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How to DNA sex birds.

The males and females of many bird species are difficult to distinguish by their appearance (peacocks are a notable exception). There are many situations where it is useful to know the sex of birds including captive breeding programmes, behavioural studies and even species delimitation in extinct taxa.

DNA sexing provides a simple and quick way to determine which birds are females and which are males. We have been using this technique for some of our bird research projects, including our study of the prion wreck of 2011. For our prion study we want to determine whether there is a gender bias in the birds that were wrecked.

So how does DNA sexing work for birds? By way of background, birds have a different chromosome system to us for determining their sex. In mammals, including us, males have an X and a Y chromosome and females have two X chromosomes. In contrast, birds have a ZW sex-determination system whereby males have two Z chromosomes and females both Z and W chromosomes.

Sex chromosomes in birds and mammals.

Sex chromosomes in birds and mammals. Figure credit: Lara Shepherd

To genetically sex a bird, DNA is first obtained from a blood, feather or tissue sample. We used tongue samples for the prions.

From these DNA samples we made lots of copies of the CHD region, a gene that occurs on both the Z and W chromosomes. Our processing of these gene copies produces a single DNA band for males (because they only have one type of chromosome) and two bands for females (representing the different CHD copies from the Z and W chromosomes).

Prion sex assignment based on the CHD region. Females have two DNA bands – the top band is from the W chromosome and the lower band is from the Z chromosome. Males just have the single Z chromosome band. The lane on the far left with multiple bands contains a size standard  with bands of DNA of known size. Photo credit Lara Shepherd

Prion sex assignment based on the CHD region. Females have two DNA bands – the top band is from the W chromosome and the lower band is from the Z chromosome. Males just have the single Z chromosome band. The lane on the far left with multiple bands contains a size standard with bands of known size. Photo credit Lara Shepherd

DNA sexing is also possible for humans, albeit using a modified method suited to our X/Y chromosome system, and is routinely used in forensics. A recent example is the detection of female DNA on the bombs used in the Boston marathon bombing.

A few more botanical highlights from the Foxton fieldtrip….and a katipo spider!

I also spent an enjoyable few days over Easter on the Wellington Botanical Society fieldtrip (see Leon’s blog about the trip). Here are a few more photos from the trip.

A carpet of tiny ‘3-leaf clover’-like leaves, growing on the shores of Lake Koitiata.  We think this is Hydrocotyle sulcata. Photo: Lara Shepherd

A carpet of tiny ‘3-leaf clover’-like leaves, growing on the shores of Lake Koitiata. We think this is Hydrocotyle sulcata. Photo: Lara Shepherd

The distinctive asymmetric flower of Selliera rotundifolia. Photo: Lara Shepherd

The distinctive asymmetric flower of Selliera rotundifolia. Photo: Lara Shepherd

The keen eyes of Bot Soc member Bev Abbott spotted the tiny fruit of sand gunnera (Gunnera arenaria). Photo: Lara Shepherd

The keen eyes of Bot Soc member Bev Abbott spotted the tiny fruit of sand gunnera (Gunnera arenaria). Photo: Lara Shepherd

It is difficult to believe that this tiny native species is in the same genus as the huge Chilean rhubarb. The leaves of this weedy exotic can be several metres in height.

Sand coprosma (Coprosma acerosa). The fruit colour of this species can vary but the sand coprosma we saw in the dunes near Foxton had striking blue striped fruit. Photo: Lara Shepherd

Sand coprosma (Coprosma acerosa). The fruit colour of this species can vary but the sand coprosma we saw in the dunes near Foxton had striking blue striped fruit. Photo: Lara Shepherd

During a break from botanizing Viv McGlynn located this Katipo spider under a piece of driftwood in the dunes. Photo: Lara Shepherd

During a break from botanizing Viv McGlynn located this Katipo spider under a piece of driftwood in the dunes. Photo: Lara Shepherd

Find out more about the endangered katipo here.

DNA sequences reveal unexpected fern relationships

Recently I have been obtaining DNA sequences from some of the fern samples collected by Te Papa Botany curator Leon Perrie on his recent trip to New Caledonia. We aim to determine the relationships of these New Caledonian ferns to other ferns around the world, including those from New Zealand.

One sample, however, gave us a surprising result. Two of the New Caledonian samples had previously been identified by Leon as members of the fern genus Dryopteris, based on their morphology. The genus Dryopteris has not previously been recorded from New Caledonia, so Leon was quite excited by these finds.

The DNA sequences established that one of these samples is indeed a Dryopteris, thus confirming that this genus is present in New Caledonia. However, the other sample unexpectedly grouped with another, albeit related, fern genus!

Watch this space as we do more work to try and establish the identity of this mystery fern.

The mystery New Caledonian fern that looks remarkably like a Dryopteris. Photo credit: Leon Perrie

The mystery New Caledonian fern that looks remarkably like a Dryopteris Photo credit: Leon Perrie.

Seabird sampling strategies: a tongue twister

Te Papa seabird scientist Sarah Jamieson measures defrosted prions from the 2011 wreck

Te Papa seabird scientist Sarah Jamieson measures defrosted prions from the 2011 wreck


Genetic research requires a small amount of tissue from animal or plant specimens to be destroyed in order to obtain DNA. Te Papa’s bird team recently pondered the best way to sample tissue for DNA whilst causing a minimal amount of damage to seabird specimens.

In July 2011 a period of unfavourable weather led to the mass mortality (‘wrecking’) of hundreds of thousands of prions.

Over 600 of the prions that died ended up in the freezer at Te Papa.

Te Papa bird scientists are researching these wrecked prions. Part of this research aims to use genetics to determine from which colonies these wrecked prions originated.

Some of these prions will be sent to a taxidermist to be made into study skins to be incorporated into Te Papa’s collections, so we wanted these bird skins and feathers to have as little damage as possible. Sarah Jamieson, Te Papa’s prion dissector, came up with the idea of using the tongue as the sample for DNA; the tongue is removed anyway during the taxidermy process.

Tongue of a defrosted broad-billed prion.

Tongue of a defrosted broad-billed prion.

A small piece of prion tongue tissue ready for DNA extraction.

A small piece of prion tongue tissue ready for DNA extraction.

We have already shown that there is sufficient DNA in the tongue tissue for our genetic work.

Is this the world’s biggest nettle leaf?

Whilst recently chasing seabirds on Titi Island we came across tree nettles (ongaonga, Urtica ferox) with super-sized leaves. The largest leaf we measured was 28 cm long, much longer than the maximum leaf length of 18 cm given for this species in the Flora of New Zealand. Perhaps the abundant seabird droppings on this island provide these nitrogen-loving plants with the fertiliser to reach such giant proportions.

Te Papa curator Colin Miskelly checks out a giant ongaonga leaf. Photo credit: Lara Shepherd

Te Papa curator Colin Miskelly checks out a giant ongaonga leaf. Photo credit: Lara Shepherd

Ongaonga leaf - note the ruler starts at 500 mm. Photo credit: Colin Miskelly

Ongaonga leaf – note the ruler starts at 500 mm. Photo credit: Colin Miskelly

Tree nettle only occurs in New Zealand and it packs a nasty punch, as anyone who has brushed into one can attest. The stinging hairs are hollow and inject a number of toxins into the skin when touched. The stings often cause a burning sensation, swelling and numbness, symptoms which can last several days. Tree nettle is known to have killed at least one person, as well as dogs and livestock. Not surprisingly we weren’t keen to test whether the large leaves on Titi Island have a worse sting than normal sized leaves!

However, not everyone avoids tree nettle – it is the favourite food for caterpillars of the New Zealand red admiral butterfly! The caterpillars roll themselves in the leaves to protect themselves from predators.

Oops-a-daisy! How many flowers do you see?

How many flowers do you see in the photo below?

Marlborough rock daisies (Pachystegia insignis). Photo credit: Lara Shepherd

Marlborough rock daisies (Pachystegia insignis). Photo credit: Lara Shepherd

Two is the obvious answer, but there are far more than two flowers in the picture. Each daisy ‘flower’ is actually made up of numerous tiny flowers, also called florets.

The Marlborough rock daisies pictured above have two types of florets. Around the outside are ray florets. Each ray floret has a single broad strap-like petal.

In the centre are the yellow disc florets, which have very reduced petals. The ray and disc florets grouped together look like the single flower found in many other flowering plants.

Marlborough rock daisy disc floret (top) and ray floret (bottom). Note the long petal on the ray floret. Photo credit: Leon Perrie

Marlborough rock daisy disc floret (top) and ray floret (bottom). Note the long petal on the ray floret. Photo credit: Leon Perrie

Such clusters of florets, called capitula (singularly, a capitulum), are typical of species in the daisy family. Other members of the daisy family include sunflowers, lawn daisies, lettuces and chrysanthemums.

Next time you find a daisy in the lawn or someone gives you a bunch of sunflowers, take a closer look.

DNA finds kiwi’s origins: Introducing Stewie

A number of biological specimens in Te Papa’s collection, particularly old specimens, lack information about when and where they were collected. This information may have been lost since the specimen was collected or was simply not recorded at the time.

However, all is not lost! Sometimes we can use DNA to determine where a specimen was collected.  We recently used DNA sequences to examine the provenance of a number of Te Papa’s unlabelled kiwi specimens.

One particularly stunning specimen we looked at is this articulated kiwi skeleton. 

Articulated kiwi skeleton from Te Papa's collection. Photo by Lara Shepherd.

Articulated kiwi skeleton from Te Papa’s collection. Photo by Lara Shepherd.

As I mentioned in an earlier blog on kiwi the bones of great spotted kiwi and the three species of brown species are very similar in size and shape and can’t be distinguished. Therefore, this kiwi skeleton could have potentially belonged to any of these four species.

To obtain bone material for our genetic analysis we drilled a small hole underneath the pelvis. Our aim was to minimize the visible damage to the skeleton.

Close-up of the hole we drilled in the pelvis to obtain bone for DNA analysis. Photo by Lara Shepherd.

Close-up of the hole we drilled in the pelvis to obtain bone for DNA analysis. Photo by Lara Shepherd.

 We compared the specimen’s DNA sequence to sequences previously obtained from kiwi from known locations around New Zealand.

The results showed that this kiwi skeleton is a Tokoeka (also known as Southern brown kiwi) from Stewart Island.  This result increases the scientific value of this skeleton and is particularly exciting because there aren’t many kiwi from Stewart Island in museum collections.

Link to our study.

When did little spotted kiwi become extinct on the New Zealand mainland?

Little spotted kiwi  only occur in New Zealand, where there are around 1500 individuals remaining.  They are the smallest kiwi species, about the size of a bantam hen, and are very susceptible to predation by introduced mammals, such as stoats and dogs.  Today they survive on predator-free offshore islands and the fenced mainland sanctuary Zealandia in Wellington.

Little Spotted Kiwi, Apteryx owenii, collected no data, New Zealand. Gift of the The Hawke's Bay Art Gallery and Museum, 1949. Te Papa

Little Spotted Kiwi, Apteryx owenii, collected no data, New Zealand. Gift of the The Hawke’s Bay Art Gallery and Museum, 1949. Te Papa

Although little spotted kiwi currently have a very restricted distribution, deposits of bones (e.g., in caves) indicate that they used to occur throughout New Zealand.  When did little spotted kiwi disappear from the mainland?

Little spotted kiwi in the North Island were already very rare when Europeans settled New Zealand. Only one or two live birds have ever been collected from the North Island mainland for museum collections, both in the 19th century.

In contrast, little spotted kiwi were common on the west coast of the South Island at this time.  When exactly they disappeared from the South Island is unclear, with misidentification with the related great spotted kiwi adding to the confusion.  However, it has been widely reported that South Island little spotted kiwi went extinct in the 1930s.  Other researchers disagree and think that little spotted kiwi were present on the west coast for much longer.

Our recent study has shed light on this debate.  We used DNA to identify to species three dead kiwi found in the South Island that post-date the 1930s.  These kiwi are now held in Te Papa’s bird collection.

Link to our study

We were able to show that a kiwi specimen found in 1952 from central Westland and two other kiwi specimens found in 1978, from NW Nelson and south Westland, were all little spotted kiwi (as opposed to juvenile great spotted kiwi).  This suggests that little spotted kiwi survived, and were widespread, in the South Island until much more recently than generally accepted.

Map of the locations where three post-1940 little spotted kiwi were found (names in black type). Today’s little spotted kiwi all derive from birds that survived on Kapiti Island (red type). Base map supplied by Geographx (http://www.geographx.co.nz/).

Map of the locations where three post-1940 little spotted kiwi were found (names in black type). Today’s little spotted kiwi all derive from birds that survived on Kapiti Island (red type). Base map supplied by Geographx (http://www.geographx.co.nz/).

Little spotted kiwi today all originate from a few individuals from Kapiti Island and are highly inbred with very little genetic diversity.  This may mean they have reduced resistance to new diseases and an increased risk of genetic defects.  If there was more certainty about the identity of the remaining mainland birds in the 1970s perhaps more effort could have been made to locate and move surviving little spotted kiwi to predator-free islands.  This would likely have boosted the genetic diversity surviving in this species today.

This result demonstrates how little we know about our native species, even the prominent ones like our (unofficial) national bird, the kiwi.  If so little was known about kiwi, then what about other reclusive members of our fauna thought to be recently extinct, such as South Island kokako, or less charismatic but equally interesting species, such as our greater short-tailed bat (Mystacina robusta)?

Learn about South Island kokako.

Learn about the greater short-tailed bat.

Using DNA forensics to determine the past distribution of the brown kiwi species rowi.

Yesterday was a special day for 20 rowi (a species of the flightless kiwi) who were flown from the South Island to their new home on Mana Island, near Wellington. It was reported that this was the first time that this species of kiwi had been in the North Island for over a century.

So how do we know that rowi used to be in the North Island?

Kiwi researcher Kristina Ramstad holding a rowi. Photo by Rachael Abbott.

Kiwi researcher Kristina Ramstad holding a rowi. Photo by Rachael Abbott.

Today kiwi are absent from large areas of New Zealand, including the southern North Island (North Island brown kiwi occur from the central North Island northwards).  We know that kiwi used to occur in the southern North Island because their bones have been found in caves and other deposits.  However, trying to identify kiwi species just by looking at the shape and size of their bones is tricky.

Little spotted kiwi is the only species that can be identified from its bones because they are much smaller than the other kiwi species.  The bones of great spotted kiwi and the three species of brown kiwi (rowi, North Island brown kiwi and tokoeka) can’t be identified to species because they overlap in size and shape.

This is the kind of puzzle that DNA can solve. As part of my PhD I examined the past distribution of each kiwi species by sequencing DNA from kiwi bones that had been collected from throughout New Zealand.  Some of these bones were up to several thousand years old, but they still contained small amounts of DNA!

Surprisingly I found that the bones in the southern North Island were most closely related to rowi, rather than the geographically closer North Island brown kiwi.  Today rowi only naturally occur in one small population at Okarito on the West Coast of the South Island and they are the rarest species of  kiwi.  My DNA work showed that they used to occur as far north as the southern Hawke’s Bay. You can read the published results here.

Caring for museum collections in a molecular world

Museums are embracing technologies, such as DNA sequencing, to both enhance understanding of their collections and showcase scientific research to the public.  Many museums around the world now have molecular laboratories.  DNA sequencing has many useful applications for museum research; for example, it can be used to distinguish new species, determine the evolutionary relationships between species and identify the region of origin of artefacts such as kahu kiwi (kiwi feather cloaks).  However, it has only recently been appreciated that care should be taken in the construction and use of museum molecular labs.  Why is the situation for museums different to universities and other institutions where molecular biology labs are commonplace?

Museums are storehouses of important biological collections and these can become contaminated with copies of DNA that are generated in molecular laboratories.  A technique routinely used in molecular labs in the polymerase chain reaction (PCR). PCR is an extremely efficient method to produce millions of copies a targeted region of DNA. If one drop (1/10th of 1 ml) of a PCR is mixed into an Olympic swimming pool then one drop of this highly diluted mixture removed it will still contain around 400 copies of DNA!

Learn more about PCR (polymerse chain reaction)

In a museum setting, if care is not taken, these millions of copies of DNA could contaminate the biological specimens in museum collections.  These specimens, such as animal bones and pressed plants, typically contain small amounts of their own DNA because DNA degrades over time, starting with the death of the organism.  It is very easy for the low levels of DNA to become swamped by the copies of DNA generated by PCR.  Future attempts to use contaminated specimens for genetic research may be compromised with the contaminating PCR products detected instead of the specimen’s own DNA. So how do we avoid this problem?

Together with Leon Perrie, a colleague in Te Papa’s Natural Environment team, I recently suggested that strict protocols should be developed for constructing and using molecular laboratories within museums.  These include having labs and collections in different buildings, or at least having separate ventilation systems for each, and having a one-way movement of people, equipment and specimens from collection areas to labs. We hope that museum researchers take up these suggestions in order to protect the research potential of their important, and often irreplaceable, collections of biological specimens.

Read our full article (requires a subscription to Nature)

Leon and Lara in the Te Papa's collections. Photo Carlos Lehnebach.Te Papa

Leon and Lara in Te Papa’s collections. Photo Carlos Lehnebach.Te Papa

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