Comparison and optimization of genetic tools used for the identification of ancient fish remains recovered from archaeological excavations and museum collections in the Mediterranean region

Abstract

Among the many fish species commercially exploited since prehistoric times, Atlantic bluefin tuna (Thunnus thynnus) is one of the most economically significant, having left an indelible imprint on several civilizations including the Phoenicians, Greeks, and Romans. Here, we describe our efforts to identify tuna specimens among the remains of 345 fish vertebrae and bones in several large collections from the Atlantic Ocean, Mediterranean Sea, and Black Sea, dating from the Late Iron Age (2nd century BCE) to the early 20th century (1911–1927). Unfortunately, ancient fish specimens are often mislabelled, which can cause a great deal of confusion among zoologists. Protocols were developed and optimized to overcome the unique challenges related to the compromised integrity of genetic material preserved in ancient bones. Three DNA isolation protocols were compared to maximize yields, and as reported for other faunal remains, a silica spin column‐based method was proven most effective. Endogenous DNA was successfully extracted from the majority of bones and amplified using polymerase chain reactions (PCRs) and an assortment of four primer pairs targeting nuclear (internal transcribed spacer) and mitochondrial sequences (cytochrome oxidase subunit 1 and control region). Protocols targeting mitochondrial markers were more successful than those focused on nuclear targets. Due to the restricted length of the extracted DNA molecules, character‐based keys containing diagnostic nucleotide substitutions were defined and used to identify 231 samples to genera, of which 171 were identified to species level. The success rate of assignment of specimens to species level varied between location and collection, reflecting variation in DNA preservation between different sites and environments. The methods detailed herein can be used to identify other ancient fish specimens and provide information about historical human diets, trade, species distribution, and biodiversity. The same tools can be applied to the analysis of processed food items with highly damaged DNA.

Ancient DNA SNP-panel data suggests stability in bluefin tuna genetic diversity despite centuries of fluctuating catches in the eastern Atlantic and Mediterranean

“…Assuming the availability of a reference genome, WGS approaches are increasingly cost-effective 9 , particularly for shallow sequencing 102 . This approach may also facilitate the recovery of data from arid Mediterranean specimens which were challenging to genotype herein due to their poor preservation 46 . Importantly, WGS would reduce ascertainment bias compared with SNP-genotyping.…”

“…Quantification was performed Ancient DNA extractions. Archival and archaeological samples underwent ancient DNA (aDNA) extraction in sterile, PCR-free conditions at the Ancient DNA Laboratory of the Department of Cultural Heritage (University of Bologna, Ravenna Campus, Italy), as part of another study which investigated their species identification via barcoding 46 . All bone specimens were sprayed with 1-2% sodium hypochlorite (bleach), left to soak for ten minutes, rinsed with distilled water and air-dried (as per 47 ).…”

Exploitation history of Atlantic bluefin tuna in the eastern Atlantic and Mediterranean—insights from ancient bones

“…Molecular identification methods, i.e. Zooarchaeology by Mass Spectrometry (ZooMS) collagen protein fingerprinting (Rick et al, 2019) or genetic barcoding (Puncher et al, 2019) were not utilized by any of the studies included, therefore, we assume that these records are in fact BFT but urge caution in their future use and interpretations.…”

“…The preser-vation of ancient DNA is of most concern among the techniques proposed. Within fish remains, it is variable (Oosting et al, 2019) and has proved to be poor in BFT bones from arid Iberian sites (Puncher et al, 2019). This may limit the use of poorly preserved remains to demographic analyses only using mitochondrial DNA (Oosting et al, 2019), which is a low-resolution marker for mod-ern population assignment (Carlsson et al, 2004;Boustanyet al, 2008) but may offer utility toward genetic diversity changes.…”

“…Fi-nally, studying growth rate changes from vertebrae is challenging, but it is still promising for the large vertebrae of BFT (Van Neer et al, 1999). As a minimum, future studies engaged in BFT zooar-chaeology would benefit by applying ZooMS (Rick et al, 2019), or genetic barcoding (Puncher et al, 2019) to provide reliable species identifications from bone and scales.…”