Ancient DNA in archaeological fish remains from the South of Portugal  1Landi M, 2Araújo A, 1Costa FO, 3 Oliveira H, 2Oliveira C, 4Morais R, 5Bernardes JP 1Centre of Molecular and Environmental Biology of Minho University; 2Centre of Chemistry of Minho University; 3CIBIO- Research Centre in Biodiversity and Genetic Resources; 4Department of HeritageStudies, FLUP, Porto University; 5UNIARQ – Algarve University INTRODUCTION Ceramic containers such as amphorae were widely used by ancient civilizations [1] to store and/or transport food items such as wine, honey, olive oil, or fish and its derivatives, such as garum [2,3]. Our study seeks to develop a molecular approach for DNA-based identification of fish and shellfish species present in fish-based products recently found in several archaeological sites in Portugal. We intend to take advantage of the known potential of next generation DNA sequencing technologies (NGS) to detect species present in complex assemblages of organisms and in environmental samples, including samples of ancient DNA [4]. A carefully curated reference library of DNA barcodes of southern European fish species [5,6] will be used as a backbone for species identification. Here we report the early steps for our study, consisting on the design and testing of mini-barcode primers for amplification of short fragments of the mitochondrial gene cytochrome oxidase I (COI), the established DNA barcode gene for animal life [8]. The full-length DNA barcode is too long (about 652 bp) for amplification from ancient DNA [7], therefore primers for amplification of shorter barcode segments (mini-barcodes) are required. Successful primers will be then selected for amplification of mini-barcodes from fish-based products, and subsequent sequencing in a NGS platform. Material and Methods Overview of the approach 2. Find appropriate regions for primer design and delimitation of relatively short fragments, preferably with some overlap 3. Conduct an in silico test for the species diagnosis capability of the mini-barcodes delimited by the potential primers selected in 2 4. Test the primer amplification success in i) recently collected samples, ii) old fish tissue samples, iii) fish-based products 5. NGS sequencing of the PCR products obtained from DNA amplification with the selected primers 6. Match NGS sequences obtained with sequence records from the reference library. 1. Select and align an assemblage of sequences representative of the most likely species to be found in fish-based products Results Primer design We selected 170 sequences from the reference library (DNA barcodes (5’ end of mtDNA COI), representing 24 marine fish and 2 marine invertebrate species. Sequences were representative of the marine species reported as target of Roman fisheries and food habits, and showed the highest intra-specific genetic diversity. We used the resultant multi-species alignment (built in MEGA v6) to seek for DNA regions showing the most conserved consensus sequence across taxa, suitable for amplifications multiple-species (Fig. 1). Figure 1 Out of the 652 bp barcoding region, 312 bp were suitable for primer design (116 bp to 428 bp). One reverse primer (M1R) was designed to pair-wise with forward primers of the COI universal primer cocktail [9], so to amplify 115 bp at 5’ end of the DNA barcode. Four additional primer pairs (M2-M5) were designed in the central region of the DNA barcode (158 bp to 428 bp). These 4 primer pairs were planned to be suitable for the amplification of overlapping DNA fragments ranging from 19 bp to 194 bp, in order to build a final DNA construct of 225 bp. Figure 2 In silico analyses of the species- and genus-level diagnostic ability of selected mini-barcodes, obtained from each of the 5 primer pairs (M1-M5). Very short mini-barcodes (19 bp) successfully identified species only in 3.8% of the cases. The highest species-level identification success was obtained when testing the 194 bp mini-barcode (primer M2), returning exact and unique matches for 69.2% of the species. At genus-level , the 51 bp mini-barcode (primer M5) yielded the highest identification success (96.2%). M1 F M2 F M3 F M5 F M2 R M5 R M3 R M4 F M4 R In silico assessment of the diagnostic ability of the mini-barcode sequences for species level identifications The ability of the 5 mini-barcode sequences to uniquely identify each of the 26 selected species was tested by submitting a representative mini-barcode sequence of each species to GenBank BLAST (www.ncbi.nlm.nih.gov/). The percentage of exact and unique matches with the species under analysis, for each of the mini-barcodes, is provided in Fig. 2. In some cases were a unique species match was not retrieved, a genus-level identification was still possible, if the top matches returned by GenBank included more than one congeneric species. Genus Species FUTURE WORK 1. Sample collection About 100 fragments are planned for the analyses, identified as belonging to Roman containers, such as amphorae, used for storage of fish-based products. Samples are available from recent discoveries in PortugaI and from collections stored in national and international Museums. 2. Molecular analyses Ancient DNA from selected fragments will be extracted in a dedicated lab, using ad hoc kit. Primers M1-M5 will be optimized on contemporary DNA extracted from different marine species, and also tested for amplification success in old fish tissue samples and DNA extracts from fragments of amphorae. Upon success rate, primers will be selected for mini-barcodes amplification and sequencing (NGS). 3. Data analyses NGS mini-barcode records will be evaluated for reliability in taxonomic identification, at species and genus level, trough a suite of specific tools implemented in public genetic databases (BOLDSystem; GenBank). Mini-barcodes with successful taxonomic resolution will allow building a reference mini-barcode library.  4. Integration of multidisciplinary studies Molecular, chemical and archaeological information will be integrated. References [1] Hansson MC, Foley BP 2008. J Archaeol Sci 35: 1169e1176; [2] Foley BP, Hansson MC, Kourkoumelis DP et al 2012. J Archaeol Sci 39: 389-398; [3] Oliveira C, Kuzniarska-Biernacka I, Parpot P et al 2013. In: Morais R, Granja H, Cerdan AM Eds. O irado Mar Atlantico. O naufragio Betico Augustano de Esposende (Norte de Portugal): 263-281; [4] Rizzi E, Lari M, Gigli E et al 2012. Genet Evol 44: 21 ; [5] Costa FO, Landi M, Martins R et al 2012. PLoS ONE: 7(4): e35858; [6] Landi M, Dimech M, Arculeo M et al 2014. PLoS ONE: In press; [7] Orlando L, Bonjean D, Bocherens H et al 2002. Mol Biol Evol 19:1920-1933; [8] Hebert PDN, Cywinska A, Ball SL et al 2003. Proc R Soc Lond B 270: 313-322; [9] Ivanova NV, Zemlak TS, Hanner RH et al 2007. Mol Ecol Notes 7: 544-548 ACKNOWLEDGMENTS This work was supported by FEDER through POFCCOMPETE and by national funds from "Fundação para a Ciência e a Tecnologia (FCT)" in the scope of the grants, F-COMP-01-0124-FEDER-010596 and Pest-C/BIA/UI4050/2011. M. Landi’s work was supported by the fellowship Ref: SFRH/BPD/45246/2008 from Fundação para a Ciência e a Tecnologia. C. Oliveira acknowledges FCT his contract under Ciência 2008 program.