fascinating stories of dna, and the kiwi’s close cousin uncovered

On Monday I was lucky enough to attend a lecture by Alan Cooper, director of the Australian Centre for Ancient DNA and one of the authors on a very recent paper that provides a new view of kiwi evolution (Mitchell et al., 2014). It was a fascinating & wide-ranging talk that started with a bit of a travelogue, as Cooper told his audience about some of the places he's visited on his search for ancient DNA (aDNA).

To do their aDNA work, he & team use well-preserved organic material – usually found  in rather cold dry places. One of these was Mylodon Cave in Patagonia, named for the extinct ground sloth, Mylodon, whose remains littered the cave – in fact, much of the floor of the cave is apparently covered with balls of ground sloth dung! In the images we saw, much of the cave looked like a bomb site: it seems this impression was close to the mark as in the past local farmers had used dynamite to excavate fossils for sale to museums.

Cooper's also worked – in the summer – in permafrost zones in northern Alaska looking for remains of mastodon & bison,  and finding mammoth bones along the way. (He didn't sound very impressed about it!) Everything they found had to be carried down braided rivers by inflatable kayaks (which don't have a good centre of gravity & makes travel a real challenge).

He's certainly got some pretty wide-ranging research interests:

• mammoth blood: looking at changes in their DNA that might be associated with haemoglobin function. With some mammoth tissue samples, a bit of genetic tweaking, and a vat full of bacteria the team were able to express mammoth haemoglobin & then look at its functioning. The oxygen-carrying protein turned out to be temperature-insensitive, releasing O2 at a steady rate regardless of temperature. This raises the possibility that in mammoths, their ears, legs & feet could cool right down without this drop in body temperature interfering with haemoglobin's ability to deliver oxygen.
• megafaunal ecology;
• other extinct animals such as the thylacine, sabre tooth lion, dodo, and Falkland Islands wolf;
• human evolution, including examining the remains of our own relatives: Neandertals and Homo floresiensis (aka the 'hobbits' – in the talk, he suggested this species may have become extinct closer to 50,000 years ago rather than the original date of around 13,000 years before present.)
• South American mummies, gaining information on human migrations & cultural changes.
• animal domestication – including using data from animal remains as a proxy for patterns of human migration & cultural change.
• evolution of disease – eg calcified bacteria, or dental calculus, on human teeth (including on australopiths) gives a record of all bacteria in an individual's mouth & allows us to track the human microbiome through time.
• palaeoenvironments and metagenomics: using information from things like stalactites, sediments, and coprolites (fossil dung eg from moa) to reconstruct ancient ecosystems.

With any work involving DNA, researchers have to avoid contamination of their samples from other sources of DNA (including themselves). When it's aDNA the problem is magnified enormously, because the initial samples are so small that the ancient signals would be swamped in the 'noise' of modern contaminants. This meant that the research team have to do all this work away from any labs regularly working with cloning and PCR, and so they work in purpose-built lab facilities, away from the main university campus. Positive air flow in the labs prevents dust etc entering. Staff can't bring anything in that's been at the university and must don gowns, masks & hoods with even more care than a surgeon (at this point I couldn't help feeling that the folks working there would need excellent bladder control, since gowning is a lengthy process & one must perforce leave the lab & ungown in order to seek a little excretory relief). Any goods coming in are 'fried' by a UV oven. The rooms where the actual research is done are 'still-air labs' within the positive pressure environment: people must move around rather slowly to minimise the creation of air currents that would spread aerosol droplets with potential to contaminate between samples. This must be a cause of some perplexity &/or amusement to those able to view the scientists from the gardens outside 🙂

Then we moved on to the ratite birds, or palaeognaths: the latter name refers to the structure of their palate, a feature that unites the tinamou, ostrich, rhea, cassowary, emu, and kiwi, along with extinct species such as moa and elephant birds. This means that there are extinct & living ratite species on all the southern continents (apart from Antarctica – but then they would be rather hard to find there given its present ice-covered state). The tinamou is unusual – it's the only living ratite that can fly.

Early ideas about ratite evolution cast them as 'primitive' species that only survive on the Gondwanan land masses because they'd been outcompeted everywhere else, and with their flightlessness as an ancient characteristic. (The tinamou, of course, was something of a feather in the ointment for this viewpoint.) Ratites became poster chicks for continental drift, because the data then available suggested that ratite adaptive radiation seemed to fit with the sequence of continental movement: Africa was the first continent to separate and ostriches were the 'oldest' of the birds.

Alan Cooper has had an interest in this story for a long time: he began his research career working on moa remains, aiming to extract & amplify mtDNA from mitochondria. (This is much easier to get than nuclear DNA (nDNA). It's also inherited down the maternal line, and he characterised it as usually neutral with respect to the overall evolution of a group of species.) Eventually he sequenced the entire mtDNA genome of two moa, and also got fragments of the genome from Madagascar's extinct elephant bird. At the same time he found that the kiwi was more closely related to cassowary & emu than to the moa, & his fragmentary data for the elephant bird also suggested that it was relatively closely related to kiw. How could this be?

Then in 2010 new research using nDNA found that tinamous were not the 'outgroup' for ratites (ie only distantly related to the rest of the species in this group), but were instead most closely related to moa (Phillips et al., 2010). Not only did this study place ostriches as the outgroup for the ratites; it also concluded that flightless had been lost independently in the various species – it was not an ancestral feature. This suggested that the various ratites must have dispersed by flying, & subsequently lost that ability.

This research didn't include elephant birds, for which there are very few skeletal remains. Most authors think there were around 7 species, but Cooper suspects they are not taking into account sexual dimorphism. This has been well documented in moa, where females are much larger than the males (a realisation that saw a reduction in the number of moa species we recognise). And large elephant birds weighed around 275kg – about 40kg heavier than the largest moa. Cooper says he spent ages looking for remains that were likely to yield DNA, eventually going back to the specimens from Te Papa that he'd used in his original research. His research team designed suitable molecular probes – short sequences of DNA based on the mtDNA of modern ratites. They also obtained a 'library' of all the elephant bird DNA that they could sequence, recognising that this probably also contained contaminants from various microbes. The probes and the elephant bird sequences were mixed, which allowed the probes to 'hybridise' wherever their sequences matched those from the birds. The hybrid mtDNA could then be pulled from the mix, thus leaving behind DNA from bacterial & fungal contamination. The result of all this was a very good coverage of elephant bird mtDNA (apparently as good as what is returned from work on modern human DNA).

These sequences were then compared to data from all other living ratites. The results identified elephant birds as the closest relatives of kiwi: they are sister taxa. The data also confirmed the moa-tinamou grouping, again placed ostriches as the outgroup for ratites, and gave relatively recent divergence dates for the various ratites that don't match the timing for the break-up of Gondwanaland; ie the order in which the Gondwanan continents separated has nothing to do with ratite distribution. Also, the additional support for the moa-tinamous sister relationship reinforces the idea that ratites were originally flighted. Cooper suggests that flying ratites were spreading around the continents following the end-Cretaceous mass extinction event that saw off the dinosaurs (and many other groups besides), and subsequently – in the absence of large mammalian predators and competitors – converged on the giant flightless form, filling the 'large herbivore' niches left vacant by the dinosaurs. (The subsequent adaptive radiation of large mammals prevented the more recent 'neognath' birds from also following this path.) In other words, it's highly likely that the ancestors of both kiwi and elephant bird came from 'somewhere else', most likely Antarctica.

The talk finished with a possible answer to the question: why is the kiwi so small, & its egg so large? The genetic data indicate that the ancestors of modern kiwi arrived in New Zealand well after the moa had expanded into the large-herbivore niche, and took on the role of a nocturnal insectivore. Similarly, tinamous arrived in South America well after the rheas had evolved. With no mammalian predators, kiwi lost the ability to fly – unlike the tinamou, in a land with a variety of mammals present. And the egg? Stephen Jay Gould suggested that kiwi had shrunk but that there was some selective advantage in retaining large egg. Cooper's explanation proposes that with a number of large avian predators around (eg Haast's eagle, laughing owl), newly-hatched ground-living kiwi chicks would be in some danger of becoming a meal. He feels that the adaptive significance of the unusually large egg is that it allows a kiwi chick to stay in the burrow for at least a week after hatching, so when it leaves the burrow's protection it's already very active and perhaps better able to avoid predation.

It was a fascinating tale, well told.

K.J.Mitchell, B.Llamas, J.Soubrier, N.J.Rawlence, T.H.Worthy, J.Wood, M.S.Y.Lee & A.Cooper (2014) Ancient DNA reveals elephant birds and kiwi are sister taxa and clarifies ratite bird evolution. Science 344 (6186): 898-900. 10.1126/science.1251981

M.J.Phillips, G.C.Gibb, E.A.Crimp & D.Penny (2010) Tinamous and moa flock together: mitochondrial genome sequence analysis reveals independent losses of flight among ratites. Systematic Biology 59(1): 90-97. doi: 10.1093/sysbio/syp079