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Gubili et al.: DNA from white shark historical samples 239
the approach outlined here allows investigation of Consistent, reproducible amplifications were ob-
genetic changes between historical and contem- tained using DNA extracted from dried finclips
porary local shark stocks and identification of the (GWMD20 and 21), jaw cartilage preserved with lac-
putative origin of individuals — aspects central to quer (GWMD15), and from dried condocranium tis-
estimates of stock viability and assessment of anthro- sue, the oldest sample used in this study (GWMD10,
pogenic impacts. collected in 1885), suggesting that tissue type and
mode of preservation impact amplification success.
Ahonen & Stow (2008) demonstrated successful Amplification of larger products may be possible
DNA extraction and PCR amplification from only with better sample preservation and from species
44% of historical jaws or teeth collected 20 to 40 yr with teeth containing a pulp cavity (Ahonen & Stow
earlier from whaler sharks (family Carcharhinidae), 2008). Whilst recovery of ancient/historical DNA is
grey nurse sharks Carcharias taurus, tiger shark technically difficult, it promises novel and potentially
Galeocerdo cuvier, and school sharks Galeorhinus important data of conservation significance for rare
galeus; teeth of all these species, except the nurse and endangered species (Alter et al. 2012).
shark, possess a pulp cavity, which generally yields
better quality DNA than that recovered from osteo- The origins, population connectivity, and genetic
dentine. From these species they reported a 608 bp diversity of white sharks in the Mediterranean are
mtDNA D-loop sequence derived from amplicons of poorly known. All satellite tagging expeditions to
ca. 700 bp obtained using generic primers designed date have been unsuccessful, and current hypo-
from a contemporary grey nurse shark. In contrast, theses rely on historical capture and sighting data
our approach uses species-specific primers to target (Fergusson 2002). The first genetic study of con-
smaller overlapping amplicons, ensuring product temporary Mediterranean material indicated this
fidelity in most historical, badly adulterated, and population exhibited little genetic diversity and
degraded material from teeth/jaws and tissue. This suggested the ancestors of these sharks came not,
strategy reliably produced 6 amplicons of between as might be expected, from the adjacent Atlantic,
135 and 286 bp for reconstituting a mtDNA D-loop but from distant Pacific stocks — perhaps a conse-
sequence of up to 874 bp from contemporary South quence of an anomalous migratory event (Gubili et
African shark teeth and 749 bp from historical mate- al. 2011). Our recent results do not counter this
rial. Yet the poorer quality and low yield of DNA from view, with 3 haplotypes apparent in the 5 historical
the osteodentine of contemporary white shark teeth Mediterranean samples, 3 individuals sharing the
suggest use of historical teeth may be possible but contemporary Mediterranean haplotype (H2), and
challenging. 2 new haplotypes (H1 & H24) clustering with con-
temporary Pacific sequences. This placement of
In contrast to poor yields from teeth, rehydration of haplotypes obtained from historical material with
historical dried tissue and brittle cartilage gave good an accepted phylogeny derived from contemporary
yields of genomic DNA. Additionally, variable success Mediterranean and Pacific samples supports the
using primers producing larger products was greatly validity of our current methodological approach.
improved by implementation of a semi-nested PCR Notably, 1 historic haplotype (H1) clusters with an
approach, often advantageous when working with Australia/New Zealand haplotype (H9), differing
degraded historical samples. To recover the full spec- by only 2 mutational steps. This is no more distant
trum of haplotypes from historical material for com- than other contemporary Australian haplotypes,
parison with contemporary samples, it was important which raises the intriguing possibility that Medi-
to use additional primers (such as those for D-loop7F), terranean white sharks have multiple, and possibly
to span areas of the sequence which were inaccessible more recent, Pacific founders. However, because
due to the strategy of using a series of small overlap- this analysis is based on less mtDNA D-loop
ping amplicons, where informative polymorphic sites sequence than reported in Gubili et al. (2011), it is
differentiating haplotypes were concealed within impossible to differentiate between the null hypo-
primer sites (e.g. Positions 386 and 451 within D- thesis of H1 evolving in situ in the Mediterranean
loop1R and D-loop3F primers, respectively) (Fig. 1). from founding stock, the most likely explanation
This highlights the importance of designing many for H24, or the alternative hypothesis that it indi-
overlapping primer sets to resolve false positives, cates multiple, and perhaps more recent, migra-
polymorphisms within the primer sequence, and PCR tions from the Pacific. It is notable that these addi-
artefacts, thereby providing sufficient fidelity to legit- tional haplotypes are present in the oldest (19th
imately compare haplotype diversity between histori- century) samples.
cal and contemporary materials.
