologia 193, 117–138. Pennings, S.C., Carefoot, T.C., Zimmer, M., Danko, J.P., Ziegler, A., 2000. Feeding preferences of supralittoral isopods and amphipods. Can. J. Zool. 78, 1918–1929.
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Peterson, B.J., Howarth, R.W., Garritt, R.H., 1985. Multiple stable isotopes used to trace the flow of organic matter in estuarine food webs. Science 227, 1361–1363. Phillips, D.L., Gregg, J.W., 2003. Source partitioning using stable isotopes: coping with too many sources. Oecologia 136, 261–269. Pinn, E.H., Atkinson, R.J.A., Rogerson, A., 1998. The diet of two mud-shrimps, Calocaris macandreae and Upogebia stellata (Crustacea: Decapoda: Thalassinidea). Ophelia 48, 211–223. Post, D.M., 2002. Using stable isotopes to estimate trophic position: models, methods and assumptions. Ecology 83, 703–718. Rau, G.H., Mearns, A.J., Young, D.R., Olson, R.J., Schafer, H.A., Kaplan, I.R., 1983. Animal 13C/12C correlates with trophic level in pelagic food webs. Ecology 64, 1314–1318. Raven, J.A., Johnston, A.M., Handley, L.L., Mc Inroy, S.G., 1990. Transport and assimilation of inorganic carbon by Lichina pygmaea under emersed and submersed conditions. New Phytol. 114, 407–417. Riera, P., 2005. δ13C and δ15N comparison among different cooccurring lichen species from littoral rocky substrata. Lichenologist 37, 93–95.
Riera, P., Stal, L.J., Nieuwenhuize, J., Richard, P., Blanchard, G., Gentil, F., 1999. Determination of food sources for benthic invertebrates in a salt marsh (Aiguillon Bay, France) by carbon and nitrogen stable isotopes: importance of locally produced sources. Mar. Ecol. Prog. Ser. 187, 301–307. Russel, G., 1991. Vertical distribution. In: Mathieson, A.C., Nienhuis, P.H. (Eds.), Ecosystems of the World 24: Intertidal and Littoral Ecosystems. Elsevier Science Publishers, Amsterdam, pp. 43–65. Sacchi, C.F., Testard, P., Voltolina, D., 1977. Recherches sur le spectre trophique comparé de Littorina saxatilis (Olivi) et de L. nigrolineata (Gray) (Gastropoda, Prosobranchia) sur la grève de Roscoff. Cah. Biol. Mar. 28, 99–505. Sacchi, C.F., Ambrogi, A.O., Voltolina, D., 1981. Recherches sur le spectre trophique comparé de Littorina saxatilis (Olivi) et de L. nigrolineata (Gray) (Gastropoda, Prosobranchia) sur la grève de Roscoff: II. Cas de populations vivant au milieu d'algues macroscopiques. Cah. Biol. Mar. 22, 83–88. Voltolina, D., Sacchi, C.F., 1990. Field observations on the feeding habits of Littorina scutulata Gould and L. sitkana Philippi (Gastropoda, Prosobranchia) of southern Vancouver Island (British Columbia, Canada. Hydrobiologia 193, 147–154.
Trophic ecology of the supralittoral rocky shore (Roscoff, France): A dual stable isotope (δ 13 C, δ 15 N) and experimental approach Sandrine Laurand, Pascal Riera ⁎ Station Biologique de Roscoff, Université Pierre & Marie Curie Paris VI-CNRS-INSUE, Place Georges-Teissier, BP 74, 29682 Roscoff cedex, France Received 30 March 2005; accepted 2 March 2006 Available online 13 March 2006
Abstract The present study investigates the trophic transfers on the upper littoral rocky shore (i.e. the supralittoral zone together with the upper midlittoral and adlittoral) of northern Brittany. The population mainly consists of four invertebrate species: the littorinids Littorina saxatilis and Melarhaphe neritoides, the isopod Ligia oceanica and the insect Petrobius maritimus. The utilisation of food sources available to these grazers was examined in a laboratory microcosm feeding experiment and a field study using stable isotopes (δ13C, δ15N). The results indicated that although Ligia oceanica preferentially occurs in the supralittoral zone, its trophic subsidies originate mostly from the adlittoral and lower intertidal zones. The stable isotope data also suggested that adlittoral terrestrial organic material may be the major food source of Petrobius maritimus. δ15N of Littorina saxatilis indicated a highly variable diet consisting of supralittoral lichens, midlittoral macroalgae and other food sources (e.g. microalgae). Both feeding experiments and stable isotope data show that only Melarhaphe neritoides has a clearly identifiable diet based on a mixture of lichens, mostly Verrucaria maura and Caloplaca marina, as estimated by an isotopic mixing model. Hence, the food web of this intertidal zone appears largely based on trophic subsidies from other habitats (i.e. upper and lower intertidal zones). © 2006 Elsevier B.V. All rights reserved. Keywords: δ13C; δ15N; Food web; Rocky shore; Supralittoral
1. Introduction Due to the diversity and abundance of benthic communities of the rocky sea shore, it is necessary to study food preferences of primary consumers to better understand the trophic functioning of these ecosystems. So far few attempts have been made to define the trophic spectrum of primary consumers inhabiting the supralittoral zone of the North–East Atlantic coasts. The supralittoral zone is rarely submerged and typically ⁎ Corresponding author. E-mail address: [email protected] (P. Riera). 1385-1101/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2006.03.002
covered by a vegetation of lichens and a fauna comprising littorinid gastropods, isopods, barnacles and insects (Russel, 1991). Along the coast of Brittany, the main invertebrates inhabiting the supralittoral rocky shore are the littorinids Littorina saxatilis and Melarhaphe neritoides, together with the isopod Ligia oceanica and the insect apterygote Petrobius maritimus (Castric-Fey et al., 1997). Littorinids are the main grazers inhabiting supralittoral and upper midlittoral rocky shores (Hawkins and Hartnoll, 1983). Most of the diet studies on littorinids have focused on feeding preferences for macroalgae (Norton et al., 1990). However, by using their taenioglosse
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radula, littorinids are able to feed on a variety of algae in several habitats (Kim and De Wreede, 1996). In Vancouver Island, Voltolina and Sacchi (1990) also found cyanobacteria and lichens in the gut contents of Littorina scutulata inhabiting the supralittoral rocky shore. Littorina saxatilis is generally thought to be omnivorous because it feeds on fresh macroalgae from its proximal environment (Sacchi et al., 1981; Hawkins and Hartnoll, 1983), but also on detritic algae (Daguzan, 1976; Norton et al., 1990) and on benthic diatoms in environments without macroalgal cover (Sacchi et al., 1977). Although Littorina saxatilis largely occupies the Verrucaria maura zone (Hawkins and Hartnoll, 1983), there have been few attempts to compare the role of lichens in its diet with that of other food sources. Melarhaphe neritoides has been reported as lichenophagous because it lives too far up to feed on algae (Colman, 1939; Daguzan, 1976). These dietary patterns were mostly based on field observations or assumptions. In addition, a potential feeding selectivity among co-occurring lichens may characterise the diet of Melarhaphe neritoides. Previous investigations on feeding preferences of Ligia spp. in different areas have yielded contrasting results. In a gut content study, Carefoot (1973) found that the main food sources of Ligia pallasii were encrusting diatoms, insect larvae, a variety of green and red seaweeds and occasional specimens of Ligia pallasii. Carefoot (1973) also found a feeding preference of Ligia pallasii for the green algae Ulva sp. rather than for brown algae. In contrast, Fucus vesiculosus was reported as the main food source of Ligia oceanica by Nicholls (1931). In experimental conditions, Pennings et al. (2000) reported a feeding preference for detrital macroalgae. However, the question of the role of the supralittoral area as a trophic habitat for Ligia oceanica has not been clearly solved. To our knowledge, the feeding ecology of the common supralittoral insect Petrobius maritimus is still unknown. Stable isotopes allow the identification of different types of organic matter as food sources (Fry and Sherr, 1984; Deegan et al., 1990), and are valuable in feeding studies when food items in the gut contents are difficult to identify due to digestion and trituration. Food items may also be digested with different efficiencies, distorting their real dietary importance (Pinn et al., 1998), and the actual ingestion of food items is difficult to observe permanently in field studies. In addition, multiple isotope methods are also powerful tools in ecological studies (Peterson et al., 1985). A recent study has indicated significant differences in δ13C and δ15N between the co-occurring lichens of the rocky coast of
Brittany (Riera, 2005). These data provided the opportunity to investigate lichen-based food chains and to demonstrate feeding selectivity among lichens. The present study investigates the trophic subsidies of the most common invertebrate species inhabiting the upper zone of an intertidal rocky shore: Littorina saxatilis, Melarhaphe neritoides, Ligia oceanica and Petrobius maritimus. Particular attention was given to the feeding utilisation of supralittoral lichens which were examined in laboratory microcosm experiments and a field study using δ13Cand δ15N. 2. Methods 2.1. Sampling and preparation The sampling station is situated in the supralittoral zone of a rocky islet facing the Perharidy head at Roscoff (France) on the northern coast of Brittany (48°43′N, 4°2′W). At this station, the upper midlittoral, supralittoral and adlittoral zones exhibit characteristic fauna and flora of rocky shores along the coasts of the North Atlantic and the Channel (Cabioc'h et al., 1992; Little and Kitching, 1996). For the stable isotopes study, sampling was carried out in March 2001. Lichens were collected by gently scraping the surface of the rocky substratum, and thalli that were free of extraneous material were selected for analysis. The lichens included five phycobiont associations: Xanthoria parietina, Caloplaca marina, Verrucaria maura, Ramalina siliquosa, Tephromela atra inhabiting the supralittoral, and the cyanobiont-alone lichen Lichina pygmaea (Raven et al., 1990) inhabiting the upper midlittoral rocky shore. The macroalgae Pelvetia canaliculata, Fucus spiralis and Enteromorpha sp. inhabiting the upper midlittoral zone, and terrestrial Gramineae present in the adlittoral zone were also collected as potential food sources. In the supralittoral zone, four common invertebrates were collected by hand: two littorinids gastropods Littorina saxatilis and Melarhaphe neritoides, the isopod Ligia oceanica and the apterygote Petrobius maritimus. The macroalgae, lichens and Gramineae were cleared of epibionts, quickly acidified with 10% HCl and rinsed with distilled water. These samples were dried (60 °C) for 48 h and ground to a fine powder using a mortar and a pestle. The invertebrates were kept alive for 24 h in the laboratory to allow evacuation of gut contents and then killed by freezing (−20 °C). The gastropods were extracted from their shell, acidified with 10% HCl to remove any residual carbonates, rinsed with distilled water, dried (60 °C, 48 h) and ground to a fine powder. Each individual isopod and insect was acidified (10%
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HCl) to remove any residual carbonates from the cuticles, rinsed with distilled water and dried (60 °C). Then, cuticles were removed from each specimen, which was ground to a fine powder using a mortar and a pestle. All samples were kept frozen (− 32 °C) until analysis. Individual analyses were performed for each consumer species. 2.2. Stable isotope measurements Carbon and nitrogen isotope ratios were determined using a Fisons CN analyser coupled on line, via a Finnigan Con-Flo 3 interface, with a Finnigan Delta S mass-spectrometer. Data are expressed in the standard δ unit notation where dX ¼ ½ðRsample =Rreference Þ 1 103; with R = 13C/12C for carbon and 15N/14N for nitrogen, and reported relative to the Vienna Pee Dee Belemnite standard (PDB) for carbon and to air N2 for nitrogen. A laboratory working standard (Peptone) was run for every ten samples. Average reproducibilities based on replicate measurements, using the Peptone standard, for δ13C and δ15N were 0.1 and 0.13‰, respectively. 2.3. Feeding experiments Laboratory feeding experiments were conducted to complete the stable isotope results. These experiments aimed to determine the food sources not or rarely used by a consumer (1) to eliminate these sources as primary food components in natural conditions, (2) to exclude these sources in the application of the mixing equations. To make the different lichens available to grazers in the same way as in natural conditions we had to keep the lichens fixed on their rocky substrates during the experiments. Consequently, any quantification of the grazing on lichens could not be performed. To obtain uniformity of response in feeding experiments and minimise dietary pre-conditioning, the consumers were starved for 6 d before the feeding experiments started. The lichens (Xanthoria parietina, Caloplaca marina, Verrucaria maura, Ramalina siliquosa, Tephromela atra and Lichina pygmaea) and macroalgae (Fucus spiralis, Pelvetia canaliculata and Enteromorpha sp.) from the sampling site were used for the feeding experiments. Lichens were taken out together with their rocky substrates using a chisel and a hammer. Rocky fragments were then hewed precisely to obtain a small piece supporting only the lichen
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species desired. Macroalgae were collected a few hours before the experiment started, immediately cleared of epibionts and examined to ensure they did not present any previous grazing marks. For each consumer species, 10 replicate experiments were performed. Each experiment involved 2, 3 and 1 individuals for Littorina saxatilis, Melarhaphe neritoides and Ligia oceanica, respectively. Preliminary investigations indicated (1) that the feeding activity of littorinids became rapidly reduced when tested individually, and (2) two or three individuals tested together produced sufficient faecal pellets to accurately evaluate feeding activity. The duration of each consumer-source exposure was 72 h for littorinids (determined in preliminary investigations), and 36 h for Ligia oceanica (as indicated by Carefoot, 1973). After each consumersource exposure, the organisms were starved until a total excretion of faeces occurred, which took 3 to 6 d for gastropods and 2 d for Ligia oceanica (determined in preliminary investigations). Then the specimens were submitted to another food source. During experiments, the macroalgae were immersed twice a day in filtered seawater, while microcosms with lichens were slightly raised twice a day. In the macroalga microcosms, the seawater was renewed every day taking care to conserve faecal pellets. Because Ligia oceanica do not support permanent immersion, the macroalgae were not immersed but vaporised twice a day to maintain humidity. The estimation of the feeding activity on the different food sources was based on the production of faeces in the microcosms. The number of faecal pellets excreted by littorinids was considered to be a measure of the feeding activity on a source due to easy identification. As littorinids produced size-constant faecal pellets (about 0.8 mm length for Littorina saxatilis and 0.4 mm for Melarhaphe neritoides), a comparison of the number of pellets produced and the different food sources can be used to estimate the preferentially ingested food. However, in contrast to the gastropods, Ligia oceanica did not produce uniform and individually identifiable faecal pellets (observed in preliminary investigations). Therefore, the food sources of Ligia oceanica were not estimated quantitatively but through the production of faeces. 3. Results 3.1. Isotopic characterisation of sources and primary consumers δ13C vs δ15N values of the different sources of organic matter and the intertidal benthic invertebrates
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Table 1 δ13C and δ15N (range of values) of lichens, algae and invertebrates from the supralittoral zone of a rocky shore δ13C (‰)
δ15N (‰)
Lichens Caloplaca sp. Tephromela atra Lichina pygmaea Ramalina silicosa Verrucaria maura Xanthoria parietina
− 21.9 to − 21.6 − 20.9 to − 20.7 − 11.4 to − 10.5 − 19.6 to − 19.2 − 17.5 to − 16.7 − 19.1 to − 19.0
− 3.4 to − 2.6 0.4 to 1.2 2.1 to 2.3 − 0.2 to 0.7 − 0.4 to 1.7 − 1.9 to 0.3
Macroalgae Enteromorpha sp. Fucus spiralis Pelvetia canaliculata Detrital Gramineae
− 10.0 to − 9.7 − 20.5 to − 19.3 − 18.6 to − 18.3 − 29.8 to − 25.9
3.9 to 4.6 2.9 to 4.2 3.6 to 4.2 − 1.3 to 1.2
3 3 3 11
Invertebrates Melarhaphe neritoides Ligia oceanica Littorina saxatilis Petrobius maritimus
− 23.8 to − 18.2 − 23.2 to − 17.9 − 23.0 to − 21.1 − 30.9 to − 22.7
0.1 to 3.1 3.9 to 7.9 0.2 to 7.8 − 2.2 to 3.0
7 8 6 11
n 3 3 3 3 5 3
n: number of individuals.
are presented in Table 1. δ13C and δ15N values for living Enteromorpha sp. (from −10.0 to −9.7‰ and from 3.9 to 4.6‰) were more 13C-enriched and 15N-enriched than stranded Enteromorpha sp. previously measured in this area by Adin and Riera (2003). Significant interspecific differences were observed for δ13C and δ15N among the lichens (Kruskall-Wallis test, p = 0.02 and
p = 0.01 for δ13C and δ15N, respectively). The lichens exhibited an isotopic pattern distribution from lowest δ13C and δ15N values for Caloplaca marina to highest values for Verrucaria maura and Lichina pygmaea. δ13C and δ15N also discriminate these lichens from the co-occurring macroalgae of the intertidal rocky substrate, particularly due to lower δ15N (Fig. 1). Among the four consumers considered, Petrobius maritimus was much more depleted in 13C than other invertebrates. Littorina saxatilis, Melarhaphe neritoides and Ligia oceanica had similar δ13C values but differed in δ15N (Kruskall-Wallis test, p = 0.125 and p = 0.003 for δ13C and δ15N, respectively) showing increasing mean δ15N from Melarhaphe neritoides to Ligia oceanica and high standard deviation (Fig. 1). 3.2. Utilisation of the different food sources For each consumer species, the number of times that an effective ingestion of a food source occurred throughout the ten replicate feeding experiments can be defined as the ingestion frequency. This measure was used to compare the utilisation of the different food sources by Littorina saxatilis, Melarhaphe neritoides and Ligia oceanica (Fig. 2). The results show that the lichens Xanthoria parietina, Tephromela atra and Ramalina siliquosa were much less frequently consumed by Littorina saxatilis than were the other food sources (Fig. 2A). These three lichens were also slightly less used by Melarhaphe neritoides, but it used all food
Fig. 1. δ13C vs δ15N (mean ± SD) for the organic matter sources and the invertebrates of the upper littoral rocky shore in March 2001. (●): Average δ13C and δ15N values corresponding to the theoretical food source of invertebrates taking into account the mean trophic enrichment of 1‰ and 3.4‰ for δ13C and δ15N, respectively. (- - -): Trophic enrichment of carbon and nitrogen.
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Fig. 2. Frequencies of ingestion of the different food sources throughout the ten replicate feeding experiments for Littorina saxatilis (A), Melarhaphe neritoides (B) and Ligia oceanica (C), defined as the number of times that an effective ingestion was observed during each feeding experiment.
sources (Fig. 2B). Verrucaria maura, Lichina pygmaea, Caloplaca marina and the macroalgae were consumed by both gastropods in almost all replicate experiments (Fig. 2A, B). Ligia oceanica fed mainly on Fucus spiralis but also consistently used Enteromorpha sp. and Pelvetia canaliculata (Fig. 2C). Except for the midlittoral Lichina pygmaea, no lichens were consumed by Ligia oceanica.
In addition to the frequencies of ingestion, the number of faecal pellets produced by the littorinids can give a more precise measure of feeding preferences (Fig. 3A, B). On this basis, the consumption intensity of the various sources was significantly different for the two gastropods (Kruskall-Wallis test, p < 0.0001 and p < 0.0001 for Littorina saxatilis and Melarhaphe neritoides, respectively). These measurements indicate
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Fig. 3. Number of faecal pellets (mean ± SD, n = 10) produced by Littorina saxatilis (A), Melarhaphe neritoides (B) throughout the ten replicate feeding experiments corresponding to each food source.
a preferential utilisation of Verrucaria maura and Lichina pygmaea by Littorina saxatilis (means 438 ± 146 and 624 ± 267 faecal pellets produced, respectively) and Melarhaphe neritoides (means 488 ± 244 and 1146 ± 739 faecal pellets produced, respectively). The other lichen species were much less intensively consumed by both gastropods, as also observed for the macroalgae (Fig. 3). 4. Discussion 4.1. Determination of the food sources most utilised by the supralittoral invertebrates The different organic matter sources had distinct δ13C vs δ15N values, which allowed their use to infer food sources for the consumers considered. The mean isotopic composition of the diet of the invertebrates can be estimated by considering a mean trophic fractionation in δ13C of 1‰ (De Niro and Epstein, 1978; Rau et
al., 1983) and a mean trophic enrichment in δ15N of 3.4‰ (De Niro and Epstein, 1981; Minagawa and Wada, 1984; Post, 2002) as a result of the assimilation of food. For each invertebrate species, the trophic enrichments in 13C and 15N are shown in Fig. 1 (dashed line) starting from the mean δ13C vs δ15N of the consumer. The closer the theoretical δ13C vs δ15N value of a consumer's food is to the one of a pure food source, the higher must be the proportion of that source in the diet of the consumer, as compared to other sources. However, the isotopic composition of these invertebrates is perhaps best explained by a mixed diet. In fact, the δ13C vs δ15N for the theoretical food could result not from a preferential utilisation of a particular source, but from a mixed diet of the main food sources potentially available. Consequently, the ability of consumers to use the different sources, as determined through feeding experiments, should help in eliminating the sources not or rarely used by the different consumers. Estimating quantitatively the contribution of the main sources to the
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diet of the consumers is possible through the utilisation of the mixing model Isosource (Phillips and Gregg, 2003). Before the Isosource analysis, the mean trophic fractionation coefficients of 1‰ for δ13C and of 3.4‰ for δ15N were subtracted from the consumers' δ13C and δ15N values, respectively. 4.2. The insect Petrobius maritimus According to this procedure, the expected δ13C and δ15N of the preferentially exploited food source by Petrobius maritimus would be ca. −27.3‰ and ca. −3.1‰ for δ13C and δ15N, respectively. These δ13C and δ15N values are clearly different from the corresponding values obtained for the lichens and macroalgae. In addition, due to the absence of food sources with more positive δ13C values, no feasible mixing solution could be modelled for this species, which is consistent with the fact that lichens and macroalgae can be excluded as main contributors to the diet of Petrobius maritimus. In contrast, the mean δ13C is close to that of C3 plants (Fry and Sherr, 1984) and to the −27‰ obtained for detrital graminae of the adlittoral zone (Fig. 1). Hence, although Petrobius maritimus largely occurs in the supralittoral zone, its diet is most likely based on terrestrial organic matter from the adlittoral zone. Consequently, because these results indicated that supralittoral lichens and macroalgae did not contribute to the diet of Petrobius maritimus, no feeding experiments including these sources were performed with this insect. 4.3. The littorinids Littorina saxatilis and Melarhaphe neritoides The isotopic differences observed between the supralittoral species Littorina saxatilis and Melarhaphe neritoides suggest differences in the utilisation of the trophic resources available in the upper rocky shore. The theoretical food source of Littorina saxatilis has mean δ13C and δ15N values of −23.1‰ and 1.4‰, respectively, which sets it apart from the area defined by supralittoral lichens (Fig. 1). The δ15N and the results of the microcosm experiment suggest that the diet of Littorina saxatilis can include a mixture of macroalgae (Pelvetia canaliculata and Fucus spiralis) and several supralittoral lichens. The experimental study excludes Xanthoria parietina, Tephromela atra and Ramalina siliquosa as major contributors to the diet of Littorina saxatilis (Figs. 2A and 3A). In contrast, Verrucaria maura and Caloplaca marina (> 400 faecal pellets produced) were consistently used by Littorina saxatilis
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(Fig. 3A). Thus, among the potential food sources considered, the diet of Littorina saxatilis is likely to include mainly Verrucaria maura and Caloplaca marina in addition to the upper midlittoral Lichina pygmaea, Enteromorpha sp., Pelvetia canaliculata and Fucus spiralis. However, based on these sources, no feasible mixing solution could be modelled for this species due the absence of a lower δ13C component. This result suggests that other less 13C-enriched sources might also be consumed, which may explain the difference observed between the δ13C of the theoretical food source of Littorina saxatilis and the inferred sources. In addition, the variation in the mean δ15N value of Littorina saxatilis indicates a high variability in its diet. This variability is consistent with the littorinid’s distribution between the lower part of the supralittoral fringe and the high midlittoral, where Pelvetia canaliculata and Fucus spiralis occur. Other sources consistently used by Littorina saxatilis could include epiphytic microalgae inhabiting mid-littoral macroalgae and/or epilithic microalgae from crevices and cracks (Sacchi et al., 1977; Voltolina and Sacchi, 1990). However, according to Sacchi et al. (1977) microphagy in this species is a necessity imposed by the trophic conditions of its habitat rather than an effective choice. Unfortunately, due to methodological constraints, these microalgae have not been extracted during this study. The theoretical mean δ13C and δ15N of the food of Melarhaphe neritoides are estimated at −21‰ and −1.9‰, respectively (Fig. 1). These δ13C and δ15N values are similar to average values of the supra-littoral lichens, in particular when considering the 15 Ndepletion of these lichens as compared to other sources. In addition, although the upper midlittoral Enteromorpha sp. and the lichen Lichina pygmaea can be used by Melarhaphe neritoides (Figs. 2B and 3B), δ13C and δ 15 N results suggest that these sources did not contribute to its diet due to much higher 13 Cenrichment, suggesting, in turn, an exclusive contribution of the supra-littoral lichens in its diet. This is consistent with the vertical distribution of Melarhaphe neritoides on the rocky shore and with previous observations of Colman (1939) and Daguzan (1976). This gastropod is generally located in rock crevices and cracks of the supralittoral zone, which act as a shelter from predators, wave impact and desiccation (Bosch and Moreno, 1986). In addition, Melarhaphe neritoides is considered not to be very mobile, covering maximum distances of about 20 cm (Daguzan, 1976). This restricts it to feeding on supralittoral lichens. As the feeding experiments indicated that the different supralittoral lichens were consumed by Melarhaphe
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neritoides (Fig. 3B), the five lichen species were considered in the application of the mixing equations. Outcomes shown in Fig. 4 indicate that Caloplaca marina played a major role in the diet of Melarhaphe neritoides (mean 62.3%) followed by lower contributions of the other lichens, from 11.2% (mean value) for Xanthoria parietina to 5.8% for Verrucaria maura. Hence, Melarhaphe neritoides can be clearly defined as lichenophagous. The variation in the utilisation of the different lichens species by Littorina saxatilis and Melarhaphe neritoides could be linked with toxic lichenic substances. For example, such substances produced by Xanthoria parietina could inhibit growth and development of the insect Spodoptera littoralis (Huneck, 1999). Lichen physionomy may also play an inhibiting role due to the encrusting thallus of Tephromela atra, or the dry thallus
of Ramalina siliquosa, which gastropods probably cannot digest. 4.4. The isopod Ligia oceanica The microcosm experiments have indicated the absence of lichens in the diet of Ligia oceanica (Fig. 2C), whereas the upper midlittoral macroalgea and Lichina pygmaea were consistently used. Consequently, these sources were considered in the application of mixing equations together with adlittoral Gramineae because Ligia oceanica can occur in this zone (Riera, pers. obs.). Mixing model results showed a decreasing contribution of Fucus spiralis and Gramineae (37.2 and 32.4%, respectively) to Pelvetia canaliculata (23%) while Enteromorpha sp. and Lichina pygmaea contributed a minor part to the diet of Ligia oceanica (Fig. 4).
Fig. 4. Mean contributions (% together with ranges in brackets) of the main organic matter sources in the diet of Melarhaphe neritoides and Ligia oceanica, calculated by a mixing model run according to Phillips and Gregg (2003) using an increment of 1% and a tolerance of 0.1 per mil.
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However, Ligia oceanica was also reported to have tendencies towards scavenging and cannibalism (Nicholls, 1931; Carefoot, 1973), which suggest a higher trophic level than primary consumers. This higher trophic level is consistent with the mean δ15N value observed for Ligia oceanica, higher than for other invertebrates inhabiting the supralittoral zone (Fig. 1). Therefore, the present results support the statute of ‘opportunistic omnivorous’ attributed to Ligia oceanica in the literature (Carefoot, 1973), its diet depending partly on small invertebrates and various fresh or detritic macroalgae. The high standard deviation for δ15N and δ13C values of Ligia oceanica confirms the variability of its diet. A similar observation was previously reported for another opportunistic crustacean, viz. Carcinus meanas (Riera et al., 1999). Hence, these results indicate that although Ligia oceanica mainly occurs in the supralittoral zone, it obtains its trophic subsidies from upper and lower intertidal zones. 5. Conclusions This study showed that the combination of stable isotope measurements based on field sampling and laboratory feeding studies can provide significant insight into food preferences of primary consumers. Our results show that a large proportion of the food web in the supralittoral zone of rocky shores is based on trophic subsidies from other habitats. Although Ligia oceanica and Petrobius maritimus occur mainly in this supralittoral, their feeding habitat is not this zone. In addition, the results underline the importance of supralittoral lichens as the major food source for Melarhaphe neritoides, while lichens were used together with macroalgae by Littorina saxatilis. Feeding preferences by these gastropods among the lichens were also observed. The dual methodological approach indicated that several sources which were easily and consistently used when individually offered to a consumer did not contribute a major part of its diet when available together with other sources in natural conditions. In particular, Lichina pygmaea and the macroalgae can be largely consumed by Melarhaphe neritoides, but they do not actually contribute to its diet, because they are not directly available in natural conditions. Among the four consumers most frequently occurring in this supralittoral zone, Melarhaphe neritoides is the only species characterised by a lichenophagous feeding mode, using the supralittoral zone as its feeding habitat. Hence, the food web of the supralittoral zone appears largely based on trophic subsidies from other habitats.
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