Trophic ecology has benefitted from the use of stable isotopes for the last three decades. However, during the last 10 years, there has been a growing awareness of the isotopic biases associated with some pre-analytical procedures that can seriously hamper the interpretation of food webs. We have assessed the extent of such biases by: (1) reviewing the literature on the topic, and (2) compiling C and N isotopic values of marine invertebrates reported in the literature with the associated sample preparation protocols. The factors considered were: acid-washing, distilled water rinsing (DWR), sample type (whole individuals or pieces of soft tissues), lipid content, and gut contents. Two-level ANOVA revealed overall large and highly significant effects of acidification for both delta(13)C values (up to 0.9 per thousand decrease) and delta(15) N values (up to 2.1 per thousand decrease in whole individual samples, and up to 1.1 per thousand increase in tissue samples). DWR showed a weak overall effect with delta(13)C increments of 0.6 per thousand (for the entire data set) or decrements of 0.7 per thousand in delta(15) N values (for tissue samples). Gut contents showed no overall significant effect, whereas lipid extraction resulted in the greatest biases in both isotopic signatures (delta(13)C, up to -2.0 per thousand in whole individuals; delta(15)N, up to +4.3 per thousand in tissue samples). The study analyzed separately the effects of the various factors in different taxonomic groups and revealed a very high diversity in the extent and direction of the effects. Maxillopoda, Gastropoda, and Polychaeta were the classes that showed the largest isotopic shifts associated with sample preparation. Guidelines for the standardization of sample preparation protocols for isotopic analysis are proposed both for large and small marine invertebrates. Broadly, these guidelines recommend: (1) avoiding both acid washing and DWR, and (2) performing lipid extraction and gut evacuation in most cases.

[1] Posidonia oceanica is a widespread coastal Mediterranean seagrass which accumulates in its subsurface large quantities of organic material derived from its roots, rhizomes and leaf sheaths embedded in sandy sediments. These organic deposits may be up to several meters thick as they accumulate over thousands of years forming the matte, whose high content in organic carbon plays a major role in the global ocean carbon cycle. In this study, very highresolution seismo-acoustic methods were applied to image the subsurface features of a P. oceanica seagrass meadow at Portlligat (Cadaqués, Girona, Spain), in the NWMediterranean Sea. Our findings yield fresh insights into the settling of the P. oceanica meadow in the study area, and define with unprecedented detail the potential volume occupied by the matte. A strong reflector, located from 4.3 to 11.7 m depth, was recognized in several seismoacoustic profiles as the substratum on which P. oceanica first settled in the study area. A 3D bathymetric model of this substratum allowed us to reconstruct the Portlligat palaeo-environment prior to the settling of P. oceanica, which corresponded to a shallow coastal setting protected from the open sea. A core drilled in the meadow at Portlligat revealed the presence of a 6 m thick dense matte composed of medium to coarse sandy sediments mixed with plant debris and bioclasts. Radiocarbon datings revealed a constant accretion rate of the matte of about 1

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Abstract. 1. Macroinvertebrate assemblages of temporary ponds are ideal model systems to explore biodiversity patterns and metacommunity ecology. In addition, the study of the environmental variables driving such biodiversity patterns is essential in establishing proper guidelines for the conservation of the singular fauna of temporary ponds, especially since such ponds are vulnerable systems.2. We analysed the macroinvertebrate assemblages and environmental characteristics of 80 ponds spread across the Doñana National Park, SW Spain to (i) analyse macroinvertebrate b-diversity and metacommunity structure; and (ii) discern the main environmental and spatial drivers of these patterns.3. The pond network was highly heterogeneous as temporary ponds were highly variable. Macroinvertebrate b-diversity partitioning showed that species replacement made the greatest contribution to total b-diversity while the contribution of nestedness was small. The macroinvertebrate community structure and b-diversity were similarly driven by: electrical conductivity (and co-variables alkalinity, pH, and ion concentrations), plant richness (and the co-variable pond surface area), maximum depth, marsh, and coastal proximity as well as two spatial descriptors extracted from Moran's eigenvector maps. The spatial descriptors indicated that large interpond distances were involved, suggesting that species dispersal limitations only take place over long distances in the area.4. Those taxa that departed from the general nested pattern, termed idiosyncratic, significantly contributed to the maintenance of high pond network diversity through the species replacement and occurred within particular environmental conditions in the pond network.5. These results reveal that environmental heterogeneity and connectivity are key factors in the preservation of high macroinvertebrate diversity in nested pond networks with high numbers of idiosyncratic species.