Extracting a century of preserved molecular and population demographic data from archived otoliths in the endangered European eel (Anguilla anguilla L.) Dirk G. Schaerlaekens a, ⁎, Willem Dekker b , Håkan Wickström c , Filip A.M. Volckaert a , Gregory E. Maes a a Laboratory of Animal Diversity and Systematics, Katholieke Universiteit Leuven, Charles Deberiotstraat 32, 3000 Leuven, Belgium b Wageningen IMARES, Institute for Marine Resources & Ecosystem Studies, P.O. Box 68, 1970 AB IJmuiden, The Netherlands c Institute of Freshwater Research, Swedish Board of Fisheries, 178 93 Drottningholm, Sweden abstract article info Article history: Received 20 August 2010 Received in revised form 8 December 2010 Accepted 13 December 2010 Available online 8 January 2011 Keywords: Otoliths Age determination Archived DNA Microsatellite genotyping European eel Anguilla anguilla Archived otolith collections represent an invaluable source of information to study demographic and genetic changes in commercially important fish populations. Studies combining both approaches are however rare and reliable extraction of molecular and population demographic data from the same collection of otoliths has never been assessed in the endangered European eel (Anguilla anguilla L.). Here we evaluate various DNA extraction protocols to compare DNA yield, microsatellite amplification success, genotype integrity and precision of age determination for eel otoliths that have been archived for 4 weeks, and for 28 and 48 years. Our results show a high amplification success and an equal genotype integrity for DNA fragments extracted from both recently sampled otoliths and high quality reference DNA tissue. Although historical samples yielded low amounts of DNA, PCR amplification was successful and genotyping reliable for short fragments, but decreased significantly with PCR fragment size. None of the extraction protocols caused physical damage to the otoliths and precision of age determination was high for both treated and untreated otoliths. Hence, the methodology can be applied as a standard for the further joint analysis of past demographic and genetic changes during the last century in the highly exploited European eel and in other fish requiring urgent conservation measures. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Over the past decade, technological improvements for extracting information from archived otolith collections yielded novel insights about demography and population structure to better understand the evolutionary consequences of anthropogenic pressure in wild fish populations (Nielsen and Hansen, 2008). Besides the increasing reliability in aging techniques, it is now feasible to use dried tissue on the rough surface of archived otoliths as a unique source of DNA (Hutchinson et al., 1999). Hence, otoliths of freshwater, anadromous and marine fishes have been analyzed genetically, resulting in several studies contrasting historical with present-day genetic diversity and evaluating the temporal stability of allele frequencies to estimate historical effective population sizes (Nielsen et al., 1997; Hoarau et al., 2005; Poulsen et al., 2006). Nevertheless, the full potential of retrospective information available in historical collections remains underutilized. The joint analysis of past demographic and genetic data lodged in the otoliths holds great potential to assess the evolutionary consequences of natural and human-induced changes on the demography, connectivity and adaptive potential of commercially important fish species (Heath et al., 2007; Nielsen and Hansen, 2008). Extracting DNA from archived otoliths is a challenging task as the DNA is often degraded and therefore more difficult to analyze than modern high quality DNA (Leonard, 2008). Further, DNA extraction from otoliths typically involves incubation of the otolith into a lysis solution containing compounds potentially harmful to its physical structure. Otolith characteristics (shape, thickness, CaCO 3 composi- tion, opacity and transparency) are species-specific and no consensus method applicable for all fish species could be found yet (Heath et al., 2007; Cuveliers et al., 2009; Therkildsen et al., 2010). Since historical otolith collections are limited and fragile, it is of critical importance to test properly how multiple research applications can be combined on the target species without damaging the archived material. The target species of this study is the European eel Anguilla anguilla (L.), a fish with a catadromous life-strategy making it completely dependent on exogenous selective pressures in both the oceanic and the continental environment (Maes and Volckaert, 2007). The species has experienced a sharp decline in both recruitment (Dekker, 2000) and stock (Dekker, 2003) levels and is now listed as critically endangered on the IUCN Red List of Threatened Species (IUCN, 2010). A management framework for the recovery of the European eel was established in 2007 by the Council of the European Union (European Commission, 2007) aiming at increasing the spawner escapement to 40% of its pristine situation. Evaluating this goal, however, is a complex task given the drastic lack of information Journal of Experimental Marine Biology and Ecology 398 (2011) 56–62 ⁎ Corresponding author. Tel.: +32 16 32 45 72; fax: +32 16 32 45 75. E-mail address: dirk.schaerlaekens@bio.kuleuven.be (D.G. Schaerlaekens). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.12.010 Contents lists available at ScienceDirect Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe