Cytochrome B diversity in the New Zealand fur seal (Arctocephalus australis forsteri)
Student: Alexander Salis
Supervised by: Professor Jon Waters and Dr Nic Rawlence
Introduction:
Arctocephalus australis forsteri, the New Zealand fur seal is considered to be a subspecies of
the southern fur seal (Arctocephalus australis) with breeding colonies distributed from
Western Australia to New Zealand, as well as on some Subantarctic islands (Yonezawa et al.,
2009; Berta and Churchill, 2012). Like all pinnipeds, New Zealand fur seals are important
marine predators; due to high abundances in certain localities and high-energy requirements,
they are considered an important component of marine ecosystems (Bowen 1997). Although
they are capable for dispersing over long distance, studies show females are largely
philopatric, returning to their birthplace for breeding (Reidman, 1990).
Like most fur seals, they underwent severe population bottlenecks due to human exploitation
in the 18th and 19th centuries (Smith, 2002), however pre-sealing population reduction
(especially in the north of new Zealand) has also been attributed to human settlement by
polynesians in New Zealand about 750 years before present (Smith, 2005). Cessation of
sealing in the 19th century has allowed significant, although slow, population recovery
(Shaughnessy et al., 1988; Gerber and Hilborn, 2001). Severe bottlenecks, such as those
caused by human exploitation have been shown to have severe consequences on genetic
variation and population structure in pinnipeds (Hoelzel, 1999; Weber et al., 2004).
This studentship was undertaken to examine the current mitochondrial variation of the
cytochrome B gene of New Zealand fur seals populations across Australia, New Zealand and
the Subantarctic and whether substantial geographic structures are present. It was predicted
that there would be significant geographic structure across the populations due to the
philopatric nature of the females.
Methods:
A 683bp fragment of the cytochrome B mitochondrial gene of 218 previously chelexextracted DNA samples of Arctocephalus australis forsteri from across the Subantarctic, New
Zealand, Tasmania and mainland Australian coast (Appendix 1) were amplified using the
primers Afos-tRNA-Glu-F12 (5’-GGAATTAACCAAGACCAATG-3’) and Afos-Cytb-R602
(5-TGGGTTATTGGATCCTGTTTCAT-3’). Each PCR reaction contained 0.3µL of MgCl2
(25mM), 2µL of Bovine Serum Albumin (10mg/ml), 1µL of 10x NH4 PCR buffer (BioLine),
1µL of dNTPs (25mM each), 0.1µL of BioLine BIOTaq DNA polymerase (BioLine), 0.5µL
of each primer (10µM), 1µL of DNA and 3.6µL of ddH2O. PCR cycling conditions were an
initial 94°C step for 2 min followed by 35 cycles of 94°C for 40s, 52°C for 40s, and 72°C for
60s, with a final extension step of 72°C for 5 min.
PCR products were visualised on 1.5% agarose SYBR safe gel to ensure that amplification
was successful. If amplification was unsuccessful, first the amount of DNA added to the PCR
reaction was increased to 2µL and if this was also unsuccessful, 1µL of a 1 in 10 dilution of
the DNA sample was added to the PCR reaction. Eventually 217 of the 218 samples were
successfully amplified. Successfully amplified PCR products were then purified using
EXOSAP (1.5 units of Exo1, 1 unit of SAP) by incubation at 37°C for 15mins and 80°C for
15mins. Following EXOSAP two sequencing reactions for each sample (one for each primer)
were setup with 3.2pmol of primer and 1ng/100bp (~7ng) of PCR product, adjusted to a
volume of 5µL with ddH2O. Sequencing reactions were sent for sequencing by to Genetic
Analysis Services (Department of Anatomy, University of Otago) in the automated ABI
3730xl DNA Analyser.
Of the 217 samples sent for sequencing, 214 (98.6%) returned readable sequence from either
the forward or reverse primer, or both. Sequences were edited in 4Peaks resulting in 642bp of
sequence and were confirmed to be Arctocephalus australis forsteri using NCBI’s Basic
Local Alignment Search Tool (BLAST). Sequences were aligned by Clustal W in MEGA
5.2.2 and consensus sequences between the forward and reverse primers for each sample were
formed. Haplotype networks were constructed by a statistical parsimony approach in
TCS1.21. Initially with 95% links were implemented, followed by a fixed connection limit at
18 steps to decipher the relationship between the two divergent clades.
Results:
Considerable genetic variation was observed with in the Arctocephalus australis forsteri
cytochrome B mitochondrial gene with 20 haplotypes across 40 variable sites observed
(Figure 1). These 20 haplotypes appeared to fall into two divergent clades separated by 16
mutations between the two most similar sequences between the two clades. The first clade
consisted of 8 haplotypes (H-1 to H-8) represented by 35% of all sequences (Figure 1). The
most common haplotype in this clade was found in New Zealand and Subantarctic samples, a
further five were found exclusively in the New Zealand samples and the remaining two
haplotypes found in the New Zealand and Tasmanian samples. The second clade consisted of
12 haplotypes (H-9 to H-20) representing 65% of the sequences and found across all four
broad regions sampled (Figure 1). The most common haplotype, H-1, was found in 30.7% of
all sequences and was predominately found in New Zealand samples with one example found
in Tasmania. A further four haplotypes were exclusively found in New Zealand samples, one
exclusively Subantarctic, one exclusively Tasmanian and another exclusively found in
mainland Australian samples. The remaining three haplotypes were a mix between mainland
Australia and New Zealand, mainland Australia and Tasmania, and New Zealand and
Tasmania. New Zealand, Tasmanian and Subantarctic samples were found across both of the
clades while Australian samples were only found in the larger second clade (Figure 1).
Figure 1: Haplotype network diagram for 642-bp cytochrome B sequences of Arctocephalus australis
forsteri. Circles represent haplotypes and sizes are scaled according to frequency. Coloured
segments represent the distribution of the haplotypes among four broad sampling regions; New
Zealand (NZ), Subantarctic (SubAnt), western and southern Australia (Aus) and Tasmania (Tas).
Vectors between haplotypes represent single nucleotide substitutions and open circles represent
hypothetical, unsampled haplotypes.
New Zealand samples showed greater diversity than the other geographic areas with 16 of the
20 haplotypes found (Figure 2). Of the New Zealand sequences, 37.5% fall into the first clade
and 62.5% in the second clade. Tasmania showed the next greatest diversity with 6
haplotypes represented across both clades (20% of sequences in the first clade, 80% in the
second). The mainland Australian sequences fell into 3 haplotypes, all in the second clade and
the Subantarctic samples fell into two haplotypes, one from the first clade (57% of sequences)
and one from the second clade (43% of sequences) (Figure 2).
Figure 2: Haplotype frequencies in four broad regions across Australia, Tasmania, New Zealand and
the Subantarctic. Haplotypes were determined from a 642-bp sequence from the mitochondrial
cytochrome B gene of Arctocephalus australis forsteri. Each haplotype is represented by a different
colour.
Discussion:
The presence of two divergent clades of cytochrome B haplotypes is consistent with previous
investigations of cytochrome B diversity in Arctocephalus australis forsteri (Lento et al.,
1997; Berry et al., 2012). Lento et al. (2007) found that one of the clades consisted of
sequences from Tasmania, New Zealand and the Subantarctic, which was similar to the first
clade in this study. The other clade they found consisted of sequences from Western
Australia, Tasmania and New Zealand, and showed greater haplotype diversity. This was
similar to the second clade found here, except that an exclusive Subantarctic clade was found,
whereas Lento et al. (1997) found no Subantarctic representatives in this clade. Berry et al.
(2012) also reported two divergent clades, with one clade restricted to New Zealand and
Tasmania, the other more diverse clade was found across Western and Southern Australia,
Tasmania and New Zealand (the Subantarctic was not included in this study).
The observation that New Zealand colonies show much greater haplotype diversity with
mainland Australia and the Subantarctic showing the least diversity, may be simply a function
of sample size, as the number of samples from New Zealand greatly outnumbers that of all
other locations. However, the observation that New Zealand and Tasmania show more
haplotype diversity that mainland Australian colonies is consistent with the findings of Berry
et al. (2012), where the sample size from mainland Australian colonies greatly outnumbered
those of Tasmania or New Zealand. This is also reflected in that Australian sequences have
only been found in one of the two divergent clades (Lento et al., 2007; Berry et al., 2012).
However, the lower haplotype diversity observed in the Subantarctic samples is likely to be
an underestimate due to low sample size, considering that of the two haplotypes observed in
this region, one is derived from each of the two divergent clades. Further sampling in the
Subantarctic would help increase resolution of haplotype diversity in this locality.
Although there appears to be geographic structure in haplotype make-up and diversity of each
location, the most striking pattern seen is the presence of the two divergent clades, with
Australian samples only represented in one clade. This implies a historical geographic
separation and the current admixture a result of secondary contact. This secondary contact
may be represented by post-sealing recolonization as suggested by Lento et al. (1997), or the
multiple dispersal events as proposed by Berry et al. (2012). Sampling of historical/ancient
specimens would be required to explain the current state of haplotype diversity by giving
insight to pre-sealing population structure.
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Appendix 1.
Sample information (specific location, broad region, identifier, number of samples and how
many samples readable sequence was obtained from).