The gang-of-four ready to take over the ant world. From left: Philip Ward, Seán Brady, Ted Schultz and Brian Fisher at the IUSSI congress in Sapporo, Japan.

It was at the XIV international meeting of the International Union for the Study of Social Insects in 2002 that the “gang of four” decided to join forces to reconstruct the phylogenetic history of ants using molecular data.  Four years later Brady et al. 2006 was published.

Now Gregory M. Erickson and co-workers published a paper in which they did just that to a specimen of one of the most famous fossil forms around: Archaeopterix. Watch Mark Norell, paleontologist from the American Museum of Natural History and co-author of the paper, explain the results:

Published online before print August 28, 2009, doi: 10.1073/pnas.0908357106

Caterpillars evolved from onychophorans by hybridogenesis

Donald I. Williamson
Marine Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom

Abstract:
I reject the Darwinian assumption that larvae and their adults evolved from a single common ancestor. Rather I posit that, in animals that metamorphose, the basic types of larvae originated as adults of different lineages, i.e., larvae were transferred when, through hybridization, their genomes were acquired by distantly related animals. “Caterpillars,” the name for eruciforms with thoracic and abdominal legs, are larvae of lepidopterans, hymenopterans, and mecopterans (scorpionflies). Grubs and maggots, including the larvae of beetles, bees, and flies, evolved from caterpillars by loss of legs. Caterpillar larval organs are dismantled and reconstructed in the pupal phase. Such indirect developmental patterns (metamorphoses) did not originate solely by accumulation of random mutations followed by natural selection; rather they are fully consistent with my concept of evolution by hybridogenesis. Members of the phylum Onychophora (velvet worms) are proposed as the evolutionary source of caterpillars and their grub or maggot descendants. I present a molecular biological research proposal to test my thesis. By my hypothesis 2 recognizable sets of genes are detectable in the genomes of all insects with caterpillar grub- or maggot-like larvae: (i) onychophoran genes that code for proteins determining larval morphology/physiology and (ii) sequentially expressed insect genes that code for adult proteins. The genomes of insects and other animals that, by contrast, entirely lack larvae comprise recognizable sets of genes from single animal common ancestors.

I think Lynn Margulis went too far this time…

The phylogenetic analysis comprises an astonishing 73,060 terminal eukaryotic taxa, 9535 molecular characters and, for good measure, they threw in 604 morphological characters. It is therefore the largest phylogenetic analysis published to date and almost six times larger than the former world record. Such feat presented many technical challenges. The logistics required the automatizing of every step in the analysis, via computer scripts, to retrieve and sort thousands of GenBank entries, to align the sequences to construct the data matrix, to perform the actual searches for the optimal solutions, and to interpretation of the mammoth-size phylogenetic trees. The crux of the analysis, the search for the optimal phylogenetic trees, was done with the powerful parsimony phylogenetic program TNT running in parallel in three multi-processor computers for 2.5 months.

Fig. 1. Pruned strict consensus tree for the combined data set (seven trees, 1879 taxa excluded). The bar shows the span of 5000 species.

The resulting phylogeny recovers most traditional taxonomic groups. This is interesting for various reasons. First, as noted about, our understanding of the tree of life is the results of many taxonomically localized efforts that have been informally pasted together¹. This is the first time a phylogeny has been reconstructed from scratch, letting the data speak unconstrained for itself without assuming that certain evolutionary relationships most be true a priori. Second, it shows that there is enough historical information contained in the data so that the optimal solution is not a complete mess or largely unresolved answer– consider that there are 9 X 10^345,593 possible tree combinations for the number of terminals included. Third, that we do have the current capacity, both in terms of software and hardware, to carry out such a large analysis. And last, but related to the previous two points, that parsimony methods for phylogenetic reconstruction are up for the task. The latter point is worth noting because early simulations, based on just a few taxa (a grand total of four actually) scared systematists into thinking that parsimony methods may result in erroneous reconstructions. Later studies using real data and a much larger collection of species has shown that this is not the case, and this 73,060 taxa analysis serves as the largest of these test cases.

The authors are no strangers when it comes to computer implementation of phylogenetic methods. James S. Farris is a pioneer in the field who developed the algorithmic foundations and produced the some of the first phylogenetic programs in the late 1960s, when the character information for each taxon to be analyzed was contained in a punch card and random addition sequence for the phylogenetic tree construction meant that the set of cards was shuffled by hand before feeding them into the terminal connected to the mainframe. Likewise, Pablo A. Goloboff has been responsible for many of the rapid search techniques developed during the 1990s up to the present, that seek to cover the searchable tree-space in a fast and efficient way.

It seems that, for phylogenetics, the only limit that remains is the availability of data.

References and notes

Goloboff, P., Catalano, S., Marcos Mirande, J., Szumik, C., Salvador Arias, J., Källersjö, M., & Farris, J. (2009). Phylogenetic analysis of 73 060 taxa corroborates major eukaryotic groups Cladistics DOI: 10.1111/j.1096-0031.2009.00255.x

Tapinoma simrothi worker. The rectangle shows the opening location of the left metapleural gland (Scanning Electron Micrograph, Roberto Keller/AMNH)

The metapleural gland is the definitive character of ants. It is unique to the family. Nothing homologous or similar is found anywhere else in insects. Within the tree of life of Hymenoptera, myrmecologists agree that the appearance of this gland provides a good cutting point to marks-out ants as a monophyletic group¹. You have it? You are an ant. You don’t? Sorry, you don’t qualify, get the hell out of here lousy wasp². It is the ultimate ant synapomorphy.

Metapleural gland opening of a Tapinoma simrothi worker, left side. Note the turf of setae (in blue) arising from inside the storage chamber (Scanning Electron Micrograph, Roberto Keller/AMNH)

The gland is a paired organ hosted inside the ant’s mesosoma. Each side consists of a cluster of glandular cells, the content of which is drained by membranous tubes into a rigid storage chamber³. The chamber is a relatively large structure in itself and its presence is usually marked on the outside as a swelling of the posterolateral corners of the thorax. The most visible evidence of its presence is, however, the wide opening of the chamber to the exterior. This has allowed us to confirm, for example, the occurrence of this glandular structure in ancient fossil ants where soft tissues are not preserved.

Storage chamber of the metapleural gland in a Tapinoma simrothi worker, left side. Internal turf of setae in blue; inner surface of cuticular skeleton in light green (Scanning Electron Micrograph, Roberto Keller/AMNH)

Upon removing parts of the mesosoma and cuticular wall of the storage chamber, we can appreciate how the atrium of this organ is formed by an invagination of the metapleuron (i. e., the lateral skeletal wall of the third thoracic segment), hence the gland’s name. We can also see that, in this species, there is a group of setae (in blue) that arise at the deepest end of the chamber and project towards its opening. I cleared off the skeleton of any soft tissue before taking this micrograph, but if you dissect an ant previously pickled in ethanol under a light stereoscope you will see the clusters of glandular cells inside as a pair of tight cotton balls (white and everything).

Now, this mass of glandular cells (of the same size as the chamber itself in this exemplar species) sits exactly at the place where the setae arise but on the inner surface of the chamber’s wall, colored here in light green (technically the chamber’s cavity is still the outside of the body). Thus, the cells float inside the body in the nutrient-rich haemolymph, anchored to the storage chamber by way of the membranous ducts. The gland’s secretion is discharged into the chamber via tiny pores in the chamber’s wall (which can be seen at higher magnification in the image below), and carried to the opening of the chamber by surface tension through the projecting setae.

Pores in the inner cuticular wall of the storage chamber (light green). Membranous ducts in white. Myrmecia brevinoda worker (Scanning Electron Micrograph, Roberto Keller/AMNH)

What does this specialized gland secretes? The gland’s secretion seems to act primary as an antiseptic against microorganism, with some ants producing considerable amounts of phenylacetic acid. Secondarily, the secretion has been demonstrated to act as an alarm signal, triggering a defensive response by nestmates⁴. Some authors attribute the ecological success of ants to this evolutionary innovation. Other social insects (bees and wasps) rely on elaborate and costly cells produced from wax and paper to rear their young in dry, protective enclosed environments that prevent the growth of harmful bacteria and fungi. Ants on the contrary will nest in wet soil and leave their larva laying down in the mud, to the dismay of concern parents. The entire ant nest is kept fairly sterilized by the constant secretion of antiseptics by the adult females, hence allowing ants to nest almost anywhere.

The metapleural gland has been lost at least twice in some arboreal and dry-wood nesting ants (In the weaver ant Oecophylla and in Camponotus carpenter ants and related genera) and in some species that are social parasites of other ants. This is consistent with the ecological success hypothesis in that the arboreal nesting provides a much drier habitat, while in parasitic life you let the host do the sterilizing for you along with filling all your other needs.

Back to structure, I know Barry Bolton was interested in the internal anatomy of the metapleural gland for taxonomy, since some years ago he showed me some dissections he had made on a couple of Ponerinae ants while I was visiting the Natural History Museum. I did a more extensive survey of the structure throughout the family during my doctorate research, but still too patchy to be useful for phylogenetic reconstruction— a comprehensive survey would require dissecting material from many groups known by a few, precious specimens in museums. But I can tell you that the morphological complexity of the metapleural gland across ants is remarkable. Compared to its true sophisticated nature, the phylogenetic information we currently derive from it is shamefully little and rough: basically if the opening is directed laterally or backwards. Oh yeah, and there is an external flap involved. My assessment is that there is great potential for cladistic characters useful at various levels of the phylogenetic hierarchy, notably between some of the subfamilies that remain grouped by tiny branches in our phylogenies.

The spherical storage chamber illustrated here with Tapinoma (and representative of what is found in Dolichoderinae, Formicinae and Aneuretinae) is as simple as this organ gets, though this is not necessarily the plesiomorphic condition for the family. From there, the morphology of the chamber goes crazy. Most other ants (e.g., Myrmeciinae, Pseudomyrmecinae, most Myrmicinae and most Ponerinae) have an elongated, sausage-shaped chamber occupying a considerable part of the mesosoma. In most dracula ants amblyoponines the chamber is a wide ball, although genera like Myopopone have a finger-like structure. In some Pachycondyla species (e.g., Pachycondyla porcata) and related genera it coils clockwise, while in Ectatomminae the chamber coils counterclockwise (see image below). Ecitoninae ants have an elongated chamber but there is a system of tracheal-like branching tubes arising midway through it.

Ectatomma tuberculatum worker (April Nobile, antweb.org)

Even the presence of a turf of setae peeking through the chamber’s opening is complex and deceiving. For example, Myrmecia ants have a group of setae that exactly correspond to the one pictured here in Tapinoma. But since the elongated chamber is three times larger the apices of the setae never reach the opening and won’t be seen by exterior inspection. Conversely, acacia ants (Pseudomyrmecinae) have an apparent group of setae coming out of the chamber, but closer inspection reveals that these setae arise right at the chamber’s opening rather than truly from the inside.

A great morphological project for anyone interested would be a detailed comparative study of the internal morphology of the metapleural gland. The result will be a reconstruction of the complex evolutionary history of an important and interesting structure that was without a doubt key in the diversification of the group.

Further reading and notes.

This is an excellent example of the way systematic papers should be. In the latest issue of the Proceedings of the National Academy of Sciences (USA), Blackledge and coworkers assembled a comprehensive data set for cladistic analysis of orb web spiders that includes six different molecular loci, 143 morphological characters and behavior in the form of characters derived from web architecture.

Hypothesis of web architecture evolution as optimized in preferred phylogeny (from Blackledge et al. 2009: fig. 2).

The resulting picture supports a single origin of aerial orb webs from irregular webs constructed in the ground. There is a subsequent evolution to more economical, irregular aerial web architectures from the more costly, regular orb types at least three times independently. And there seems to be an instance of evolution towards the simplified aerial web spun by bolas spiders.

However, apart from the nice evolutionary story, the real treat in this paper is hidden in the small text and supplementary information. All the data compiled would have been useless if analysed with poor methods. Instead the authors performed a series of sophisticated phylogenetic techniques that, besides vanilla Bayesian and parsimony analyzes, included implied weighting for the morphology partition and direct optimization for the molecular data. Morphology and molecular data were analyzed separately and in combination for an impressive total of 64 different types of phylogenetic analyzes.

Implied weights and direct optimization analyzes are worth remarking here because they are not well known and still rarely used in flashy high ranked papers. While in a regular parsimony analysis all characters are given equal weights regardless of how well or how poorly they fit a given tree, in implied weights analysis characters are downweighted as a function of the amount of homoplasy (extra steps) that is required to explain their distribution on any given tree topology during the tree-search phase. It is an a posteriori type of character weighting. One of the rationales behind this methods is that one extra step in a character that already performs very poorly (is very homoplasious), should not be counted equally as one extra step in a character with almost perfect fit. The method was developed in the early 1990′s but is has had a resurrection of late for morphological data, curiously because with the rise of molecular analyzes many authors have noticed that the results of analyzes of morphology under implied weights mirror more closely the molecular phylogenies.

For the molecular data, the use of direct optimization techniques is simply a way to push the limits on finding the optimal correspondences among the positions of DNA sequences under comparison. Multiple sequence alignment methods tend to find just one of the many possible optimal solution (if not suboptimal) from which a regular phylogenetic analyzes (parsimony, maximum likelihood or Bayesian) is then performed during a second phase. In contrast, direct optimization side-steps alignment altogether by searching for the optimal correspondences among sequences during tree search in a simultaneous single step process, thus performing a much more aggressive evaluation of the multiple possible alternatives. It is computational intensive, but this is hardly an excuse for good science as exemplified in this paper.

The result of applying these methods is a well supported phylogeny that allow the authors to make a rigorous reconstruction of the evolution of spider silk, bringing together information from silk chemistry, spider morphology and behavioral ecology.

Don’t forget to take a look at the supplementary information. There are lots of nice pictures showing all the types of web architectures. Oh, and its open access, so no subscription required.

Reference

Blackledge, T., Scharff, N., Coddington, J., Szuts, T., Wenzel, J., Hayashi, C., & Agnarsson, I. (2009). Reconstructing web evolution and spider diversification in the molecular era Proceedings of the National Academy of Sciences, 106 (13), 5229-5234 DOI: 10.1073/pnas.0901377106

Courtesy of Rabeling & Verhaagh/PNAS. Used with permission.

The recent description of the new and unusual ant species from Brazil Martialis heureka, caused furor in the popular media. It was entertaining to watch how, like the children’s game of Chinese whispers, the report rapidly deteriorated and became increasingly sensationalistic as it spun through news agencies around the globe. Reports ranged from accurate and informative to down right silly, with some newspapers almost claiming that the species actually originated in Mars (You can read more about it at Myrmecos blog and comments therein).

I have to say, I appreciate the medias attention to insect science no matter how distorted it gets. But now that the news storm has settle we can point out some other good news about Rabeling, Brown, and Verhaagh’s paper.  News that may not make for a good newspaper headline but that are nevertheless relevant to specialists in ant systematics.

Martialis heureka is the first ant species to be placed into our existing scheme of classification for the ant family in a strict phylogenetic way, with the use of quantitative methods, since the publication of the large scale phylogenetic projects for ants by Moreau et al. (2006) and Brady et al. (2006). That is, we now have a system in place, with enough molecular markers across enough ant taxa, that makes possible taking an ant species from which we know nothing about and “plug it in” to find out its evolutionary position relative to the rest of the family. These are good news also because it shows that the large scale Tree-of-life efforts for Formicidae are working as intended: rather than been the final saying about ant phylogeny, they created a platform from which our taxonomic knowledge can grow phylogenetically as more species are added to the pool of data.

One can agree or disagree about M. heureka position as sister to the rest of extant ants (I have my reservations, subject for another post), but that is beyond the point. The data is there and the methods are transparently laid out for anyone to scrutinize, reproduce and tinker with if so one wishes. This contrast with the still common way of doing taxonomy base of notions of overall similarity or favoring some characters over another without a clear method. These taxonomic decisions can be very well informed guesses, made by specialists with decades of experience, but they are guesses nevertheless, and as such difficult to assess (or challenge) by the rest of the community.

Not all is glossy though. The paper on M. heureka also highlights a big piece that is missing in our system: morphology. Our vast knowledge of ant morphology is not yet structured into a coherent system equal to the molecular one. Ideally, such system would allows us to plug-in a species by comparing its anatomical features to a pool of existing morphological data, score characters, and combine it with molecular markers for phylogeny reconstruction.

Courtesy of Rabeling & Verhaagh/PNAS. Used with permission.

The impact of this vacuum is apparent in M. heureka‘s paper. What constitute unique derived features in this species and which ones are shared primitive (and potentially ancestral to all ants) remains speculative- surely those specialized mandibles (unique among ants) are derived, but it is not clear that having one tibial spur per leg (as opposed to two) is an apomorphy for this lineage, for example, since this character occurs on and off scattered across the family.  Such uncertainty greatly impacts our attempts to reconstruct how ants first evolved and how they looked like, even when we have what seems to be a vital piece of the puzzle. And I am not even bringing up the f-word.

All in all, ant systematics seems to have gotten into great shape in the last couple of years and the publication of M. heureka by Rabeling and coworkers is a welcome attest to this.

References

Rabeling, C., Brown, J. M., and Verhaagh, M. (2008). Newly discovered sister lineage sheds light on early ant evolution. Proceedings of the National Academy of Sciences. doi: 10.1073/pnas.0806187105

Brady SG, Fisher BL, Schultz TR, Ward PS (2006). Evaluating alternative hypotheses for the early evolution and diversification of ants. Proc Natl Acad Sci USA 103:18172–18177. doi: 10.1073/pnas.0605858103

Moreau CS, Bell CD, Vila R, Archibald SB, Pierce NE (2006). Phylogeny of the ants: Diversification in the age of angiosperms. Science 312:101–104. doi: 10.1126/science.112489