Functional Ecology
Structure and functioning of intertidal food webs along an avian flyway: a comparative approach using stable isotopes
Teresa Catry, Pedro M. Lourenço, Ricardo J. Lopes, Camilo Carneiro, José A. Alves,
Joana Costa, Hamid Rguibi-Idrissi, Stuart Bearhop, Theunis Piersma, José P.
Granadeiro
Supporting Information
Appendix S1. Methodological protocols
Sample collection and processing
Water samples for particulate organic matter (POM) analysis were collected during high-tide either with a Van Dorn Bottle sampler or an alternative device, at a minimum depth of 50 cm, thus avoiding the superficial water layer. Approximately 20 l of water were pre-filtered through a 200 μm mesh size net to eliminate large zooplankton and detrital particles and then filtered again through pre-combusted Whatman GF/C filters until enough material was retained. Filters were subsequently dried in an oven at 60ºC and stored until further processing.
Samples of the upper layer (~1 cm) of sediment were collected for organic matter (SOM) analysis. Visible detrital particles were removed before drying samples for storage.
Textile panels (6-8 panels approximately 10x10 cm; Whatman, grade 105) were laid in the sediment surface in order to collect the microalgae that migrate to the surface during low tide. About 2h after deployment, panels were carefully taken into plastic containers, then rinsed with estuarine/marine water and decanted in order to separate the microalgae from the sediment attached to the panels. The supernatant was then filtered onto pre-combusted Whatman GF/F filters and dried afterwards.
Leaves from around 10 individuals of dominant plant species (mainly saltmarsh, mangrove and seagrass) were collected by hand. Rhizomes of seagrass and pneumatophores of mangrove were also sampled at Banc d’Arguin and Bijagós, respectively. Parts of macroalgae from at least five individuals were also collected by
Structure and functioning of intertidal food webs along an avian flyway: a comparative approach using stable isotopes. Catry T, Lourenço PM, Lopes RJ, Carneiro C, Alves JA, Costa J, Rguibi-Idrissi H,
Bearhop S, Piersma T, Granadeiro JP

Functional Ecology hand. Epiphytes and mud were carefully scrapped and washed from algae and plants before further processing. All samples were dried before further analysis.
Zooplankton were collected by towing a 200 μm mesh size net for approximately 30 mins, either using a zodiac cruising at low speed (ca. 2 knots) or slowly walking in medium-deep water. Samples were sorted in the same day under a binocular lens (to separate zooplankton individuals from potential debris) and subsequently dried.
Macroinvertebrate sampling was directed towards potential shorebirds’ prey. At
Tejo estuary and Sidi Moussa, where more information on shorebird diet was already available (e.g. Kersten et al. 1981, Moreira 1994, Lourenço et al. 2008, Catry et al.
2012a, Martins et al. 2013, authors’ unpublished data), sampling of macroinvertebrates was less extensive than at the remaining areas. Macroinvertebrates were collected by hand or using sampling cores and 0.5-1mm sieves, and kept alive for 24 hours in estuarine/marine seawater to clear their guts. We preferentially sampled macroinvertebrates in the range of sizes potentially consumed by shorebirds. Shells of bivalves and gastropods and exoskeleton of larger crustaceans (shrimps and crabs) were discarded and only soft tissues were kept for analysis. Chironomidae larvae (Insecta) were sampled at saltpans in the Tejo estuary and Sidi Moussa. Each replicate of macroinvertebrates consisted of a pool of a variable number of individuals (Table S1).
Samples were dried at 60º C before further processing.
Shorebirds were mainly captured at roosting areas. Between 1 and 2 mm of toenail were clipped from three to four toes of each bird using sharp scissors and stored in individual plastic bags. Sampling of toenails of shorebirds took place mainly between late December and February, to ensure that isotopic signatures of toenails were exclusively representing from their local wintering diet (assuming that birds have been in their wintering grounds since early November: preliminary authors’ unpublished data indicates half-lives of 27 and 35 days for δ13C and δ15N in toenails of captive dunlins; see also Bearhop et al. 2003).
Sample preparation for stable isotope analysis
All samples (except bird toenails) were ground into a homogeneous powder using mortar and pestle. Lipid extraction was performed in macroinvertebrate samples, which
Structure and functioning of intertidal food webs along an avian flyway: a comparative approach using stable isotopes. Catry T, Lourenço PM, Lopes RJ, Carneiro C, Alves JA, Costa J, Rguibi-Idrissi H,
Bearhop S, Piersma T, Granadeiro JP

Functional Ecology were immersed in a 2:1 chloroform/methanol solution with a solvent volume 3–5 times larger than sample volume (Logan et al. 2008). Samples were then mixed for 30 s, left undisturbed for approximately 30 min, and centrifuged for 10 min at 3400 rpm, and the supernatant containing solvent and lipids was removed. We repeated this process at least three times until the supernatant was clear and colourless following centrifugation.
Samples were re-dried at 60ºC for 24 h to remove any remaining solvent.
Samples of sediment, zooplankton, bivalves, gastropods and crustaceans, were tested for the presence of carbonates by adding a few drops of 10% HCl to a subsample.
Whenever a reaction (bubbling) was observed, some more drops of acid were added; the sample was then centrifuged to remove excess acid and finally washed with distilled water. This process was repeated three times or until bubbling stopped (Vinagre et al.
2008). Given that acidification can affect δ15298 N signatures, a non-acidified subsample was always kept for separate nitrogen isotope analysis (Carabel et al. 2006).
Toenails were washed in double baths of 0.25 N sodium hydroxide solution alternated with baths of double distilled water to remove adherent contamination, and then dried at 50° C for 48 h (Catry, Martins & Granadeiro 2012b).
References
Bearhop, S., Furness, R.W., Hilton, G.M., Votier, S.C. & Waldron, S. (2003) A forensic approach to understanding diet and habitat use from stable isotope analysis of (avian) claw material. Functional Ecology , 17 , 270-275
Carabel, S., Godínez-Domínguez, E., Verísimo, P., Fernández, L. & Freire, J. (2006) An assessment of sample processing methods for stable isotope analyses of marine food webs. Journal of Experimental Marine Biology and Ecology , 336 , 254-261.
Catry, T., Alves, J.A., Gill, J.A., Gunnarsson, T.G. & Granadeiro, J.P. (2012a) Sex promotes spatial and dietary segregation in a migratory shorebird during the nonbreeding season. PLoS ONE , 7(3) , e33811.
Structure and functioning of intertidal food webs along an avian flyway: a comparative approach using stable isotopes. Catry T, Lourenço PM, Lopes RJ, Carneiro C, Alves JA, Costa J, Rguibi-Idrissi H,
Bearhop S, Piersma T, Granadeiro JP

Functional Ecology
Catry, T., Martins, R.C. & Granadeiro, J.P. (2012b) Discriminating geographic origins of migratory waders at stopover sites: insights from stable isotope analysis of toenails.
Journal of Avian Biology , 43 , 79-84.
Kersten, M., Piersma, T., Smit, C. & Zegers, P. (1981) Netherlands Morocco expedition
1981 – some preliminary results . RIN-report, Texel, The Netherlands.
Logan, J.M., Jardine, T.D., Miller, T.J., Bunn, S.E., Cunjak, R.A. & Lutcavage, M.E.
(2008) Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods. Journal of Animal Ecology , 77, 838-846.
Lourenço P.M., Silva, A., Santos, C.D., Miranda, A.C., Granadeiro, J.P. & Palmeirim
J.M. (2008) The energetic importance of night foraging for waders wintering in a temperate estuary. Acta Oecologica , 34 , 122-129.
Martins, R.C., Catry, T., Santos, C.D., Palmeirim, J.M. & Granadeiro, J.P. (2013)
Seasonal variations in the diet and foraging behaviour of dunlins Calidris alpina in a
South European Estuary: improved feeding conditions for northward migrants. PLoS
ONE , 8 (12) , e81174.
Moreira, F. (1994) Diet and feeding rates of knots Calidris canutus in the Tagus estuary
(Portugal). Ardeola , 82 , 133-136.
Vinagre, C., Salgado, J., Costa, M.J. & Cabral H.N. (2008) Nursery fidelity, food web interactions and primary sources of nutrition of the juveniles of Solea solea and S. senegalensis in the Tagus estuary (Portugal): a stable isotope approach. Estuarine,
Coastal and Shelf Science , 76 , 255-264.
Structure and functioning of intertidal food webs along an avian flyway: a comparative approach using stable isotopes. Catry T, Lourenço PM, Lopes RJ, Carneiro C, Alves JA, Costa J, Rguibi-Idrissi H,
Bearhop S, Piersma T, Granadeiro JP