Page 3 - DNAfromhistorical
P. 3
Gubili et al.: DNA from white shark historical samples 235
In this study, we report on the first attempt to tine was placed in a 1.5 ml microcentrifuge tube.
extract and amplify mitochondrial DNA (mtDNA) of Osteodentine from each tooth (averaging 0.12 g) was
a series of small overlapping contiguous sequences initially washed, and subsequently crushed to pow-
(Fulton & Stiller 2012) from the osteodentine of der on a Spex 6750 freezer mill (Spex SamplePrep).
contemporary white shark teeth from South Africa.
We have also used this method to recover se- Fragments (0.10 to 0.25 g) of historical tissue and
quences from dried skin and jaw cartilage of Medi- jaw cartilage from Mediterranean, Atlantic and Pa-
terranean and Pacific Ocean white sharks collected cific Ocean white sharks were rehydrated for 24 h in
34 to 129 yr ago. This resource was used to test 1× TE buffer (10 mM Tris pH 7.5, 1 mM EDTA) to re-
whether the prevailing Pacific origin hypothesis duce aerosol contamination and increase rate of di-
(Gubili et al. 2011) of Mediterranean white sharks gestion. A standard phenol extraction protocol (Sam-
can be refuted by sequencing the D-loop of more brook et al. 1989) was employed to obtain genomic
individuals. DNA from all samples, modified by an additional
40 µl of Proteinase K added prior to final overnight
MATERIALS AND METHODS incubation (55°C). Finclip DNA extractions for verifi-
cation of tooth-derived sequences from contempo-
Sample preparation and extraction protocols rary SA individuals were performed in a different
laboratory.
Jaw samples and small pieces of fins were taken
from 3 individuals caught in beach protection nets Contamination controls
(Cliff et al. 1989) at different locations along the east
coast of South Africa (SA; KwaZulu-Natal) between Stringent measures employed in successful ancient/
2004 and 2005 (Table 1). In addition, we assembled a historical DNA projects (Valentine et al. 2008,
collection of museum jaw cartilage and uncurated, McMenamin & Hadly 2012) were instigated to elimi-
dried tissue samples from 7 white sharks collected 33 nate contamination risks. All DNA extractions, in-
to 128 yr ago from the Mediterranean Sea and cluding those from teeth, took place in laboratories
Atlantic and Pacific Oceans (Table 1). free of contemporary shark material, and separate
tools were designated for use on historical tissue and
Teeth (n = 3–4) from each of the South African jaws cleaned with 25% sodium hypochlorite solution be-
were detached, cleaned, and washed overnight with tween samples. Three tissue extractions were per-
Virkon (Day-Impex Limited). After drying, teeth formed for each sample, with the addition of a nega-
were cut by a rotary power tool (FMTC 140HTK, Per- tive control consisting of only reaction reagents. PCR
formance TM). Tool, cutting parts, surfaces, and vice took place in a separate DNA-free location and in-
were washed with 70% ethanol and 10% sodium cluded 2 negative controls. Replicate amplifications
hydroxide. Enamel was removed, and the osteoden- (n = 3–5) were performed for each sample, of which 2
Table 1. Carcharodon carcharias. Details of each sampled South African white shark (supplied by the KwaZulu-Natal Sharks
Board), and historical material (obtained from museum and private collections). Tissue type used in analysis is also provided.
Haplotype refers to the 749 bp partial mitochondrial DNA D-loop sequence haplotype referred to in Fig. 2. F: female; M: male;
TL: total length; −: unknown
Sample ID Capture date Capture location Sex TL (mm) Mass (kg) Tissue type Haplotype
Contemporary samples Durban, South Africa F 2210 100 Teeth & finclip −
DUR04039 4 Nov 2004 Richards Bay, South Africa M 2598 170 Teeth & finclip −
RB05086 24 Oct 2005 Ispingo, South Africa M 2570 134 Teeth & finclip −
ISP05004 8 Sep 2005
−
Historical samples −
~6000
GWMD3 1 May 1900 Atlantic − − − Jaw cartilage −
−
GWMD10 1885 Port Jackson, NSW, Australia M − − Chondocranium H20
−
GWMD11 10 Dec 1891 Liguria, Monterosso, Italy F − Jaw cartilage H24
GWMD12 1900 Toscana, Lucca, Viareggio, Italy F − Finclip, full specimen H1
GWMD20 29 May 1953 Tunara, Favignana, Italy F − Dried fin, trophy board H2
GWMD21 4 Apr 1980 Tunara, Favignana, Italy F − Dried fin, trophy board H2
GWMD15 − Palermo, Sicily, Italy − − Jaw cartilage H2
