Organising Life

This second long-overdue post is a talk I gave a few months ago. As a practice for presenting our dissertations in September, we were asked to present any paper relevant to the course. Anything at all. So I found one about pigeons. Enjoy!

Note: I've pasted in text, which has resulted in some amusing font issues. I haven't worked out how to fix them. Such is life.

My paper is ‘The mysterious spotted green pigeon and its relation to the dodo and its kindred’, by Heupink, Grouw and Lambert. They used ancient DNA analysis to find out a bit more about a very mysterious bird and shoehorn in a bit about the dodo, because dodos are cool. But I think that the pigeon itself is an interesting story in its own right.

The spotted green pigeon, Caloenas maculata, is extinct. It was described in 1783 based on two specimens. One was collected by Sir Joseph Banks, one by General Thomas Davies. But then the Banks specimen was lost...

... Leaving us with just one. And it lives in the World Museum...

... in Liverpool. In fact, its other common name is the Liverpool pigeon. Unfortunately, nobody knows where either specimen originally came from.

But both Banks and Davies did most of their collecting in Oceania, so probably from there.

Here's a reconstruction. It's a fairly standard tropical pigeon. It could definitely fly. Apparently its short, rounded wings suggest that it lived on islands. But to infer more about it, it would be helpful to know what its closest relatives are.

Unfortunately, the spotted green has rather jumped around the pigeon tree. The big group containing European pigeons is Columbinae at the top. Raphinae has the huge diversity of tropical pigeons.

Based on morphology, the spotted green has been placed somewhere in Ptilinopini, sister to the imperial, mountain or fruit pigeons.

On the other hand, it’s also very similar to the Nicobar pigeon, which sits in the extended dodo clade (green). Some think the spotted green is a sister species to the Nicobar; others even think they’re conspecific, with the spotted green being a juvenile or otherwise ‘abnormal’ Nicobar.

What technique can help resolve taxonomic questions based only on morphology? DNA analysis! Which is where Heupink and colleagues come in.

The team managed to extract DNA from two feathers. Because that one remaining specimen is a couple of hundred years old, they used ancient DNA techniques, which included lots of contamination precautions and lots of controls.

Unfortunately, the first PCR failed to amplify anything. The fragments they had were too short to make it through. So they made some changes to their methods, having mostly just followed a kit the first time round.

There were two key improvements, both on the final step of DNA extraction, when you bind the DNA to a membrane and wash away impurities.

First, they used a binding buffer with more isopropanol. This encouraged the shorter DNA molecules to actually bind. Another group had already tried this and it worked for them. The second thing was to wash with the buffer in the kit multiple times, and also with phenol. This removed more PCR inhibitors, quantitatively and qualitatively.

So finally...

They succeeded in getting detectable, amplifiable DNA! Unfortunately...

The fragments were super short. Like less than 30 base pairs long.

The quantity was really low.

And for one of the two feathers, the DNA was super fragmented too.

But at least they had DNA. The idea had been to analyse a barcode from the mitochondrial 12S gene. Their reference dataset was over a hundred 12S sequences from pigeons, downloaded from GenBank.
But the spotted green fragments were too short for a typical barcode...

... so the team designed three mini-barcodes instead. These turned out to work really well. They sequenced successfully and consistently, and...

... a BLAST search said that the sequences came from an unknown pigeon, which was exactly what they wanted.

Using the mini-barcodes, and all of the reference sequences, they built a maximum likelihood tree for each feather, and they were pretty much identical, which was a relief. All but one of the other taxa ended up where they were expected to - the pigeon tree isn’t completely stable yet, but this was consistent with previous findings. But the only support metric they’ve used there is bootstrap, which sticks in my mind from the cladistics course as something you shouldn’t rely on too hard. It would have been nice to see another measure there too.

But anyway, This is a zoom in on one of them. the spotted green pigeon comes out closest to the Nicobar with high bootstrap support (it's out of 100), for what it’s worth. They are very similar after all.

But could it be the same as the Nicobar? Is it really a different species? I’m sure you all remember how tricky species delimitation can be, and we can’t look at within-species variation for the spotted green because we only have one of them. But Hupink’s team did look at simple pairwise identity - how similar the spotted green mini-barcodes were to sequences for other taxa.

There was an average of 90.5% pairwise identity between the spotted green and the Nicobar. This translated to about 0.11 substitutions per site. The average difference within species was 0.01 substitutions per site, and the average difference between species of the same genus was 0.09.

T-tests showed that the spotted-Nicobar average was significantly different from within-species, but not from within-genus.

So, the spotted green shares a genus with the Nicobar pigeon but is different enough to be its own species.

But that’s not quite the end. There is actually a third species in the genus: Caloenas canacorum, the Kanaka pigeon. Why didn’t the team include it? They talked about this.

The Kanaka pigeon is extinct, and is only known from a handful of subfossils found on New Caledonia and Tonga: hot, humid, beautiful places that turn DNA into soup. The boundaries of ancient DNA keep being pushed, but it’s not there yet.

Also, the Nicobar is bigger than the spotted green, and the Kanaka is bigger than the Nicobar, so we can be pretty sure it’s not the same species as the spotted green. Leaving it out doesn’t increase confusion over what the spotted green is.

Now remember that Caloenas is in the extended dodo clade. The dodo and its also-extinct sister taxon, the Rodrigues solitaire, form the subfamily Raphinae.

Apart from Raphinae (in the box), all species in this clade fly and come from Oceania. They’re all tropical island-hoppers. People guessed this from the spotted green pigeon’s morphology, but now we have phylogenetic evidence to back that up. Adding another Oceanic island-hopper also adds some more support to the island-hopping hypothesis of how the dodo and Rodrigues solitaire got to their islands - that their ancestors could fly until they arrived.

It also reinforces how morphologically weird Raphinae is, and how much morphological variation the clade shows. In fact, it’s probably not surprising that it was so hard to place the spotted green by morphology.

So, the paper starts with a lone specimen of unknown providence and uses ancient DNA to confirm that it was an island-hopper from Oceania and a proper species in its own right. On top of that, they contributed to DNA extraction methodology and say a bit about the dodo.

References
Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. PNAS 110, 15758–15763 (2013). Heupink, T.

H., van Grouw, H. & Lambert, D. M. The mysterious Spotted Green Pigeon and its relation to the Dodo and its kindred. BMC Evolutionary Biology 14, 136 (2014).

Pereira, S. L., Johnson, K. P., Clayton, D. H. & Baker, A. J. Mitochondrial and Nuclear DNA Sequences Support a Cretaceous Origin of Columbiformes and a Dispersal-Driven Radiation in the Paleogene. Syst Biol 56, 656–672 (2007).

The IUCN Red List of Threatened Species. Available at: http://www.iucnredlist.org/. (Accessed: 6th May 2017)

Image credits by slide

31 Didunculus strigirostris https://upload.wikimedia.org/wikipedia/commons/c/cf/Didunculus_strigirostris.jpg
Goura scheepmakeri https://upload.wikimedia.org/wikipedia/commons/6/69/Goura_scheepmakeri_sclaterii_1_Luc_Viatour.jpg
32 Map, modified from https://upload.wikimedia.org/wikipedia/commons/thumb/f/f2/World_pacific_0001.svg/2000px-World_pacific_0001.svg.png

Allow me to introduce one of the most exciting pieces of lab machinery I've had the pleasure to work with over the past few months: the Precellys 24 tissue homogeniser:

It doesn't have a name. They only name PCR machines in this lab.

It breaks up tissue samples. The idea is that you put your sample in a tube with some liquid and some very hard beads - the ones I used were chips of garnet. Then the machine shakes the tubes vigorously, so that the beads and sample slosh together and the sample gets ground up, and you end up with a sample soup that's much easier to do chemistry with. Here's how the manufacturer describes it:

The machine is about 50 cm tall and shaped like a can of beans. You lift that thick, heavy lid to get to the ring of holes where your tubes sit. You take off the white cog-shaped cap, slot your tubes in, put the cap back on, close the massive lid, hit start and retreat at speed to a safe distance. After a couple of seconds warming up, the machine gives a sudden WHRROOAARRRR, like a small JCB, the sample holder becomes a grey blur and the whole 30 kg lot shudders along the bench towards the closest edge. This continues for a minute or so. It takes a break and then goes again, however many times you've asked it to. Mercifully, it tends to stop travelling after a while.

When you dare come back in the room, you lift the lid and take off the plastic cap and remove your sample tubes. They feel hot through your gloves. My samples were soil and poo, and they came out of the homogeniser looking like frothy chocolate milkshakes. Needless to say, it's ruthlessly effective; only the garnets survived as solid material and I'm amazed that the tubes never broke.

But the most surprising thing is the white cap. In the instruction book, it calls it a "patented system" used for "quick and safe locking". But I've been generous calling it a cap. It's no more than a thin, flat piece of plastic that sits on top of the sample holder. There's no click, nothing holding it down other than gravity. It's comparable in weight to a large grape. And yet it stays on, and the sample tubes do not fly out of their holes.

Bravo Precellys, for a truly remarkable piece of engineering.

Tuesday, 8 August 2017

The mysterious spotted green pigeon

Monday, 7 August 2017

The tissue homogeniser

Searching for ancient microscopic worm eggs