Genetic data show that Carcharhinus tilstoni is not confined to the tropics, highlighting the importance of a multifaceted approach to species identification.

Abstract

This study shows a range extension for the Australian blacktip shark Carcharhinus tilstoni, which was believed to be restricted to Australia's tropical waters, of >1000 km into temperate waters, revealing its vulnerability to a wider commercial fishery.

Full text

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Journal of Fish Biology (2010) 77, 1165–1172
doi:10.1111/j.1095-8649.2010.02770.x, available online at wileyonlinelibrary.com
Genetic data show that Carcharhinus tilstoni is not
confined to the tropics, highlighting the importance
of a multifaceted approach to species identification
J. J. Boomer*†, V. Peddemors‡ and A. J. Stow*
*Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia and
‡Cronulla Fisheries Research Centre of Excellence, P.O. Box 21, Cronulla,
NSW 2230, Australia
(Received 26 June 2010, Accepted 9 August 2010)
This study shows a range extension for the Australian blacktip shark Carcharhinus tilstoni, which
was believed to be restricted to Australia’s tropical waters, of >1000 km into temperate waters,
revealing its vulnerability to a wider commercial fishery.
Journal compilation © 2010 The Fisheries Society of the British Isles
© 2010 The Authors
Key words: blacktip shark; Carcharhinus limbatus; mtDNA; shark fishery; species distribution.
Sharks are proving to be especially vulnerable to anthropogenic pressures, in part this
is a response to the K-selected natural-history traits that characterize many species
along with pressure from targeted and non-targeted fishing activities (Stevens et al.,
2000). In many cases, species distributions span state and international management
boundaries, across which protection and management processes may vary for any
given species (Last & Stevens, 2009). Therefore, effective conservation and manage-
ment require knowledge of species distributions. Despite the size and notoriety of
sharks, distributions of some species remain uncertain due to limited opportunities
for observation or difficulties with species identification.
Sharks, especially those in the carcharhinid family, can be very difficult to identify
to species level using morphological features (Chan et al., 2003). Distinguishing
features for some species are few, and sharks caught by commercial fishers often
have important features removed (e.g. head and teeth) before being landed ashore,
making reliable identification very difficult (Chan et al., 2003; Last & Stevens, 2009).
The fin trade exacerbates problems with identifying harvested sharks. A solution
has been found with the development of DNA markers for species identification.
Over the last decade, rapid improvements in DNA technology have seen a suite of
markers developed which require only a small sample of tissue for reliable species
identification (Chapman et al., 2003; Greig et al., 2005; Ward et al., 2008). The
use of this technology has resulted in further taxonomic revision and improved
†Author to whom correspondence should be addressed. Tel.: +61 2 9850 8143; fax: +61 2 9850 7972;
email: jessica.boomer@mq.edu.au
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J. J. BOOMER ET AL.
knowledge of shark species distributions (Gardner & Ward, 2002; Quattro et al.,
2006; Corrigan et al., 2008).
There are two species of shark referred to by the common name blacktip shark, the
common blacktip shark Carcharhinus limbatus (M¨ uller & Henle) and the Australian
blacktip shark Carcharhinus tilstoni (Whitley). These species are distinguished by
vertebral counts and forensic methods (Lavery & Shaklee, 1991; Last & Stevens,
2009). Carcharhinus limbatus has a global distribution while C. tilstoni is believed
to be restricted to the tropical waters off northern Australia (Fig. 1) where it occurs
in sympatry with C. limbatus (Last & Stevens, 2009). Blacktip sharks are taken in
commercial fisheries worldwide. It is generally accepted that C. limbatus is taken in
most regions while in northern Australia both C. limbatus and C. tilstoni are taken by
commercial fisheries (Ovenden et al., 2010). At present, no external morphological
features are available to distinguish these species and therefore poor knowledge of
catch rates compromises the development of sustainable fisheries. DNA technologies
provide a solution to this problem (Shivji et al., 2002).
Blacktip sharks are within the top five shark species commercially harvested in
temperate waters of New South Wales (NSW) on the Australian east coast (Macbeth
et al., 2009). Additionally, they are caught in this region by the shark beach meshing
programme, which operates between September and May off 51 popular beaches
between Newcastle and Wollongong (Green et al., 2009a). Historically, blacktip
sharks occurring in NSW waters have been classified as C. limbatus (Green et al.,
2009a). The species identity of blacktip sharks occurring in NSW, however, has
Sydney
N
km
0 312·562512501875 2500
Fig. 1. Currently recognized distribution of Carcharhinus tilstoni ( ), which is known from Thevenard Island
(Western Australia) to Rockhampton (Queensland) along the continental shelf of tropical Australia
(Last & Stevens, 2009). ( ), area from which samples for this study were collected.
© 2010 The Authors
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never been thoroughly investigated. In this study, genetic approaches were used to
identify species of blacktip sharks occurring off Sydney, NSW, Australia.
Tissue samples were collected from sharks caught in the NSW Shark Meshing
(Bather Protection) Program (NSWDPI, 2009) (Fig. 1). DNA was extracted using a
modified salting out technique (Sunnucks & Hales, 1996). Two mitochondrial genes
(COI and ND4) were amplified. The cytochrome oxidase I (COI) gene was ampli-
fied with the primers FishF1Mod 5?ACC AAC CAC AAA GAY ATY GGC AC
3?(modified from Ward et al., 2005) and FishR15?TAG ACT TCT GGG TGG
CCA AAG AAT CA 3?(Ward et al., 2005). The sodium dehydrogenase subunit 4
(ND4) gene was amplified with primers designed in this study MaND4F 5?ACC
MAA AGC YCA CGT WGA AGC 3?and MaND4R 5?TCT TGC TTG GAG TTG
CAC CA 3?. These genes were selected for analysis because they have previously
been successfully applied to distinguish C. limbatus and C. tilstoni (Ovenden et al.,
2010). The COI gene was used to initially screen samples. A total of 54 samples
identified by field observers as some form of carcharhinid shark were screened, 13
of these were identified as C. limbatus and five as C. tilstoni based on COI. As a
prior study suggested that C. limbatus and C. tilstoni may share haplotypes at the
COI gene (Wong et al., 2009), these 18 samples were further investigated using the
ND4 gene to confirm their species identity. For both genes, PCR reactions were
in 15 μL volumes and contained ×1 PCR buffer, 2 mM MgCl2, 0·2 mM of each
deoxynucleoside triphosphate (dNTP), 0·5 μM of each primer, 1 U Taq DNA poly-
merase (Promega; www.promega.com) and 1 μL DNA template. Cycling conditions
consisted of an initial denaturation step at 94◦C for 3 min, followed by a touch-
down PCR with six cycles, decreasing the annealing temperature by 1◦C per cycle.
Denaturation was at 94◦C (30 s), annealing at 60 to 55◦C (1 min) and extension
at 72◦C (1 min). A cycle of 94◦C (30 s), 55◦C (30 s) and 72◦C (1 min) was
then repeated 30 times, followed by a final extension of 72◦C for 5 min. The PCR
product was purified with EXOSAP-IT (USB; www.usbweb.com) and sequenced in
an ABI 377 automated DNA sequencer.
DNA sequences were assembled and aligned in MEGA version 4 (Tamura et al.,
2007) with reference sequences for C. tilstoni and C. limbatus as well as reference
sequences for the graceful shark Carcharhinus amblyrhynchoides (Whitley) and the
bull shark Carcharhinus leucas (M¨ uller & Henle). Carcharhinus amblyrhynchoides
has been shown to be very closely related to C. limbatus and was included in analyses
to ensure this species was not present in the sample set (Ward et al., 2008; Ovenden
et al., 2010). Reference sequences for each of these species were obtained for the
COI region from GenBank (GenBank accession numbers: C. tilstoni, DQ108283;
C. limbatus, EU39862; C. amblyrhynchoides, EF609307 and C. leucas, EF609311).
The reference sequences for the ND4 gene for each species except C. leucas were
obtained from Ovenden et al. (2010) (GenBank accession numbers: C. limbatus,
GQ227272; C. tilstoni, GQ227268; C. amblyrhynchoides, GQ227276). The ND4 ref-
erence sequence for C. leucas was obtained as part of this study. The ND4 and COI
sequences were concatenated for phylogenetic analysis giving a final sequence of
1353 base pairs (bp). Phylogenetic relationships were inferred using the neighbour-
joining (NJ) distance method implemented in MEGA 4 (Tamura et al., 2007) and
the Bayesian method implemented in MrBayes 3.1 (Ronquist & Huelsebeck, 2003).
For the NJ approach, the best model of nucleotide substitution was determined to
be a Tamura–Nei model (TrN) using Akaike information criterion with the software
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J. J. BOOMER ET AL.
FindModel (LANL, 2009), which is an internet-based application of the programme
ModelTest (Posada & Crandall, 1998). Reliability of tree nodes for the NJ tree was
assessed using 10000 bootstrap replicates. The Bayesian approach to tree building
was completed in MrBayes 3.1 (Ronquist & Huelsebeck, 2003) using a GTR + G
substitution model. Four chains were run for 500000 generations and a consensus
tree was constructed. Carcharhinus leucas was used as the out-group for each of
these phylogenetic analyses.
Commercially harvested sharks, particularly species with slow growth and low
fecundity such as the carcharhinids, are in dire need of well-developed management
plans to slow population declines (Field et al., 2009). Of fundamental importance
is the species identification. This study highlights the benefits of applying genetic
approaches to species identification. The results show that the range of C. tilstoni
extends into temperate waters, >1000 km further south than previously known, and
that at least two species of blacktip sharks (C. limbatus and C. tilstoni) occur in
the temperate waters off Sydney, Australia. The topology of the phylogenetic trees
clearly supports the presence of these two species using both the NJ and Bayesian
approaches. Two clades of NSW blacktip sharks belonging to each of the two species
were supported by high bootstrap values in the NJ tree (Fig. 2) and high probabilities
NSW 25
NSW 98
NSW 105
NSW 17
NSW 177
NSW 64
NSW 228
NSW 276
NSW 291
NSW 244
NSW 188
NSW 18
NSW 336
Carcharias limbatus
Carcharias amblyrhynchoides
NSW 23
NSW 154
Carcharias tilstoni
NSW 191
NSW 126
NSW 132
Carcharias leucas
86
99
99
0·01
Fig. 2. Neighbour-joining tree of mitichondrial (mt)DNA sequences. Reference sequences are designated by
species name. Bootstrap values are given where values are >80.
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of clade credibility in the Bayesian tree (>95%). One clade consisting of 13 indi-
viduals grouped with the reference sequence for C. limbatus and the other with five
individuals grouped with the reference sequence for C. tilstoni. No samples from
NSW were found to group with the reference sequence from C. amblyrhynchoides.
Three COI haplotypes were found, two of which were associated with C. limbatus
and one with C. tilstoni. Five ND4 haplotypes were found, two of which were associ-
ated with C. limbatus and three with C. tilstoni. All mutations were single nucleotide
substitutions and predominantly transitions (Tables I and II). Between C. limbatus
and C. tilstoni, 13 fixed differences at ND4 and three fixed differences at COI were
found. Ten of the ND4 characters and two of the COI characters correspond to the
fixed differences identified by Ovenden et al. (2010), therefore supporting the use
of these characters as diagnostic features. The remaining differences were not fixed
between species (Ovenden et al., 2010).
The overall level of sequence divergence between C. tilstoni and C. limbatus fur-
ther supports species identification. For the 1353 bp of sequence data, an average
sequence divergence of 1·5% occurred between NSW samples of C. tilstoni and
C. limbatus. Average sequence divergence between NSW C. tilstoni and the refer-
ence sequence for C. tilstoni was low (0·2%) as was that between NSW C. limbatus
and the C. limbatus reference sequence (0·2%).
Sampling as a part of the NSW shark meshing programme has predominantly been
opportunistic to date and as such accurate predictions about the relative abundance
of these two species in NSW waters are unable to be made. Proportions of each
species collected in this study, however, do suggest that C. limbatus may be more
abundant of the two near Sydney. Because C. limbatus and C. tilstoni differ in
aspects of their growth and reproduction (Last & Stevens, 2009), they may require
different management strategies. As both of these species probably comprise part
of the catch in commercial fisheries in NSW waters, a more detailed study of the
relative composition of these species is urgently required to evaluate the sustainability
of current shark fishing practices.
This work illustrates how difficulties involved with identifying carcharhinids on
the basis of morphology can be resolved with a genetic approach. The finding of
an additional species of carcharhinid in temperate waters off Australia suggests
Table I. Nucleotide substitutions at the cytochrome oxidase I (COI) gene. The position of
each of these diagnostic substitutions has been adjusted so that the base pair count corresponds
to those presented by Ovenden et al. (2010). GenBank accession numbers are given for
reference haplotypes and haplotypes new to this study. New haplotypes identified in this
study are indicated in bold
COI
Accession
number256292394520
n
Carcharhinus limbatus COI 1
C. limbatus COI 2
C. limbatus GenBank
Carcharhinus tilstoni COI 1
C. tilstoni GenBank
CCC
T
G 12
HM231107
EU398625
1
T
T
T
T
A
A
5
DQ108283
n, the number of individuals with each haplotype.
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Table II. Nucleotide substitutions at the sodium dehydrogenase subunit 4 (ND4) gene. The position of each of these diagnostic substitutions has
been adjusted so that the base pair count corresponds to those presented by Ovenden et al. (2010). Carcharhinus limbatus haplotypes are designated
C. lim and Carcharhinus tilstoni haplotypes are designated as C. til. Reference sequences used for construction of phylogenetic trees are designated
C. lim ref and C. til ref. GenBank accession numbers are given for each haplotype. New haplotypes identified in this study are indicated in bold
ND4
Accession
number
76 85 115 211 217 232 259 283 293 298 308 325 358 367 388 463 523 531 550 671 775
n
C. lim 1
HM231104
T
C
C
T
C
T
C
T
C
C
C
C
C
T
A
G
C
A
T
C
C
12
C. lim 2
GQ227273
T
G
1
C. lim ref
GQ227272
T
A
G
C. til 1
GQ227271
T
T
C
C
T
T
T
T
C
C
A
T
G
C
T
T
3
C. til 2
HM231105
C
T
T
C
T
C
T
T
T
T
C
C
A
G
C
T
T
1
C. til 3
HM231106
C
T
T
C
T
C
T
C
T
T
T
C
C
A
G
C
T
T
1
C. til ref
GQ227268
C
T
T
C
C
T
T
T
T
C
C
A
G
C
T
T
n, the number of individuals with each haplotype.
© 2010 The Authors
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taxonomic uncertainty with carcharhinid catches worldwide. Furthermore, this study
demonstrates how scientific benefits can be gained from shark meshing programmes
and fisheries by the low cost practice of collecting and preserving small amounts of
tissue.
We would like to thank D. Reid and the NSW shark meshing observers for the collection of
tissue samples, S. Dixon for assistance in the laboratory, J. Ovenden, J. Morgan and I. Field
for providing sequence data and valuable advice and K. Rowling for useful comments on the
manuscript. This work was funded by NSW Industry and Investment (formerly NSW DPI).
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