AWC Summer Project Completion Report. By Charlotte Till, University of Otago.

Project Title:

Genetic investigation into New Zealand blue eyed shags and other coastal birds.

Over the period Nov 14th 2011 through to Feb 4th 2012 a genetic investigation was undertaken into modern and prehistoric shag samples from New Zealand, and Sub Antarctic and South America. This work followed on from previous research undertaken in 2011 which resulted in my genetics honours level dissertation. As such I already had all primer pairs that would be needed and had a solid grasp on the research area.

In order to address the aims of my summer project new modern shag samples were obtained from Te Papa as well as from Martyn Kennedy. Ancient shag samples were obtained from Paul Scofield and collected on a recent field trip to the Nelson region. A summary of samples investigated over the summer can be seen below.

Sample Type / Shag Name / Obtained from / Identifier
Modern / Falkland Island / Falkland Island 2008 / Falk
Modern / Stewart Island shag / Karitane, Otago, 2011 / SIS K
Modern / Stewart Island shag / Warrington Beach, Otago, 2011 / SIS W
Modern / Guanay shag / Martyn Kennedy / GBN41
Modern / Kerguelen shag / Te Papa 1985 / 89/a
Modern / Kerguelen shag / Te Papa 1985 / 11/b
Modern / Kerguelen shag / Te Papa 1985 / 12/b
Modern / Kerguelen shag / Te Papa 1985 / 79/a
Ancient / Labelled as King shag / Paul Scofield, Red Cliffs, Canterbury / AV158036
Ancient / Labelled as King shag / Paul Scofield, Red Cliffs, Canterbury / AV16267
Ancient / Labelled as King shag / Paul Scofield, Wairau Bar, Marlborough / AV10995
Ancient / Labelled as King shag / Paul Scofield, Marfells Beach, Marlborough / AV 3042
Ancient / Labelled as King shag / Paul Scofield, Marlborough / AV12878
Ancient / Labelled as King shag / Paul Scofield, Doubtless Bay, Northland / AV6564
Ancient / Labelled as King shag / Catherine Collins & Jon Waters, Delaware Bay, Nelson / Broken
Ancient / Labelled as King shag / Catherine Collins & Jon Waters, Delaware Bay, Nelson / Whole

Note: Samples from Paul Scofield are from Canterbury Museum. A distribution map of

ancient samples and Stewart Island shag distribution is shown on page five.

All modern samples were extracted using the DNeasy extraction method and PCR was carried out using a selection of primers previously used to amplify across Control Region one of the mitochondrial genome (Till, 2011). The Falkland Island and two Stewart Island shag samples were the only modern samples to be successfully amplified and sequenced. Despite fresh extractions being carried out using both the DNeasy method and standard Chelex method no further modern samples (i.e., the Guanay and Kerguelen shags) were able to be successfully amplified for any primer combinations tested.

All ancient samples were extracted under strict aDNA protocols and conditions in the aDNA lab (Anthropology Department, University of Otago). The two Red Cliff samples and the Wairau Bar sample were successfully amplified using overlapping aDNA primer pairs (designed during my honours) and successfully sequenced. The sequencing results were confirmed by independent replication. Of the remaining ancient samples amplification for one of the primer pairs has been successful for three samples (13042, AV6564 & AV12878) but despite one attempt this result has yet to be successfully replicated, so sequencing of these samples was not undertaken. Access to the aDNA lab is highly restricted and time constraints with other lab users limited the amount of time that could be spent there. In the coming year I aim to try to get successful replication of these samples and later sequence them.

Results

The two Stewart Island shag samples fit within the northern cluster of Stewart Island shags as was predicted (see Figure 1). The Falkland Island sample also fits nicely into the South American / Sub Antarctic group.

Of the three successfully sequenced ancient samples all were labelled as King shag, but sequencing of these samples reveals that AV158036 and AV10995 are Stewart Island shag remains and that AV16267 is most likely a Pied shag (see Figure 2; samples with the same haplotype have been collapsed to representative samples in this tree).

Conclusions

The results of the aDNA portion of my summer research are two fold. 1) Physical identification of bones can be flawed resulting in misrepresentation and 2) the historic range of the Stewart Island shag could have been at least as far north as Wairau Bar near Blenheim.

The results of the modern sequencing support the bigger picture previously found during my honours work of post glacial colonisation of South America and the Sub Antarctic while NZ and its close off shore islands remained ice free. Also evident is a North – South split within the mitochondrial lineages of the Stewart Island shag, with the northern cluster more closely aligned with the Chatham Island shags.

Acknowledgements

I would like to thank Martyn Kennedy for his direction and assistance in the lab, and Catherine Collins for all he help in the aDNA lab. Also thanks must go to Catherine Collins, Jon Waters and Paul Schofield for providing samples. Lastly a very big thank you to the AWC for the opportunity to undertake this research which helps to fill in some of the ‘gaps’ I was not able to address in 2011.

References

Till, C. 2011. BSc Hons (Genetics), University of Otago. Species, sub species or new species? A phylogenetic analysis of the Blue eyed shag complex.


Figure 1.


Figure 2.


Red Cliffs

Doubtless Bay

Delaware Bay

Wairau Bar (also Marfells Beach, and general Marlborough area)

Modern boundary between Northern and Southern Stewart Island shag groups

Approximate modern range extent of Stewart Island shags, extending from Southland and Stewart

Island up to Oamaru.

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