Finally, that sponge ParaHox gene

ParaHox genes are a bit like the underappreciated sidekicks of Hox genes. Or little sisters, as the case may be, since the two families are closely related. Hox genes are probably as famous as anything in evo-devo. Being among the first genes controlling embryonic development to be (a) discovered, (b) found to be conserved between very distantly related animals, they are symbolic of the late 20^th century evo-devo revolution.

ParaHoxes get much less attention despite sharing some of the most exciting properties of Hox genes. Like those, they are involved in anteroposterior patterning – that is, partitioning an embryo along its head to tail axis. Also like Hox genes, they are often neatly clustered in the genome, and when they are, they tend to be expressed in the same order (both in space and time) in which they sit in the cluster*. Their main ancestral roles for bilaterian animals seem to be in patterning the gut and the central nervous system (Garstang and Ferrier, 2013).

There are three known types of ParaHox gene, which are generally thought to be homologous to specific Hox subsets of Hox genes – by the most accepted scheme, Gsx is the closest sister of Hox1 and Hox2, Xlox is closest to Hox3, and Cdx to Hox9 and above. It is abundantly clear that Hoxes and ParaHoxes are closely related, but there has been a bit of debate concerning the number of genes in the ancestral gene cluster that gave rise to both – usually called “ProtoHox” (Garcia-Fernàndez, 2005).

Another big question about these genes is precisely when they originated, and in this regard, ParaHox genes are proving much more interesting than Hoxes. You see, there are plenty of animals with both Hox and ParaHox genes, which is what you’d expect given the ProtoHox hypothesis, but there are also animals with only ParaHoxes. If there really was a ProtoHox gene/cluster that then duplicated to give rise to Hoxes and ParaHoxes, then lone ParaHoxes (or Hoxes for that matter) shouldn’t happen – unless the other cluster was lost along the way.

So a suspiciously Gsx-like gene in the weird little blob-creature Trichoplax, which has nothing remotely resembling a Hox gene, was a big clue that (a) Hox/ParaHox genes might go back further in animal evolution than we thought, (b) the loss of the entire Hox or ParaHox cluster is totally possible**, despite how fundamental these genes appear to be for correctly building an animal.

I wrote (at length) about a study by Mendivil Ramos et al. (2012), which revealed that while Trichoplax had no Hox genes and only one of the three types of ParaHox gene, it preserved the more or less intact genomic neighbourhoods in which Hox and ParaHox clusters are normally situated. One of the more interesting results of that paper was that the one sponge genome available at the time – that of Amphimedon queenslandica, which had no trace of either Hox or ParaHox genes – also contained statistically significant groupings of Hox and ParaHox neighbour genes, as if it had a Hox neighbourhood and a ParaHox neighbourhood, but the Hoxes and ParaHoxes themselves had moved out.

That study thus pointed towards an intriguing hypothesis, previously championed by Peterson and Sperling (2007) based solely on gene phylogenies: sponges once did have Hox and ParaHox genes/clusters, which at least some of them later lost. This would essentially mean that the two gene clusters go straight back to the origin of animals if not further***, and we may never find any surviving remnant of the ancestral ProtoHox cluster, since the closest living relatives of animals have neither the genes nor their neighbourhoods (that we know of).

Hypotheses are nice, but as we know, they do have a tendency to be tragically slain by ugly facts. Can we further test this particular hypothesis about sponges? Are there facts that could say yay or nay? (Of course there are. I wouldn’t be writing this otherwise 😉 )

I keep saying that we should always be careful when generalising from one or a few model organisms, that we ignore diversity in the animal kingdom at our own peril, and that “distantly related to us” = “looks like our distant ancestors” is an extremely dodgy assumption. Well, here’s another lesson in that general vein: unlike Amphimedon, some sponges have not just the ghosts of vanished ParaHox clusters, but intact, honest to god ParaHox genes!

It’s calcareous sponges again. Sycon ciliatum and Leucosolenia complicata, two charming little calcisponges, recently had their genomes sequenced (alas, they weren’t yet public last time I checked), and since then, there’s been a steady stream of “cool stuff we found in calcisponge genomes” papers from Maja Adamska’s lab and their collaborators. I’ve discussed one of them (Robinson et al., 2013), in which the sponges revealed their rather unhelpful microRNAs, and back in October (when I was slowly self-destructing from thesis stress), another study announced a couple of delicious ParaHoxes (Fortunato et al., 2014).

(Exciting as it is, the paper starts by tickling my pet peeves right off the bat by calling sponges “strong candidates for being the earliest extant lineage(s) of animals”… I suppose nothing can be perfect… *sigh*)

The study actually covers more than just (Para)Hox genes; it looks at an entire gene class called Antennapedia (ANTP), which includes Hoxes and ParaHoxes plus a handful of related families I’m far less interested in. Sycon and Leucosolenia don’t have a lot of ANTP genes – only ten in the former and twelve in the latter, whereas a typical bilaterian like a fruit fly or a lancelet has several times that number – but from phylogenetic analyses, these appear to be a slightly different assortment of genes from those present in Amphimedon, the owner of the first sequenced sponge genome. This picture is most consistent with a scenario in which all of the ANTP genes in question were present in our common ancestor with sponges, and each sponge lineage lost some of them independently. (You may not realise this until you start delving into the history of various gene families, but genes come and go a LOT in evolution.)

Sadly, many of the branches on these gene trees are quite wonky, including the one linking a gene from each calcisponge to the ParaHox gene Cdx. However, somewhat fuzzy trees are not the only evidence the study presents. First, the putative sponge Cdxes possess a little motif in their protein sequences that is only present in a handful of gene families within the ANTP class. If you take only these families rather than everything ANTP and make trees with them, the two genes come out as Cdx in every single tree, and with more statistical support than the global ANTP trees gave them. Another motif they share with all Hoxes, ParaHoxes and a few of their closest relatives, but not with other ANTP class families.

Second, at least the gene in Sycon appears to have the right neighbours (Leucosolenia was not analysed for this). Since the Sycon genome sequence is currently in pieces much smaller than whole chromosomes, only four or so of the genes flanking ParaHox clusters in other animals are clearly linked to the putative Cdx in the sponge. However, when the researchers did the same sort of simulation Mendivil Ramos et al. (2012) did for Amphimedon, testing whether Hox neighbours and ParaHox neighbours found across all fragments of the genome are (a) close to other Hox/ParaHox neighbours or randomly scattered (b) mixed or segregated, they once again found cliques of genes with little overlap, indicating the once-existence of separate Hox and ParaHox clusters.

Fortunato et al. (2014) also examined the expression of their newfound Cdx gene, and found it no less intriguing than its sequence or location in the genome, although their description in the paper is very limited (no doubt because they’re trying to cram results on ten genes into a four-page Nature paper). The really interesting activity they mention and picture is in the inner cell mass of the young sponge in its post-larval stages – the bit that develops into the lining of its feeding chambers. Which, Adamska’s team contend, may well be homologous to our gut lining. In bilaterians, developing guts are one of the major domains of Cdx and ParaHox genes in general!

So at least three different lines of evidence – sequence, neighbours and expression – make this picture hang together quite prettily. It’s incredibly cool – the turning on their heads of long-held assumptions is definitely the most exciting part of science, I say! On the other hand, it’s also a little disheartening, because now that everyone in the animal kingdom except ctenophores has definitive ParaHox genes and at least the empty seats once occupied by Hox genes, are we ever going to find a ProtoHox thingy? May it be that it’ll turn up in one of the single-celled beasties people like Iñaki Ruiz-Trillo are sequencing? That would be cool and weird.

The coolest twist on this story, though, would be to discover traces of ProtoHoxes in a ctenophore, since solid evidence for ProtoHox-wielding ctenophores would (a) confirm the strange and frankly quite dubious-sounding idea that ctenophores, not sponges, are the animal lineage farthest removed from ourselves, (b) SHOW US A FREAKING PROTOHOX CLUSTER. (*bounces* >_> Umm, * cough* OK, maturity can suck it 😀 ) However, given how horribly scrambled at least one ctenophore genome is (Ryan et al., 2013), that’s probably a bit too much to ask…

***

Notes

*Weirdly, the order of expression in time is the opposite of that of the Hox cluster. In both clusters, the “anterior” gene(s), i.e. Hox1-2 or Gsx, are active nearest the front of the embryo, but while anterior Hox genes are also the earliest to turn on, in the ParaHox cluster the posterior gene (Cdx) wakes up first. /end random trivia

**Of course we’ve long known that losing a Hox cluster is not that big a deal, but previously, all confirmed losses occurred in animals with more than one Hox cluster to begin with – a fish has plenty of Hox genes left even after chucking an entire set of them.

***With the obligatory ctenophore caveat…

***

References

Fortunato SAV et al. (2014) Calcisponges have a ParaHox gene and dynamic expression of dispersed NK homeobox genes. Nature 514:620-623

Garcia-Fernàndez J (2005) The genesis and evolution of homeobox gene clusters. Nature Reviews Genetics 6:881-892

Garstang M & Ferrier DEK (2013) Time is of the essence for ParaHox homeobox gene clustering. BMC Biology 11:72

Mendivil Ramos O et al. (2012) Ghost loci imply Hox and ParaHox existence in the last common ancestor of animals. Current Biology 22:1951-1956

Peterson KJ & Sperling EA (2007) Poriferan ANTP genes: primitively simple or secondarily reduced? Evolution and Development 9:405-408

Robinson JM et al. (2013) The identification of microRNAs in calcisponges: independent evolution of microRNAs in basal metazoans. Journal of Experimental Zoology B 320:84-93

Ryan JF et al. (2013) The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science 342:1242592

Hi, real world, again!

The Mammal has emerged from a thesis-induced supermassive black hole and a Christmas-induced food coma, only to find that in the month or so that she spent barely functional and buried in chapters covered in the supervisor’s dreaded Red Pen, things actually happened in the world outside. This, naturally, manifested in thousands of items feeling thoroughly neglected in RSS readers and email inboxes. (Jesus. How many times have I vowed never to neglect my RSS feed again? Oh well, it’s not like unemployment is such a busy occupation that I can’t deal with a measly two and a half thousand articles 😛 )

… earlier tonight, the paragraph here said I wasn’t doing a proper post yet, “just pointing out” a couple of the cooler things I’ve missed. Then somehow this thing morphed into a 1000+ word post that goes way beyond “pointing things out”. It’s almost like I’ve been itching to write something that isn’t my thesis. >_>

So the first cool thing I wanted to “point out” is the genome paper of the centipede Strigamia maritima, which is a rather nondescript little beast hiding under rocks on the coasts of Northwest Europe. This is the first sequenced genome of a myriapod – the last great class of arthropods to remain untouched by the genome sequencing craze after many genomes from insects, crustaceans and chelicerates (spiders, mites and co.).  The genome sequence itself has been available for years (yay!), but its “official” paper (Chipman et al., 2014) is just recently out.

Part of the appeal of Strigamia – and myriapods in general – is that they are considered evolutionarily conservative for an arthropod. In some respects, the genome analysis confirms this. Compared to its inferred common ancestor with us, Strigamia has lost fewer genes than insects, for example. Quite a lot of its genes are also linked together similarly to their equivalents in distantly related animals, indicating relatively little rearrangement in the last 600 million years or so. But this otherwise conservative genome also has at least one really unique feature.

Specifically, this centipede – which is blind – has not only lost every bit of DNA coding for known light-sensing proteins, but also all known genes specific to the circadian clock. In other animals, genes like clock and period mutually regulate one another in a way that makes the abundance of each gene product oscillate in a regular manner (this is about the simplest graphical representation I could find…). The clock runs on a roughly daily cycle all by itself, but it’s also connected to external light via the aforementioned light-sensing proteins, so we can constantly adjust our internal rhythms according to real day-night cycles.

There are many blind animals, and many that live underground or otherwise find day and night kind of irrelevant, but even these are often found to have a functioning circadian clock or keep some photoreceptor genes around. However, based on the genome data, our favourite centipede may be the first to have completely lost both. The authors of the genome paper hypothesise that this may be related to the length of evolutionary time the animals have spent without light. Things like mole rats are relatively recent “inventions”. However, the geophilomorph order of centipedes, to which Strigamia belongs, is quite old (its most likely sister group is known from the Carboniferous, so they’re probably at least that ancient). Living geophilomorphs are all blind, so chances are they’ve been that way for the last 300+ million years.

Nonetheless, the authors also note that geophilomorphs are still known to avoid light – the question now is how the hell they do it… And, of course, whether Strigamia has a clock is not known – only that it doesn’t have the clock we’re used to. We also have no idea at this point how old the gene losses actually are, since all the authors know is that one other centipede from a different group has perfectly good clock genes and opsins.

In comparison with fruit flies and other insects, the Strigamia genome also reveals some of the ways in which evolutionary cats can be skinned in multiple ways. There is an immune-related gene family we share with arthropods and other animals, called Dscam. The product of this gene is involved in pathogen recognition among other things, and in flies, Dscam genes are divided into roughly 100 chunks or exons, most of which are are found in clusters of variant copies. When the gene is transcribed, only one of these copies is used from each such cluster, so in practical terms the handful of fruit fly Dscam genes can encode tens of thousands of different proteins, enough to adapt to a lot of different pathogens.

A similar arrangement is seen in the closely related crustaceans, although with fewer potential alternative products. In other groups – the paper uses vertebrates, echinoderms, nematodes and molluscs for comparison – the Dscam family is pretty boring with at most one or two members and none of these duplicated exons and alternative splicing business. However, it looks like insects+crustaceans are not the only arthropods to come up with a lot of DSCAM proteins. Strigamia might also make lots of different ones (“only” hundreds in this case), but it achieved this by having dozens of copies of the whole gene instead of performing crazy editing feats on a small number of genes. Convergent evolution FTW!

Before I paraphrase the entire paper in my squeeful enthusiasm (no, seriously, I’ve not even mentioned the Hox genes, and the convergent evolution of chemoreceptors, and I think it’s best if I shut up now), let’s get to something else that I can’t not “point out” at length: a shiny new vetulicolian, and they say it’s related to sea squirts!

Vetulicolians really deserve a proper discussion, but in lieu of a spare week to read up on their messiness, for now, it’s enough to say that these early Cambrian animals have baffled palaeontologists since day one. Reconstructions of various types look like… a balloon with a fin? Inflated grubs without faces? I don’t know. Drawings below (Stanton F. Fink, Wikipedia) show an assortment of the beasts, plus Yunnanozoon, which may or may not have something to do with them. Here are some photos of their fossils, in case you wondered.

They’re certainly difficult creatures to make sense of. Since their discovery, they’ve been called both arthropods and chordates, and you can’t get much farther than that with bilaterian animals (they’re kind of like the Nectocaris of old, come to think of it…).

The latest one was dug up from the Emu Bay Shale of Australia, the same place that yielded our first good look at anomalocaridid eyes. Its newest treasure has been named Nesonektris aldridgei by its taxonomic parents (García-Bellido et al., 2014), and it looks something like this (Diego García-Bellido’s reconstruction from the paper):

In other words, pretty typical vetulicolian “life but not as we know it”, at first glance. Its main interest lies in the bit labelled “nc” in the specimens shown below (from the same figure):

This chunky structure in the animal’s… tail or whatever is a notochord, the authors contend. Now, only one kind of animal has a notochord: a chordate. (Suspicious annelid muscle bundles notwithstanding. Oh yeah, I also wanted to post on Lauri et al. 2014. Oops?) So if this thing in the middle of Nesonektris’s tail is a notochord, then at the very least it is more closely related to chordates than anything else.

Why do they think it is one? Well, there are several long paragraphs devoted to just that, so here goes a summary:

1. It’s probably not the gut. A gut would be the other obvious ID, but it doesn’t fit very well in this case. Structures interpreted as guts in other vetulicolians – which sometimes contain stuff that may be half-digested food – (a) start in the front half of the body, where the mouth is, (b) constrict and expand and coil and generally look much floppier than this, (c) don’t look segmented, (d) sometimes occur alongside these tail rod-like thingies, so probably aren’t the same structure.

2. It positively resembles modern half-decayed notochords. The notochords of living chordates are long stacks of (muscular or fluid-filled) discs, which fall apart into big blocks as the animal decomposes after death. Here’s what remains of the notochord of a lamprey after two months for comparison (from Sansom et al. (2013)):

This one isn’t as regular as the blockiness in the fossils, I think, but that could just be the vetulicolians not being quite as rotten.

There is, of course, a but(t). To be precise, there are also long paragraphs discussing why the structure might not be a notochord after all. It’s much thicker than anything currently interpreted as such in reasonably clear Cambrian chordates, for one thing. Moreover, it ends right where the animal does, in a little notch that looks like a good old-fashioned arsehole. By the way, the paper notes, vetulicolian tails in general don’t go beyond their anuses by any reasonable interpretation of the anus, and a tail behind the anus is kind of a defining feature of chordates, though this study cites a book from the 1970s claiming that sea squirt larvae have a vestigial bit of proto-gut going all the way to the tip of the tail. (I suspect that claim could use the application of some modern cell labelling techniques, but I’ve not actually seen the book…)

… and there is a phylogenetic analysis, in which, if you interpret vetulicolians as deuterostomes (which impacts how you score their various features), they come out specifically as squirt relatives whether or not you count the notochord. I’m never sure how much stock to put in a phylogenetic analysis based on a few bits of anatomy gleaned from highly contentious fossils, but at least we can say that there are other things – like a hefty cuticle – beyond that notochord-or-not linking vetulicolians to a specific group of chordates.

Having reached the end, I don’t feel like this paper solved anything. Nice fossils either way 🙂

And with that, I’m off. Maybe next time I’ll write something that manages to be about the same thing throughout. I’ve been thinking that I should try to do more posts about broader topics rather than one or two papers (like the ones I wrote about ocean acidification or homology versus developmental genetics), but I’ve yet to see whether I’ll have the willpower to handle the necessary reading. I’m remarkably lazy for someone who wants to know everything 😀

(Aside: holy crap, did I ALSO miss a fucking Nature paper about calcisponges’ honest to god ParaHox genes? Oh my god, oh my GOD!!! *sigh* This is also a piece of incredibly exciting information I’ve known for years, and I miss it when it actually comes out in a journal bloody everyone reads. You can tell I’ve been off-planet!)

References:

Chipman AD et al. (2014) The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima. PLoS Biology 12:e1002005

García-Bellido DC et al. (2014) A new vetulicolian from Australia and its bearing on the chordate affinities of an enigmatic Cambrian group. BMC Evolutionary Biology 14:214

Lauri A et al. (2014) Development of the annelid axochord: insights into notochord evolution. Science 345:1365-1368

Sansom RS et al. (2013) Atlas of vertebrate decay: a visual and taphonomic guide to fossil interpretation. Palaeontology 56:457-474

Thornbushes

When I discussed sponge microRNAs last week, I said deep animal phylogeny was difficult. Quite fortuitously, another paper went online recently that explores exactly this difficulty (Nosenko et al., 2013). Following on from the microRNA post, I’ll use this paper as an excuse/guide to discuss the tangled relationships of animals.

First of all, let’s recap the problem. My trusty old family tree of animals just so happens to be an excellent illustration:

When I first made this tree to explain what the hell I was talking about re: the Cambrian creature Nectocaris, I put in some question marks mostly out of laziness. To illustrate why the “old” Nectocaris didn’t make sense, I only needed the relationships of bilaterians among themselves. Everything outside the Bilateria was irrelevant to the little creature’s mystery, so I decided to forgo reading up on them and stay on an uninformed fence.

But, in fact, said fence is not just my half-arsed perch. I appear to share it with an entire, very much whole-arsed field. While now there’s a reasonable agreement over ecdysozoans and deuterostomes and all that jazz, the non-bilaterians still wander all over the place depending on how you do your analysis. Nosenko et al. cite a number of recent large-scale studies, and point out that they totally fail to agree where to put poor Trichoplax and jellies of various kinds. The other thing they fail at is deciding how many branches sponges actually represent (the problem the microRNA study I discussed tried to tackle). To illustrate the extent of the chaos, I sketched the phylogenies six recent studies cited by Nosenko and colleagues came up with (sponge lineages are marked by dots):

Remarkably, all six studies agree on the basic deuterostome-ecdysozoan-lophotrochozoan arrangement inside Bilateria in spite of using different sets of bilaterian species. In contrast, the non-bilaterian animals – sponges of all kinds, cnidarians, ctenophores and Trichoplax – appear in pretty much every conceivable configuration.

A plethora of pitfalls

Why? What makes these questions so difficult that datasets made of 100+ genes from dozens of species representing all major animal groups and using the best available methods have this much trouble answering them?

Time is probably not the issue, or at least not in the simple sense of “it all happened too long ago”. The Nosenko paper brings up the example of fungi, which are roughly as ancient (or, in the context of all living things, as young) as animals. Studies that tried to use the exact same set of genes to analyse the relationships within each group could apparently produce a nice clear tree for fungi. Animals? A whole lot of noise.

Perhaps the “tree” of animals is really more like Rokas and Carroll’s (2006) evolutionary bushes, with its base branching so quickly that genes didn’t have time to accumulate many informative changes between one split and the next. Perhaps it even happened so fast that ancient within-species sequence variation was carried through several such events, resulting in what population geneticists call incomplete lineage sorting, a situation where the history of genes is not the same as the history of species.

Perhaps we haven’t got a good enough sample of genes, animals, or both.

If early animal evolution was bush-like, only a large amount of good data has any hope of accurately resolving how it went. But finding suitable genes for phylogenetic analysis is not easy. They have to be known in all of our species. They should have unambiguous identities so we know we’re actually comparing the same gene across species. They should evolve slowly enough that chance hasn’t had time to wash away their records of relatedness.

Likewise, picking suitable species can be difficult. Aside from the availability of sequences, the two greatest problems are taxon sampling and long branches. Good taxon sampling means covering the diversity of a group. So for example, if you have to pick three vertebrates, you don’t want them all to be mammals. A mammal, a shark and, say, a bony fish would be a much more representative sample.

Long branches are the bogeyman of phylogenetics. “Long” here means many evolutionary changes compared to other lineages in your sample. Similarities in gene/protein sequences are not always due to shared ancestry: because there’s a limited number of letters in the DNA and protein alphabets, sometimes they happen just by chance. If you have two unusually long branches, they might have a lot of these chance similarities, many more than either of them shares with its true relatives by common ancestry. Some of the newer changes might also have overwritten the older similarities linking them with their real families, a problem known as saturation. The overall outcome is that long branches attract each other.

Last but not least, perhaps the assumptions we put into our analyses don’t actually fit the data. All phylogenetic analyses are based on a model of evolution. For molecular data, these models specify, for example, how likely different sequence changes are, and which bases or amino acids are commonest and rarest. All analyses also need a way of picking the best tree, which range from simply choosing the one with the fewest changes to choices based on complicated probability theory. Sometimes, models and methods still work reasonably well when their assumptions are violated, but, as you might expect, counting on that is generally a stupid idea.

Nosenko et al. (2013) come to the conclusion that the issue of non-bilaterian animal phylogeny is plagued by pretty much the whole package.

Dissecting the Problem

First, studies may have increased the size of their datasets by incorporating less than ideal genes. To test the effect of gene sampling, Nosenko et al. (2013) divided their collection of 122 genes into two parts. One consisted of genes involved in protein synthesis, mostly genes encoding ribosomal proteins, which all evolve very slowly. The other was a mixed bag of non-ribosomal genes with all sorts of functions and evolutionary rates.

Perhaps not surprisingly, the latter set displayed a much higher level of saturation. Accordingly, when they analysed the ribosomal dataset with models of evolution that are more prone to errors due to saturation, they got the same trees they’d seen using more accurate models on the non-ribosomal data. Clearly, saturation, gene and model choice are affecting the answers they’re getting, and they are all problems that would affect your average phylogenomic study.

Second, the authors found every indication of a serious long-branch problem. In most phylogenetic trees, the longest branch is the outgroup. Outgroups are organisms outside your group of interest (the ingroup). Similarities between the outgroup and members of the ingroup are likely to have evolved before the origin of the ingroup, therefore they can be used to locate the root of the ingroup tree. However, outgroups are rarely sampled as well as ingroups, hence they tend to form long branches, making them a liability.

In the case of animals, removing the outgroup cleared the disagreements between the different gene sets, demonstrating that some of them had been due to long-branch artefacts. (Of course, without an outgroup you don’t know which animal lineages split first, which makes this solution not much use at all for important evolutionary questions like what the common ancestor of all animals looked like.)

Likewise, using a more distant outgroup changed the trees considerably. Ctenophores are worth special mention here. When Dunn et al. (2008) placed these jellyfish-like creatures as the sister group to all other animals, it was an odd, unexpected result. Well, ctenophore genomes evolve ridiculously fast, and there’s a good chance that their position “way out there” is an artefact of that. In Nosenko et al.‘s analyses, they ended up in the Dunn position when the more saturated non-ribosomal data were used – or when the ribosomal dataset was analysed with a more distant outgroup. When everything possible was done to reduce long-branch issues, they stayed deep in the crown of the tree next to cnidarians.

Fourth, the assumptions of even the best evolutionary model don’t take into account an annoying property of protein sequences: their overall amino acid compositions can differ across lineages. Changing the entire makeup of an organism’s protein complement involves changes in evolutionary patterns that none of the models account for. Once again, those damned ctenophores are one of the problem taxa with “deviant” sequence compositions. (The even worse news is that the closest available outgroups also differ from typical animals in this respect.)

Fifth, taxon sampling is influencing what you get. For example, the more sponges Nosenko et al. included, the more support they got for sponges being a single lineage. Ctenophores probably also suffer from this problem. For one thing, they’re very poorly known in almost every way that is relevant to picking species for phylogenetic analysis.

For another, they may actually have an additional problem that is literally impossible to crack – phylogenetic analysis of ctenophores themselves and a look at their fossil record hint that most ctenophore lineages have died out, with existing species all coming from a relatively recent common ancestor. That would make the entire phylum incurably long-branched no matter how many living species you throw at your datasets!

And finally, the ribosomal dataset that was the least prone to long-branch artefacts and the most informative about the deepest branches in animal phylogeny comes with a big caveat: it’s not a random selection of genes. In fact, all of these genes are interacting parts of a single system, which means they might not evolve independently (in the statistical sense). Are they all affected by a common set of biases, and does it render them unsuitable for recovering the true history of animals? We don’t yet know.

Hope dies last…

Being the phylogeny nut that I am, I really enjoyed this dissection of a thorny problem. At the same time, the results are kind of depressing. (Especially if, like me, you’re interested in early animal evolution.) No matter how carefully you set up your analysis, biases lurk around the corner waiting to jump on you and destroy your conclusions. You have a choice between not knowing where to root the tree of animals and being screwed by the outgroup. Well-worn measures of statistical confidence can support contradictory hypotheses. Ctenophores are fucking hopeless.

Is there anything we can do about this conundrum? Nosenko et al. conclude their paper on a somewhat hopeful note. There are other methods in molecular phylogenetics than simple sequence comparison. Although they’ve been no more helpful so far than traditional sequence analysis, we’re getting more and more full genome sequences from all over the animal kingdom. There’s more to look at than ever. Perhaps, one day, we’ll find a tool that can trim this thorny beast of a bush (or bush of beasts?) into shape.

Meanwhile, the quandary of deep animal phylogeny stands as a reminder that science is not all-powerful. The universe is a puzzle, but we have no reason to assume that nature left us enough information to solve it all. Which, as far as I’m concerned, shouldn’t stop us from trying. 😉

***

References:

Dunn CW et al. (2008) Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452:745-749

Erwin DH et al. (2011) The Cambrian conundrum: early divergence and later ecological success in the early history of animals. Science 334:1091-1097

Nosenko T et al. (2013) Deep metazoan phylogeny: when different genes tell different stories. Molecular Phylogenetics and Evolution (in press), doi: 10.1016/j.ympev.2013.01.010

Philippe H et al. (2009) Phylogenomics revivew traditional views on deep animal relationships. Current Biology 19:706-712

Pick KS et al. (2010) Improved phylogenomic taxon sampling noticeably affects nonbilaterian relationships. Molecular Biology and Evolution 27:1983-1987

Rokas A & Carroll SB (2006) Bushes in the tree of life. PLoS Biology 4:e352

Schierwater B et al. (2009) Concatenated analysis sheds light on early metazoan evolution and fuels a modern “urmetazoon” hypothesis. PloS Biology 7:e20

Sperling EA et al. (2009) Phylogenetic-signal dissection of nuclear housekeeping genes supports the paraphyly of sponges and the monophyly of Eumetazoa. Molecular Biology and Evolution 26:2261-2274

Sponges never yield

Ah, those pesky sponges again. Although their lives are rather low on action, these strange animals have found lots of other ways to fascinate me. Borrowed skeletons, mysterious lost Hox genes, wonderful alien shapes and perhaps the oldest fossils that might be animals – sponges have no trouble supplying us with stories.

Sponges are also something of a headscratcher for evolutionary biologists. These days it’s generally agreed that they can be divided into four major groups, the glass sponges (hexactinellids), the demosponges, the calcareous sponges and the homoscleromorphs. My impression is that the close relationship of the first two is also well-established. However, to this day biologists haven’t quite managed to agree whether the three are one lineage to the exclusion of other animals, or two or three separate lineages with some of them being closer to non-sponge animals. The trees below illustrate two possibilities:

This is kind of important if you want to know what kind of organism gave rise to the diversity of animals. If sponges are paraphyletic (some sponges closer to non-sponges, as in the right-hand tree), then the mother of all animals was likely sponge-like, sitting on the seafloor and driving water through its porous body to capture food. In such scenarios, two or three of the deepest branchings of the animal tree run into sponges on one side. The simplest explanation for that is that the ancestors at these branching points were themselves sponge-like.

If, however, sponges are monophyletic (their last common ancestor only gave rise to sponges, as in the left-hand tree), then the last common ancestor of animals immediately branched into sponges and non-sponges, whose living descendants are very different from sponges. Suddenly, guessing what Mummy Metazoa might have been like becomes much harder.

The deepest phylogeny of animals is difficult. We are talking about lineages that diverged over 600 million years ago even by conservative estimates (Peterson et al., 2004), and it’s also likely that their early divergences followed each other in rapid succession. That combination is depressingly good at eroding the useful information in gene and protein sequences (Rokas and Carroll, 2006). So what can a phylogeneticist do?

Picking your genes carefully is one solution – use as many as you can to maximise the information you can glean from them, but don’t use genes that evolve so fast their “information” is basically all noise. Another option is to use so-called rare genomic changes. These are things like gaining and losing bits of genes or insertions of parasitic DNA.

Their advantage is that they are unlikely to occur twice in the same way. If two animals have the same viral sequence between genes A and B, it’s far more likely to indicate relatedness as opposed to chance similarity than having the same letter at position 138 in the sequence of gene A. The principle of parsimony (choose the simplest explanation) is a shitty way of interpreting sequence similarity because there’s a high chance of any given change occurring more than once. It works much better for such unlikely events as gaining a virus in the same spot.

microRNAs look like a pretty good source of rare genomic changes. They are small RNAs encoded in the genome, and they play crucial roles in gene regulation in most animals. Their sequences evolve extraordinarily slowly, so it’s relatively easy to identify them across species despite their tiny size. There’s loads and loads of them – miRbase, the microRNA database, lists 1600 different miRNA genes for humans, yielding over 2000 mature miRNAs after processing. The miRNAs in our genome include everything from ancient types with origins in the mists of the Precambrian to young sequences confined to our close relatives. On top of all that, they are thought to be very difficult to lose. All in all, perfect phylogenetic markers.

Perfect for some cases, that is. Their presences and absences may paint a coherent evolutionary picture for most animals, but don’t ask them about sponges. Robinson et al. (2013) tried…

In their introduction stands this depressing summary of current animal miRNA lore, based on many non-sponge genomes plus that of the demosponge Amphimedon queenslandica:

“None of the thousands of miRNAs thus far discovered in eumetazoans are present in the genome [of] A. queenslandica and none of the eight silicisponge‐specific miRNAs have been described in any eumetazoan (or any other eukaryotic group for that matter).”

(Eumetazoans = all animals except sponges and the Blob; silicisponges = glass sponges + demosponges)

However, that’s only two of the four sponge groups. What about the other two? Are they any more helpful? Might they have silicisponge-like repertoires, supporting sponge monophyly? Or might they be hiding some “eumetazoan” miRNAs, arguing for one history or another involving sponge paraphyly? This is what the authors wanted to find out.

They looked for miRNAs by collecting and sequencing small RNAs from calcareous and homoscleromorph sponges. Two species of the former and one of the latter also have genome projects going, which allowed the researchers to verify the RNAs they found as bona fide miRNAs (miRNA genes have a particular structure that doesn’t all show in the mature RNA product) as well as look for the protein components of the editing machinery miRNAs need to reach maturity.

(Below: the three sponges with newly sequenced genomes. Sycon ciliatum from the Adamska group, Leucosolenia complicata from habitas.org.uk, Oscarella carmela & Oscarella sp. from the Nichols lab.)

Well, the calcarean genomes certainly contained genes for miRNA-processing enzymes, which is a good sign that they also have miRNAs somewhere. So what do those look like?

Overall, the results of Robinson et al.‘s search are a bit disappointing. They used strict criteria to identify miRNAs, since there are plenty of other kinds of small RNA molecules floating around doing stuff in animal cells. According to these criteria, only one miRNA was confidently identified in the calcareous sponges. This was present in both Sycon and Leucosolenia, but the niggardly bastards didn’t share it with either silicisponges or other animals. Leucosolenia may have a second one. A bunch of eumetazoan-like sequences also showed up, but these were probably contamination from actual eumetazoans, since the Sycon genome, which was obtained from squeaky clean lab-grown sponges, had none of them.

Oscarella only yielded two possible miRNAs, neither of which was known from anything else (including the other homoscleromorphs in this study!) Worse, they couldn’t even find the two processing enzymes in the Oscarella genome – they only recovered a small fragment of one. Maybe the genome sequence is just incomplete, which wouldn’t be very surprising. Then again, maybe Oscarella genuinely doesn’t have a functioning miRNA system, and that could be quite interesting.

Either way, now we know something about microRNAs in all the great sponge lineages. It doesn’t look like they’re going to help us sort out deep animal phylogeny, but maybe the very absence of similarities is telling us something. The reason many microRNAs are so conserved in other animals is that they play important roles in fundamental developmental processes, such as specifying cell types. If sponges aren’t really fussed about keeping them, then maybe their development just doesn’t depend heavily on miRNAs. So what do they do with theirs? Why the difference? Questions, questions…

(Incidentally, here’s yet another reason not to just look at one species and make sweeping claims about “sponges”. Here is also a reason to thank the gods for next generation sequencing. Without the ability to quickly and cheaply [for certain values of “cheap”] sequence tons of DNA and RNA from any creature you fancy, half of the story of animal evolution would be hidden in undeciphered strings of DNA in animals too few people care about for a sequencing project. Yay for technology, yay for diversity!)

[P.S.: there’s so much I’ve wanted to write about and didn’t recently. Since I came back from my Christmas break, I’ve spent most of my time buried in work-related literature. Reading more literature was the last thing I wanted to do with my free time. I’m almost done with that, though. No promises, but I’m almost done ;)]

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References:

Peterson KJ et al. (2004) Estimating metazoan divergence times with a molecular clock. PNAS 101:6536-65451

Robinson JM et al. (2013) The identification of microRNAs in calcisponges: independent evolution of microRNAs in basal metazoans. Journal of Experimental Zoology B, advance online publication available 24/01/2013, doi: 10.1002/jez.b.22485

Rokas A, Carroll SB (2006) Bushes in the tree of life. PLoS Biology 4:e352

Another man after my own heart

It’s not terribly hard to turn me into a squealing fangirl. One of the ways is to agree with me eloquently and/or share my pet peeves. Another is to give me lightbulb moments. A third is to disagree with me in a well-reasoned, intelligent way. And finally, if I see you thoughtfully examining your own thinking, you are awesome by definition. Michaël Manuel’s monster review of body symmetry and polarity in animals (Manuel, 2009) did all of the above.

(In case you wondered, that means a long, squeeful meandering >.>)

Manuel writes about the evolution of two fundamental properties of animal body plans [1]: symmetry and polarity. You probably have a good intuitive understanding of symmetry, but here’s a definition anyway. An object is symmetrical if you can perform some transformation (rotation, reflection, shifting etc.) on it and get the same shape. Polarity is a different but equally simple concept – it basically means that one end of an object is different from the other, like the head and tail of a cat or the inner and outer arcs of a rainbow.

I can’t say that I’d thought an awful lot about either before I came across this review, so it’s not really surprising that I had lightbulbs going off in my head left and right while I was reading it. Because I didn’t think deeply about symmetry and polarity and complexity, I basically held the mainstream view I – and, I suspect, most of the mainstream – mostly picked up by osmosis.

That meant I fell victim to my own biggest pet peeve big time – I believed, without good reason and without even realising, that the body plan symmetries of major lineages of living animals represented successive increases in complexity. Sponges are kind of asymmetrical, cnidarians and ctenophores are radially symmetrical, and bilaterians such as ourselves have (more or less) mirror image symmetry, and these kinds of symmetry increase in complexity in this order. Only… they aren’t, and they don’t.

It turns out that this guy not only shares my pet peeve but uses it to demolish my long-held hidden assumptions. Double fangirl points!

Let there be light(bulbs)!

Problem number one with the traditional view – aside from ignoring that evolution ain’t a ladder – is that the distribution of symmetry types among animals is a little more complicated. Most importantly, most kinds of sponges are not asymmetrical. Most species may be, but that’s not the same thing. You see, most sponge species are demosponges, which make up only one of the four great divisions among sponges. Demosponges do have a tendency towards looking a bit amorphous, but the other three – calcareous sponges, glass sponges and homoscleromorphs – usually are some kind of symmetrical. All in all, the evidence points away from an asymmetrical animal ancestor. (Below: calcareous sponges being blatantly symmetrical, from Haeckel’s Kunstformen der Natur.)

The second problem is that my old view ignores at least one important kind of symmetry. Some “radially” symmetrical animals are actually closer to cylindrical symmetry. To understand the difference, imagine rotating a brick and a straight piece of pipe around their respective long axes. You can rotate the pipe as much or as little as you like, it’ll look exactly the same. In contrast, the only rotation that brings the brick back onto itself is turning it by 180° or multiples thereof. A pipe, with its infinitely many rotational symmetries, is cylindrically symmetrical, while the brick has a finite number of rotational symmetries [2], making it radially symmetrical.

Problem number three is that bilateral symmetry is actually no more complex than radial symmetry! What does “complexity” mean in this context? Manuel defines it as the number of coordinates required to specify any point in the animal’s body. In an animal with cylindrical symmetry, you only need a maximum of two: where along the main body axis and how far from the main body axis you are. Everything else is irrelevant, since these are the only axes along which the animal may be polarised. (Add any other polarity axis, and you’ve lost the cylindrical symmetry.)

Take a radially symmetrical creature, like a jellyfish. These also have a main rotational axis and an inside-outside axis of polarity. However, now the animal’s circumference is also divided up into regions, like slices in a cake. How does a skin cell around a baby jelly’s mouth know whether it’s to grow out into a tentacle or contribute to the space between tentacles? That is an extra instruction, an extra layer of complexity. We’re up to three. (Incidentally, here’s some jellyfish symmetry from Haeckel’s Kunstformen. [Here‘s photos of the real animal] A big cheat he may have been, but ol’ Ernst Haeckel certainly had an eye for beauty!)

And with that, jellies and their kin essentially catch up to the basic bilaterian plan. Because what do you need to specify a worm? You need a head-to-tail coordinate, you need a top-to-bottom one, and you need to say how far from the plane of symmetry you are. Still only three! Many bilaterians, including us, added a fourth coordinate by having different left and right sides, but that’s almost certainly not how we started when we split from the cnidarian lineage. (Below: radial symmetry doesn’t hold a monopoly on beauty! Three-striped flatworm [Pseudoceros tristriatus] by wildsingapore.)

Not only that, but Manuel argues that there’s very little evidence bilateral symmetry evolved from radial symmetry. By his reckoning, the most likely symmetry of the cnidarian-bilaterian common ancestor was cylindrical and not radial (more on this later, though). Thus the (mostly) radial cnidarians and the (mostly) bilateral bilaterians represent separate elaborations of a cylinder rather than stages in the same process.

There were a bunch more smaller lightbulb moments, but I’m already running long, so let’s get on to other things.

Respectful disagreement

I think my disagreements with Manuel’s review are more of degree than of kind. Our fundamental difference of opinion comes back to the symmetries of various ancestors and the evidence for them. He argues that key ancestors in animal phylogeny – that of cnidarians + bilaterians, that of cnidarians + bilaterians + ctenophores, and that of all animals – were cylindrical. (Below is the reference tree Manuel uses for his discussion, with symmetry types indicated by the little icons.)

I think he may well be correct in his conclusions, but I’m not entirely comfortable with his reasons. For example, he infers that the last common ancestor of cnidarians and ctenophores was cylindrical. One of his main arguments is that the repeated structures that “break up the cylinder” to confer radial symmetry are not the same in these two phyla. I think this is an intelligent point a smart guy who knows his zoology would make, so disagreement with it becomes debate as opposed to steamrolling [3].

Why I still disagree? As I said, it comes down to degrees and not kinds. Manuel considers the above evidence against a radially symmetrical common ancestor. I consider it lack of evidence for same. The situation reminds me of Erwin and Davidson (2002), which is also one of my favourite papers ever. They raise perhaps the most important point one could make about comparative developmental genetics: homologous pathways could have been present in common ancestors without the complex structures now generated by those pathways being there. Likewise, I think, radial symmetry could have been there in the common ancestor of cnidarians and ctenophores while none of the complex radially symmetrical structures (tentacles, stomach pouches, comb rows etc.) in the living animals were. Perhaps there were simpler divisions of cell types or whatnot that gave rise to the more overt radial symmetry of jellyfish, sea anemones and comb jellies.

In a related argument, Manuel discusses the homology (or lack thereof) of the dorsoventral axis in bilaterians and the so-called directive axis in sea anemones. Sea anemones actually show hints of bilateral symmetry, which prompted some authors (e.g. Baguñà et al., 2008) to argue that this bilateral symmetry and ours was inherited from a common ancestor (i.e. the cnidarian-bilaterian ancestor was bilateral).

I agree with Manuel that the developmental genetic evidence for this is equivocal at best. I even agree with him that developmental genetics isn’t decisive evidence for homology even if it matches better than it actually does in this case. But again, once the genetic evidence is dismissed as inconclusive, he relies on the non-homology of bilaterally symmetrical structures to conclude non-homology of bilateral symmetry. Again, I think this is a plausible but premature inference. Since I’m not sure whether homology or independent origin of bilateral symmetry is the better default hypothesis in this case, and I don’t think the evidence for/against either is convincing, I actually wouldn’t come down on either side as of yet.

But I can see his point, and that’s really cool.

Why else you’re awesome, Michaël Manuel…

Because you have a whole rant about “basal lineages”. I grinned like a maniac throughout your penultimate paragraph. Incidentally, you might have given me another favourite paper – anything with “basal baloney” in its title sounds like it’s worth a few squees of its own!

Because you apply critical thinking to your own thinking. See where we disagreed, non-homology of structures vs. symmetries, evidence against vs no evidence for, and all that? After you made the argument from non-homology of structures, I expected you to leave it at that. And you didn’t. You went and acknowledged its limitations, even though you stood by your original conclusions in the end.

Because you reminded me that radial symmetry is similar to metamerism/segmentation. I’d thought of that before, but it sort of went on holiday for a long time. Connections, yay!

Because you were suspicious about sponges’ lack of Hox/ParaHox genes. And how right you were!

*

Phew, that turned out rather longer and less coherent than I intended. And I didn’t even cover half of the stuff in my notes. I obviously really, really loved this paper…

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[1] Or any body plan, really…

[2] Astute readers might have noticed that a brick has more than one axis of symmetry, plus several planes of symmetry as well. So it’s not only radially but also bilaterally symmetrical. The one thing it certainly isn’t is cylindrical 😉

[3] Not to say I don’t enjoy steamrolling obvious nonsense, but I also like growing intellectually, and steamrolling obvious nonsense rarely stretches the mind muscles…

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References:

Baguñà J et al. (2008) Back in time: a new systematic proposal for the Bilateria. Philosophical Transactions of the Royal Society B 363:1481-1491

Erwin DH & Davidson EH (2002) The last common bilaterian ancestor. Development 129:3021-3032

Manuel M (2009) Early evolution of symmetry and polarity in metazoan body plans. Comptes Rendus Biologies 332:184-209

Beautiful aliens

When you think nature can’t get any more weird and wonderful, count on the deep sea to prove you wrong. Just saw this guy via I fucking love science on Facebook, and wow. What a beauty! (Figure from Lee et al., 2012)

This deep water dish drainer is a harp sponge (Chondrocladia lyra), a newly described member of a group of weird carnivorous sponges. Like so many other creatures at the bottom of the Northeast Pacific, it was captured by the cameras of the Monterey Bay Aquarium Research Institute (here’s the story at MBARI’s own website). Their robotic explorers brought two of the animals to the lab for flesh and blood scientists to take apart and examine in detail, and captured several more on video.

The harp sponge’s slender branches are equipped with tiny hooks that capture small crustaceans for the sponge to engulf and digest. At the branch tips, sperm is made in those little bulbs and then released into the water. The prey-catching snare of branches also intercepts sperm, and it seems that this might trigger the maturation of eggs in the recipient (Lee et al., 2012). Making eggs only when you’ve got hold of some quality sperm is a nice way of conserving energy in a notoriously resource-poor environment!

(… and I think the creature looks totally Ediacaran, even if it doesn’t really resemble any particular Ediacaran fossil. Don’t ask how I make these associations 😛 Either way, it’s gorgeous!)

Incidentally, I was googling for images of other Chondrocladia species, and quite unexpectedly I stumbled on a lovely piece of art inspired by one. This might be even cooler than space Anomalocaris:

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Reference:

Lee WL et al. (2012) An extraordinary new carnivorous sponge, Chondrocladia lyra, in the new subgenus Symmetrocladia (Demospongiae, Cladorhizidae), from off of northern California, USA. Invertebrate Biology early view, available online 18/10/2012, doi: 10.1111/ivb.12001

An ode to sponges, skeletons and bacteria

Sponges are not what you’d normally think of as “exciting” animals. They are simple creatures that spend the entirety of their adult lives sitting around, patiently sifting immense amounts of water for microscopic food. The closest most of them get to “doing” anything is popping out a few babies every now and then. (Exception: deadly shrimp-killin’ predators :o) However, these (mostly) placid filter feeders have a lot to offer once we move past the usual coolness filters that make our inner ten-year-old a Velociraptor fan*.

I’ve been getting quite fond of sponges recently. It’s mostly a byproduct of the reading I do for my work, which partly concerns the mineralised hard parts of animals. All sponges have skeletons, and the majority of them make hard(ish) skeletons from one of two minerals: either amorphous silica (think glass) or calcium carbonate (think chalk, limestone, clam shell, etc.) (The rest, including bath sponges, use proteins.) Siliceous sponges in the class Hexactinellida (= glass sponges proper) can have beautiful, intricate skeletons like this one from a Venus’s flower basket (Euplectella sp. by NEON ja, Wikimedia Commons):

They are not only gorgeous, but, at least in some cases, also insanely strong and bendy – nature’s fibreglass fishing rods, if you like. See this photo from sponge guru Werner Müller’s group for a demonstration. That glass rod is the skeleton of Monorhaphis chuni, a deep-sea glass sponge that anchors itself with the largest known single structure made of silica in the living world. This “giant spicule” can be up to 3 m long, and flexible enough to bend around in a circle (Levi et al., 1989).

Some sponges have both glassy and calcareous (or “chalky”, if you like) skeletons. And such sponges are giving me all kinds of squee moments lately. Something I’ve only learned recently is that sponges often live in close association with a variety of bacteria. Now it turns out that these symbiotic bacteria contribute to their skeleton-building abilities!

Last year, Dan Jackson and his team published evidence that a sponge species stole a gene it uses to make its calcareous skeleton from a bacterium (Jackson et al., 2011). The gene in question occurs only in bacteria – and sponges. While the sponge species used in the study does harbour bacteria in the cells that produce the calcareous portion of its skeleton, multiple lines of evidence indicate that the gene in question sits in its own genome, and has done so for a long time. It is only active in the skeleton-forming cells, and its protein product is present in bits of skeleton isolated from the animal, suggesting that it does in fact function in building the skeleton. (As of that study, its exact role is still unknown.)

(Above: Astrosclera willeyana, coralline sponge and convicted gene thief. The living animal forms a crust over an ever-growing bulk of dead skeleton. From Jackson et al. [2011])

Most recently, another “spongy” research team found that members of a different sponge lineage have the actual bacteria in their cells make their skeletons for them. Uriz et al. (2012) examined three species of crater sponges, belonging to the “siliceous” sponge genus Hemimycale. In certain cells of the animals, they saw tiny round objects that molecular genetic tests revealed to be bacteria. The bacterial cells were surrounded by a coat of varying thickness that, when the researchers probed its elemental composition using X-rays, proved to be made of calcium carbonate. According to their observations, the bacteria live and divide inside membrane-enclosed vacuoles. They accumulate calcareous material as they mature, and finally the host cell spits them out to form a mineral crust around the animal. (Below: colonies of Hemimycale columella, one of the three species used in the study, from the Encyclopedia of Marine Life of Britain and Ireland via Encyclopedia of Life)

The bacteria look like they’ve had a long-standing partnership with their host sponges. They were abundant in all examined individuals of all three species. Unlike free-living bacteria, they appear to lack cell walls. They are also inherited by baby sponges. Mother Hemimycale sponges nurture their embryos in their bodies (apparently this is common among sponges). Sponges provide their embryos with so-called nurse cells, which, in the case of these species, contain some mineral-making bacteria. The young sponge eventually eats the nurse cells, thereby acquiring the bacteria. By the time it becomes independent and settles on a comfortable rock, its body is littered with tiny mineral spheres made by its inherited symbionts.

On closer examination, it seems that Hemimycale is far from the only sponge genus to harbour similar hired skeleton-builders. Uriz and colleagues tell us that they have found previously overlooked evidence of such “calcibacteria” in several other sponges – one of which is only distantly related to Hemimycale. Could calcibacteria be ancient partners of these animals, inherited by many different sponges from a distant common ancestor? Could bacteria even hold the key to the origin of calcareous animal skeletons?

(FWIW, I don’t really buy the second idea. As far as I know, all non-sponge animals that have been investigated make their skeletons with their own genes – nothing suspiciously bacterial-like the way Jackson et al.‘s spherulin is. [Caveat: there remain plenty of groups that haven’t been investigated in sufficient molecular detail.] However, the idea that sponges as a whole may have acquired their calcareous skeletons this way is fascinating. Incidentally, though the ID isn’t 100% certain yet, the calcibacteria may belong to the same bacterial class as mitochondria and these insidious bastards. Do alpha-proteobacteria have a special knack for endosymbiosis?)

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*Not to say Velociraptor isn’t cool, but being a vicious toothed, raptor-clawed killer bird is, well, not the only road to coolness 😛

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ETA: 42nd post, yay! (Also yay: random Hitchhiker’s Guide reference in completely unrelated post :D)

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References:

Jackson DJ et al. (2011) A horizontal gene transfer supported the evolution of an early metazoan biomineralization strategy. BMC Evolutionary Biology 11:238

Levi C et al. (1989) A remarkably strong natural glassy rod: the anchoring spicule of theMonorhaphis sponge. Journal of Materials Science Letters 8:337-339

Uriz MJ et al. (2012) Endosymbiotic calcifying bacteria: a new cue to the origin of calcification in Metazoa? Evolution early online view, doi: 10.1111/j.1558-5646.2012.01676.x

The shadow of a skeleton

Sponges are in a generous mood these days, as far as exciting discoveries are concerned! First Otavia breaks the record for oldest known animal, and now Coronacollina (what a pretty name!) shows up with what looks like the oldest hard skeleton in the animal kingdom.

Hard skeletons* are a real success story in the history of life. From the tough organic support structures of trees to our own strong and versatile bones, they’ve revolutionised (or, in the case of trees, pretty much created) ecosystems. (We also owe them some gorgeous landscapes.) Skeletons really came into fashion during the Cambrian explosion, when incorporating minerals into shells, spikes and other hard parts became commonplace among animals. However, there are a few examples of animals with hard parts that are older, mainly from the very end of the Ediacaran period just before the dawn of the Cambrian. Our spiny new friend does one better than those, hailing from the heyday of Ediacaran creatures.

Coronacollina acula (Clites et al., 2012) is described as a smallish creature similar to the Cambrian sponge Choia. Its 300+ specimens were preserved as imprints that show every sign of having come from a fairly solid animal. The body is kind of cone-shaped with what appears to be threefold symmetry. Most intriguing are the traces of long, thin spikes that radiate from the main body of many specimens. There are up to four of them, fewer than Choia had, and they were clearly made of a hard material in life: the grooves they left are straight as arrows, narrow and sharply defined, unlike a trace left by a soft structure. Like the more numerous spikes of Choia, they may have acted as stabilisers/struts to keep the living sponge from being upended by waves.

(From my perspective, it’s a pity that only the imprints were preserved. I have an occupational interest in biomineralisation, so I’d really like to know what the spicules were originally made of. If Coronacollina is a relative of Choia, odds are they were either organic or, if they were mineralised at all, made of silica. Interestingly, the authors bet on some sort of mineral because the spicules broke so often, as though they were quite brittle. I would’ve thought that mineralised structures would leave more than imprints, but apparently the chances of silica or calcium carbonate skeletons being preserved in coarse sandstone aren’t that great. You learn something every day…)

Clites and colleagues consider the creature important for two reasons: first, because it is the oldest known example of an animal with a hard skeleton. The shadows of its long thin spikes in the rocks foreshadow, so to speak, of the age of skeletons that came with the Cambrian. Second, finding an Ediacaran animal that can be related to something outside its own weird contemporaries is always worth a little celebration! 😉

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Note:

*The word “skeleton” is used in a very loose sense here. It includes any hardened structure that gives support and/or protection to some part of an organism. Bones, shells, armour plates, teeth, perhaps even the protein meshwork that gives bath sponges shape, can belong here.

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Reference:

Clites E. et al. (2012) The advent of hard-part structural support among the Ediacara biota: Ediacaran harbinger of a Cambrian mode of body construction. Geology advance online publication (doi: 10.1130/G32828.1)

Of really old maybe-sponges, molecular clocks and common ancestors

If you’ve ever visited this blog before, you probably know that the early evolution of animals is one of my many random interests. You could say it’s my main interest, though that may be less obvious from my posting record so far. Well, knowing that, you could imagine my face when my labmate pointed me to this National Geographic news piece.

It doesn’t surprise me much that the earliest known animal would be like a sponge. Although for what they do, their construction is nothing short of ingenious, sponges are comparatively simple animals. While it’s possible that they weren’t always like that, it appears that their genomes are devoid of lots of the genes other animals have added to the “toolkit” that fashions their complex bodies (Larroux et al., 2008). They also retain morphological features that were probably present in the ancestors of animals and lost in pretty much every other animal lineage alive today. Notably, their food-capturing cells look an awful lot like the cells of choanoflagellates, which are thought to be the closest living relatives of animals and perhaps similar in appearance to our distant ancestors.

What looks positively amazing about the newly described sponge-like thingies, who go by the deceptively Italian-sounding name of Otavia (they’re actually named after the Otavi Group of rock formations in Namibia), is their age. The oldest ones, apparently, are close to 760 million years old, perhaps 180 million years older than the earliest occurrences of the famous and mysterious Ediacaran animals (Narbonne, 2005). (By the way, that difference is about the length of the “age of dinosaurs”!) The news, and Brain et al. (2012), point out that this date also precedes some events that were thought to set the stage for the rise of animals: the giant ice ages known as Snowball Earths, and the rise in atmospheric oxygen levels towards the end of Precambrian times.

We could talk about the significance of that, I suppose, but the issue the whole discovery brought to my mind is, strangely, molecular clocks.

Let’s face it, the Precambrian fossil record of animals is not brilliant. It’s getting better, as more Ediacaran fossils are dug up and analysed with more sophisticated methods, but it still raises as many questions as it answers, and the earliest history of animals is still shrouded in mystery. For one thing, when did animals even evolve? All we know from fossils is that it must have been “before”. If a particular fossil is not only an animal, but member of an identifiable subgroup of animals, it means that the branch separating that subgroup from all other animal lineages must have split by that time. A number of Precambrian animals may be members of groups that are many such splits into the animal family tree, and things that look like the predecessors of those splits are difficult to identify in the fossil record. So where did they come from? Where did it all begin? Kind of hard to say based on the bunch of hard to interpret blobs, fronds and strange fractal bodies that is the Ediacaran biota.

When the fossil record speaks gibberish, people sometimes query another keeper of deep evolutionary history: DNA. Molecular clock methods date splits between lineages by counting differences between their living members. The basic idea is this: if most mutations have no effect on fitness, then most mutations are created equal, with the same chance of fixing themselves in the gene pool. If that is true, then genomes change at roughly constant rates – dependent only on mutation rate – over time. Using that assumption and lineages whose divergence time is known (usually, from good fossil records), you can translate the genetic differences between two or more groups into evolutionary time.

The problem is that real life is not so simple as that. Evolution is not always neutral. The same gene may behave like a clock in one lineage or during one time period and not another. Part of a gene may be a good clock while another part isn’t. Even if all of a gene evolves in a clock-like manner in all lineages under study, there’s no guarantee that the clock will tick at the same rate in all of them. Different genes or parts of a gene can tick at different rates, and this can vary over time. If we’re trying to measure very long times, it can be hard to correctly estimate the amount of change in a gene. There can be error in the fossils used for calibration, or the calibrating lineages may evolve differently from the ones we’re interested in. And so on.

And thus, published estimates for early divergences among animals range from numbers that make reasonable sense with the fossil record (e.g. Peterson et al., 2004), to some that throw another billion years on top of those numbers (see Chapter 11 in Knoll [2003] for an accessible discussion).

The problem, as I see it, is this. With a billion-year margin of error, some of those estimates must be wrong. As Andrew Knoll noted, they all require that animals began much earlier than their fossil record (at least as it was known at the time). How can we trust any of them? Even for the ones that match what we think of the fossil record – well, stopped clocks are accurate twice a day. For a scientist, being accidentally right is no better than being wrong.

I suppose Otavia, if it’s really a sponge-ish creature, fits the Peterson & co. estimates quite neatly. Fairly certain bilaterians like Kimberella are known from the White Sea assemblage of the Ediacaran, somewhat under 560 million years ago (Narbonne, 2005). The origin of bilaterians is somewhere between two and four splits[1] after sponges diverged from other animals. Peterson and colleagues estimated it between 573 and 656 million years ago – so if sponges are indeed a conservative bunch, sponge-like animals must have been around quite a bit earlier, but perhaps >1 billion years ago is really stretching it. 760 million sounds kind of nice, farther back than the Kimberellas and Dickinsonias but not too far.

Kind of. But, seeing as we’ve had to wait this long for a maybe-sponge that old, who’s to say even older animals aren’t hiding in some unexplored fossil bed? Who’s to say that the next “oldest animal” find won’t validate some of the more outlandish estimates?

The other thing I’m wondering about re: Otavia is: is it a sponge (assuming it’s an animal at all), or could it belong to a lineage ancestral to both sponges and other animals? (Were early sponges ancestral to other animals? The idea has been played with in phylogenetic circles…) I guess we’ll never know for certain. I still think it’s worth raising the question. Creatures that might be ancestral to more than one phylum are extremely valuable to evolutionary biologists, but they might be very hard to recognise for what they are. Part of the problem with Ediacaran animals is that many if not most of them lack features associated with living phyla – but that’s exactly what we would expect from creatures that preceded the divergence of those phyla! Given how little, say, a jellyfish and a snail have in common, what on earth would their common ancestors look like? Would they have any fossilisable characteristics at all that could give us a hint as to their family ties?

And I guess I’ll close today’s musings with that question. If I spent more time reading Brain et al. (2012), there’d probably be a lot more to discuss, but after doing lab work all day and spending an extra couple of hours writing this, my brain doesn’t feel up to it 🙂

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[1] You can refer to the rough animal phylogeny in the Nectocaris post for the moment. Being slightly out of the loop in this area, I wouldn’t hazard a guess as to the relationships of ctenophores, cnidarians and placozoans, hence my uncertainty. It’s possible that these three all form a single branch with bilaterians on the other side. Or they could represent three different branching events, or anything in between. I really should make an animal phylogeny page, I think, since I keep finding myself wanting to talk about bilaterians and lophotrochozoans and things that don’t make much sense unless you know at least the basics shown in tree I made for Nectocaris…

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References:

Brain CK et al. (2012) The first animals: ca. 760-million-year-old sponge-like fossils from Namibia. South African Journal of Science 108:658; doi:10.4102/sajs.v108i1/2.658

Knoll AH (2003) Life on a Young Planet. Princeton University Press.

Larroux C et al. (2008) Genesis and expansion of metazoan transcription factor gene classes. Molecular Biology and Evolution 25:980-996

Narbonne GM (2005) The Ediacara biota: Neoproterozoic origin of animals and their ecosystems. Annual Review of Earth and Planetary Sciences 33:421-442

Peterson KJ et al. (2004) Estimating metazoan divergence times with a molecular clock. PNAS 101:6536-6541