title: Within-otolith variability in chemical fingerprints: implications for sampling designs and possible environmental interpretation.
abstract: Largely used as a natural biological tag in studies of dispersal/connectivity of fish, otolith elemental fingerprinting is usually analyzed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). LA-ICP-MS produces an elemental fingerprint at a discrete time-point in the life of a fish and can generate data on within-otolith variability of that fingerprint. The presence of within-otolith variability has been previously acknowledged but not incorporated into experimental designs on the presumed, but untested, grounds of both its negligibility compared to among-otolith variability and of spatial autocorrelation among multiple ablations within an otolith. Here, using a hierarchical sampling design of spatial variation at multiple scales in otolith chemical fingerprints for two Mediterranean coastal fishes, we explore: 1) whether multiple ablations within an otolith can be used as independent replicates for significance tests among otoliths, and 2) the implications of incorporating within-otolith variability when assessing spatial variability in otolith chemistry at a hierarchy of spatial scales (different fish, from different sites, at different locations on the Apulian Adriatic coast). We find that multiple ablations along the same daily rings do not necessarily exhibit spatial dependency within the otolith and can be used to estimate residual variability in a hierarchical sampling design. Inclusion of within-otolith measurements reveals that individuals at the same site can show significant variability in elemental uptake. Within-otolith variability examined across the spatial hierarchy identifies differences between the two fish species investigated, and this finding leads to discussion of the potential for within-otolith variability to be used as a marker for fish exposure to stressful conditions. We also demonstrate that a 'cost'-optimal allocation of sampling effort should typically include some level of within-otolith replication in the experimental design. Our findings provide novel evidence to aid the design of future sampling programs and improve our general understanding of the mechanisms regulating elemental fingerprints.
authors.affiliation: Université de Nice Sophia-Antipolis, Faculté des Sciences, EA 4228 ECOMERS, Nice, France; Laboratory of Conservation and Management of Marine and Coastal Resources, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), University of Salento-CoNISMa (Consorzio Nazionale Interuniversitario per le Scienze del Mare), Lecce, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Di Franco
authors.affiliation: Dipartimento di Biologia, Università di Pisa, Pisa, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Bulleri
authors.affiliation: Laboratorio di Spettrometria di massa analitica ed isotopica, Dipartimento di Beni Culturali, University of Salento, Lecce, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pennetta
authors.affiliation: Laboratorio di Spettrometria di massa analitica ed isotopica, Dipartimento di Beni Culturali, University of Salento, Lecce, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: De Benedetto
authors.affiliation: Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth, United Kingdom.
authors.forename: K Robert
authors.initials: KR
authors.lastname: Clarke
authors.affiliation: Université de Nice Sophia-Antipolis, Faculté des Sciences, EA 4228 ECOMERS, Nice, France; Laboratory of Conservation and Management of Marine and Coastal Resources, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), University of Salento-CoNISMa (Consorzio Nazionale Interuniversitario per le Scienze del Mare), Lecce, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Guidetti
mesh_terms: D063147:Animal Distribution; D000818:Animals; D004777:Environment; D005399:Fishes / Q000502:physiology; D019083:Mediterranean Region; D010037:Otolithic Membrane / Q000737:chemistry*; D062206:Spatial Analysis