Population Genetics of New Zealand Fur
Seals – Establishing a Framework to
Examine Fisheries Bycatch
Allan Wilson Centre Summer Studentship Report 2013-2014
Will Stovall
Supervisor: Neil Gemmell
Introduction
The New Zealand fur seal (Arctocephalus forsteri), a member of the mammalian order
Pinnipedia, is a relatively common feature of rocky coasts around the South Island of New
Zealand. The species is showing strong recovery following exploitation to near extinction in the
19th century, but still faces several direct and indirect threats from humans. In the last few
decades, the threat of fisheries bycatch has dramatically increased in scope, and of all marine
mammals in New Zealand, A. forsteri remains the most frequently caught by commercial net
fisheries. Populations on the West Coast of the South Island appear most adversely affected by
this pressure, where population growth has been low or declining for much of the last decade.
In a 2005 study[1], the Gemmell lab utilized microsatellite markers to develop an
understanding of fur seal population structure, immigration/emigration frequency, and the
relationships among individuals within populations. Although this experiment imparted some
understanding of the genetic structure of extant populations, and was able to assign bycaught
individuals to broad geographic regions, it is likely that more modern genetic analysis methods
could reveal further information. Our current project principally focuses on the utilization of
single nucleotide polymorphisms (SNPs) to assess population structure.
New sequence-based approaches, such as genotyping-by-sequencing (GBS), could
provide a more efficient and economical means of obtaining genotypic information than previous
SNP chip technologies have offered. The GBS approach primarily focuses on the construction of
libraries through reduction of genome complexity using restriction enzymes. With the
emergence of next generation sequencing methods, it is possible to perform such procedures on
high-diversity, large-genome species. We aim to explore the utility of this technology for
investigating New Zealand fur seal population dynamics.
Methods
The samples we will utilize in evaluating population structure were collected as part of
the Robertson and Gemmell ’05 study from six South Island colonies and one colony in the
Wellington region of the southern North Island (See [1] for names and geographic locations). At
the current phase of the experiment, several DNA extractions have been performed in addition to
concentration checks, gel electrophoresis, and restriction enzyme digestions. Herein I describe
the procedures performed thus far, followed by an explanation of the GBS methods to be
employed in future stages of the project.
DNA Extractions & Electrophoresis
DNA was extracted from twelve tissue samples obtained from Ohau Point near
Kaikoura in accordance with the Gemmell and Akiyama[2] lithium chloride DNA extraction
protocol. This exercise was performed primarily as practice for future extractions and
digestions. The tissues used in this analysis were liver, lung, blood, and feces (3 of each)
obtained from a single deceased pup. After extraction was complete, the DNA
concentration of the samples were checked using a NanoDrop system. Blood and fecal
samples yielded very low concentrations, while liver and lung samples showed promising
amounts of nucleic acid material. Electrophoresis revealed, however, that all samples had a
high percentage of degraded, low molecular weight DNA (likely due to samples being
collected post-mortem).
To counteract degradation, the next round of extractions was performed on skin
samples obtained from live individuals at Victory Beach on the Otago Peninsula. A total of
thirty-six extractions were conducted, and concentrations were evaluated using a Qubit
System rather than NanoDrop. All of these samples yielded relatively high DNA
concentrations (≈600ng/mL), and high molecular weight fragments were revealed upon
electrophoresis.
Restriction Enzyme Digestions
The genotyping-by-sequencing protocol to which we aim to adhere is derived from a
2011 study performed by Rob Elshire and colleagues[3]. The authors of this study were the
first to design and apply the GBS approach, and specifically discuss within their publication
the application of GBS to high-diversity species. Though several of their experiments were
performed on plants using the restriction enzyme ApeK1, they outline that the enzyme
PST1 is especially suitable for digest of mammalian samples. Thus far, the twelve deceased
pup samples have been digested using this enzyme and have cut relatively well despite
being degraded. The next item on the agenda is to perform this procedure on the thirty-six
skin samples from Victory Beach.
GBS Methodology
In late February of this year, I attended a GBS conference in Palmerston North
accompanied by Ma Lei and Kim Rutherford, two bioinformaticians from the Gemmell Lab.
The workshop was led by Rob Elshire and the Cornell group, and was geared toward
demonstrating the applications of the method and educating individuals who hope to
employ GBS in their research. This section focuses on the series of steps that compose
genotyping-by-sequencing which will be executed in later stages of our project. A graphic
representation of this process can be observed in Figure 1 (Obtained from Elshire et al.
2011).
After samples from each of the seven colonies have been plated and digested with
the PST1 enzyme (Steps 1&2), specific barcode and common adapters, which allow for
recognition of fragments during sequencing, will be ligated to either end of the genomic
DNA fragments(Step 3). The samples are then “cleaned-up” by application to a sizeexclusion column to remove unreacted adapters(Step 4).
Figure 1: Steps in GBS Library Construction (Obtained from Elshire et al. 2011)
After unreacted adapters have been removed, appropriate primers are added and
PCR is performed to increase the fragment pool (Step 5). Lastly, the PCR products are reapplied to a size exclusion column and fragment sizes of the resulting library are evaluated
using a BioRad Experion or similar instrument (Step 6&7). These final fragment sizes can
subsequently be sequenced and mapped, allowing for the development of population
profiles for each specific fur seal colony.
Discussion
In their 2011 and subsequent studies, Elshire and the Cornell group demonstrate
that GBS is highly reproducible, and can reach previously inaccessible regions of the
genome. They also assert that the approach is exceptionally useful for conservation
studies, as it can help infer population structure in the absence of a reference genome or
prior knowledge of diversity in the species[3]. In our study, pinpointing the origin of
bycaught individuals may provide insight into which specific colonies are most threated by
commercial bycatch, the rate of individual displacement due to competitive interactions,
and the genetic similarity within and between populations.
Through constructing population profiles of each A. forsteri colony and crossreferencing these data with the DNA of bycaught individuals, it may be possible to establish
a direct link between population growth decline in certain colonies and commercial
fisheries. If successful, this research may provide incentive for further genetic evaluation
of New Zealand fur seals, and pave the way for similar studies on more endangered species
of Pinnipeds.
References
[1] Robertson, B. C., & Gemmell, N. J. (2005). Microsatellite DNA markers for the study of
population structure in the New Zealand fur seal Arctocephalus forsteri. New Zealand
Department of Conservation.
[2] Gemmell, N. J., & Akiyama, S. (1996). An efficient method for the extraction of DNA from
vertebrate tissues. Trends in Genetics, 12(9), 338-339.
[3] Elshire, R. J., Glaubitz, J. C., Sun, Q., Poland, J. A., Kawamoto, K., Buckler, E. S., & Mitchell,
S. E. (2011). A robust, simple genotyping-by-sequencing (GBS) approach for high
diversity species. PloS one, 6(5), e19379.