Population Genetics of New Zealand Fur Seals Establishing a Framework to Examine Fisheries

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 (Arctocephalusforsteri), 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. forsteriremains 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], theGemmell labutilized 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 size-exclusion column to remove unreacted adapters(Step 4).

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 re-applied to a size exclusion column and fragment sizes of the resulting library are evaluated using a BioRadExperion 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 cross-referencing 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 Arctocephalusforsteri. 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.