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Gubili et al.: DNA from white shark historical samples 237
cluding 4 contemporary Mediterranean sequences primer sets. D-loop2 was initially used on historical
(HQ540294 to HQ540296,Gubili et al. 2011; JF715925) material, but discarded due to the poor quality of the
and 91 sequences from worldwide locations sequence produced. No correlations between the age
(AY026196 to AY026224, Pardini et al. 2001; GU00 of sample and semi-nested PCR success rate were
2302 to GU002321, Jorgensen et al. 2010; HQ414073 detected for each primer pair (R = −0.356 to 0.373).
to HQ414086, Blower et al. 2012; KC914387, Chang et All forward and reverse sequences were identical
al. 2014; KC511601 to KC511626). These specifically and confirmed as Carcharodon carcharias by BLAST
included sequences from proximal populations (such searches. Low-quality amplification for the D-loop4
as South Africa and North West Atlantic) which may amplicon from the historical Atlantic sample (GW
be considered as potential source populations for the MD3) meant it was excluded from the final analysis.
Mediterranean. Base pair positions with gaps/missing However, when a smaller segment (510 bp) of se-
data were excluded from analysis. Haplotypes, haplo- quence was analysed, this sample displayed a unique
typic diversity, and average pairwise sequence differ- haplotype found within the Atlantic/South African
ences were obtained using DnaSP 5.10.1 (Librado & grouping (data not shown). Assembled contigs from
Rozas 2009). A haplotype genealogy was constructed each of 6 remaining historical samples produced in
in HAPVIEW following the method of Salzburger et each case a 749 bp partial sequence of the mtDNA
al. (2011) using a phylogenetic tree derived in PhyML control region (Fig. 1).
v3.0 (Guindon & Gascuel 2003, Guindon et al. 2010)
following 10 000 bootstraps using GTR+G+I as the The historic samples were aligned to the 96
evolutionary model inferred by JMODELTEST known contemporary white shark mtDNA se-
(Posada 2008). quences available in GenBank, revealing 88 poly-
morphic sites distinguishing 55 different haplo-
RESULTS types. These haplotypes were assembled into a
network with 2 main lineages separated by a mini-
DNA amplification from contemporary teeth mum of 30 nucleotide substitutions: one lineage
was composed mainly of North West Atlantic and
D-loop PCR products were recovered for each South African sequences, while the other included
SA individual, although not all amplifications were all Pacific haplotypes (Fig. 2). Contemporary Medi-
equally successful. All sets of primers yielded a terranean samples (n = 4; GenBank HQ540294 to
PCR product of the expected size in at least 1 indi- HQ540296, Gubili et al. 2011; JF715925) exhibited
vidual. PCR using primer sets D-loop1, D-loop2, a single haplotype (H2), shared with 3 historical
and D-loop4 yielded a product of the expected size Mediterranean samples (GWMD15, 20, and 21;
in all samples (100%). Larger fragments produced Fig. 2). Two additional historical Mediterranean
by D-loop3 (286 base pairs [bp]) and D-loop5 haplotypes (H1, GWMD12, Toscana, Italy; H24,
(245 bp) primers were successful in only 1 (Durban) GWMD11, Monterosso, Italy) were identified from
in 3 samples (33.33%), indicating that amplification single individuals. Mediterranean haplotypes show-
success is dependent upon size of the target frag- ed little differentiation from those of Pacific sharks;
ment due to the poor quality of template DNA (p < for example, only 3 mutational steps separate the
0.05; X227 = 40.4). When aligned, the 5 overlapping common Mediterranean haplotype (H2) from the
sequences amounted to 874 bp (from Position 268 Northeast Pacific/Australia/New Zealand (H19)
to 1142) of the D-loop. Sequences obtained from haplotype, and 6 steps separate it from the South-
tooth samples were identical to those generated west Pacific (Taiwan) haplotype (H6) (Fig. 2). The
from the fin tissues. newly described historical Mediterranean haplotype
H24 was separated from the common Mediterran-
MtDNA amplification from historical material ean haplotype (H2) by only 3 mutational steps, and
from Northeast Pacific/Australia/New Zealand
DNA was extracted from 7 historical samples (H19) and Southwest Pacific (Taiwan) haplotypes
(Table 1). The semi-nested PCR protocol was 74% (H6) by 4 steps and 7 steps, respectively (Fig. 2).
successful across all amplicons, improving on the However, the historical Mediterranean haplotype
much lower success rate (26%) and poor repro- H1, while separated by 6 mutational steps from the
ducibility of non-nested reactions across all D-loop common Mediterranean haplotype H2, was only 2
steps removed from contemporary Australian/New
Zealand sequences (H9); placing it firmly with
contemporary Pacific haplotypes. Estimates of ave-
