Evolution of Vertebrates through the eyes of parasitic flatworms

Evolution of Vertebrates through the eyes of parasitic flatworms Renee Hoekzema June 14, 2011 Essay as a part of the 2010 course on Vertebrate Evolution by Wilma Wessels Abstract In this essay we give a brief introduction to the subject of parasite-host cophylogeny and review a specific study in this field, namely the research done by Verneau et al. on the sequenced rdna of twenty-six species of parasitic flatworm of the family Polystomatidae that infest aquatic and amphibious tetrapod hosts [1]. The parasite phylogeny that is derived in this study gives us independent evidence for a few basic divergences in the phylogeny of the tetrapod hosts and the authors have used the molecular clock dating method to give time estimates for these divergence events. They fix their molecular clock at the Actinopterygii- Sarcopterygii split at 425 Ma, and then give the following estimates: 353 ± 26 Ma for the amniote-lissamphibian divergence, 191 ± 40 Ma for the diversification of chelonians, 246 ± 11 for the Archeobatrachia-Neobatrachia split that is usually associated with the break-up of Gondwanaland, and 92± 12 Ma for a specific divergence in the Neobatrachian parasites that they associate with the separation of South-America and Africa. They note that the time that seems to have elapsed between the origin of amniote parasites and the diversification of chelonians parasites might indicate the presence of an amniote in the water at an earlier time than we presently know of. We discuss the results of this study and the benefits and downsides of host-parasite cophylogenetic research in general. Introduction Cophylogeny is the study of comparing the evolutionary tree of different species or families and looking for the event of cospeciation: simultaneous divergence. In this essay we specifically discuss cophylogeny in the system of a parasite and its host. The evolution of the host can, certainly in the case of vertebrate hosts, be inferred from the palaeontological record. For the parasites we often have little or no fossil record, but we can use modern techniques such as the sequencing of a strand of ribosomal DNA to both determine the phylogeny of the parasite and give a time estimate of evolutionary divergences using the molecular clock. If cospeciation has occurred, then this molecular clock gives us a completely independent time estimate for the divergence in the evolution of the host, which can then be matched to the time estimate derived from the palaeontological data. Furthermore, the phylogeny of the parasite might in some cases be able to shed some light on the shady areas in the evolutionary tree of the host, giving us a clue on what missing links we might yet excavate, or telling us something about the ecology of extinct taxa. Determining whether cospeciation has occurred, however, is a highly non-trivial assignment. Parasites can both speciate and go extinct within the lineage of a single host. They can also infest a variety of hosts at the same time, if these have enough interaction or a medium is present that can sustain the parasite - like lice and flees that inhabit gregarious 1

mammals or parasitic invertebrates that experience a larval phase in water before infesting an amphibian or fish. Over geological periods of time, a parasite might expand its range of hosts and make what is called a horizontal transfer to a another host that is possibly not directly related to the previous one. These processes make it hard to determine the historical relationships between the parasite and host with any certainty. Luckily there are certain cases, such as the emergence of a geographical barrier, in which the divergence of host and parasite has the same cause and cospeciation can be determined conclusively. In this essay we will look at the 2001 study by Verneau et al. [1] in which the partial 18S rdna sequences of twenty-nine modern species of parasitic flatworm are considered. These worms have probably always infested tetrapods, as they have been found on a great number of amphibious tetrapods as well as on teleost fish and lungfish. Therefore they are very well suited for a cophylogenetic study of early vertebrate evolution. Parasitic flatworms The Monogenea is a class of fish parasites within the phylum Platyhelminthes (flatworms). Polystomatidae is the only family within the Monogenea to infest tetrapod hosts, which makes them ideally suited for a study of tetrapod evolution. Verneau et al. selected twenty-six polystomatids, three nonpolystomatid monogeneans and three tapeworms as a comparative outgroup, and partially sequenced their 18S ribosomal DNA. With a computer program they created a minimum evolution tree from this data set, the result of which is shown in figure 1. This diagram represents independent evidence for a few basic notions in our present understanding of the evolutionary tree of early tetrapods and amphibians, inferred from palaeontological evidence. We see a split between Actinopterygii and Sarcopterygii and the origin of tetrapods from fish. A little later we see a split between parasites that infest amniotes (chelonians) and amphibians. Then we see the split between Archaeobatrachia and Neobatrachia as a consequence of the breakup of Gondwanaland, which is very likely a cospeciation event. The authors admitted that the placement of the only salamander (Caudata) parasite Sphyranura oligorchis could not be conclusively determined from their data. Molecular clock Verneau et al. used the molecular clock method to give time estimates for the divergence events in figure 1. They assumed the rate of change of the ribosomal DNA to be constant in time, though possibly different for different lineages, allowing for faster and slower evolving lineages. They anchored the rate of change at two points, namely the Actinopterygii-Sarcopterygii split at 425 Ma and the present time. To estimate the time of a divergence, they considered all the lineages descending from that specific divergence except for the ones that were shown to evolve exceptionally fast or slow, and averaged over the different time estimates that followed from the linear interpolation for each lineage. This procedure gives the following results. The amniote-lissamphibian divergence is estimated at 353 ± 26 Ma, which is congruent with the current estimate for the evolution of aminotes - the oldest known amniote-like tetrapod is from the early Carboniferous [2]. The diversification of chelonians is placed around 191 ± 40 Ma. Note that this probably implies that the parasite has survived on some unknown aquatic amniote before the turtles appeared in the water and diversified. The early diversification of amphibians with the split between Archeobatrachia and Neobatrachia (connected to the split-up of Gondwanaland), was estimated at 246 ± 11, although a later analysis of completely sequenced 18S and partial 28S rdna pushed the estimate up to around 300 Ma [3, 4], which is also more consistent with recent molecular datings of the amphibians themselves [5, 6]. Finally, the divergence in parasites of Neobatrachia that divides the Polystoma group and the Eupolystoma/Sundapolystoma 2

that only infest African and Asian species, was estimated at 92±12 Ma and is probably related to the separation of South-America and Africa, which ended around 100 Ma. The Polystoma group is currently wide spread on frogs from all across the different continents, but this can be explained by the recent dispersal event of the hosts from America to Eurasia during the upper Cenozoic which is probably related to the glacial sea level minima, combined with host switching events. All these estimates are summarized in figure 2. Figure 1: Minimal evolution tree from [1]. The star represents the event to which molecular clock is gauged, the split of Actinopterygii and Sarcopterygii at ca. 425 Ma. Lengths represent the relative change in the sequence. On the right we see the hosts that the species infests. 3

Figure 2: A sketch of the parasite evolutionary tree, adapted from [1], with the time estimate and related host-divergence of several of the divergence events. The dotted line represents the unknown aquatic amniote hosts that carried the parasite before the chelonians arrived but after the origin of amniote parasites. Discussion The study by Verneau et al. shows us that by studying the DNA of parasitic organisms, we can find new information about the evolution, palaeobiogeography and ecology of their hosts, and new evidence for previous conclusions. The parasitic flatworm shows independent evidence for the broad outline of the evolutionary tree of amphibian vertebrates, such as the Actinopterygii-Sarcopterygii split 1, the amniote/non-amniote divergence and the Archaeobatrachia-Neobatrachia divergence. Furthermore, we now know that there was probably an aquatic amniote carrying the parasite in the period between the evolution of amniotes and the arrival of chelonians in the water. This is because we can see that the lineages split long before the chelonians evolved and radiated, so we are probably not dealing with the horizontal transfer directly from a lissamphibian host to the chelonians. We can be quite sure, however, that the parasite could not have infected a terrestrial animal, so there must have been an amniote in the water in the meantime. The estimate from the study for the split or transfer of the parasite to amniotic hosts is 353 ± 26 Ma, while currently the 1 The new sequences in [4] show more clearly that the tetrapods are indeed more closely related to the Sarcopterygii. 4

earliest known fully aquatic amniotes are from the Upper Carboniferous [7, 8, 9], so this might suggest the existence of other aquatic amniotes before these. It is also not excluded that the parasite could have infested an amniote with an amphibious lifestyle. It is in some cases quite hard to determine the lifestyle of early amniotes from the palaeontological data, and it has even been suggested that all amniotes were amphibious or aquatic until after the Carboniferous [7]. There have recently also been palaeontological studies by Laurin et al. that tried to determine the lifestyle of early amniotes more accurately using the microanatomical features of the radius or humerus [10, 11], but thus far not many extinct amniotes have been subjected to the study. As we can see, the results from Verneau et al. would support the theory that amniotes were present in the water from a very early time on. There are downsides to cophylogenetic research, and we can also see this in study by Verneau et al. One problem is the fact that the molecular clock is a very unreliable dating method. We can see from the different branchlengths in figure 1 that different lineages have a very different rate of ribosomal evolution, and since all these lineages have common ancestors, there is no reason to assume that the rate of change is constant along a lineage. Of course, the estimated time of a particular divergence does become more reliable if there are more lineages originating from the divergence and if the divergence is closer to one of the fixed points. Note that the uncertainty of the fixed point (in this case Actinopterygii-Sarcopterygii split at 425 Ma) also has to be taken into account when calculating the uncertainty in the molecular time estimates. The authors do not comment on this. We notice, however, that the authors may have underestimated their own error: in [3], Verneau, Du Preez and Badets adjust their estimate for the Archeobatrachia- Neobatrachia split to 300 Ma, a value that is about five times the estimated error bar higher than their first estimate of 246 ± 11. Another problem with this kind of research is that, because of the complicated philogenetic properties of the host-parasite system, the conclusions that we draw concerning the evolutionary history of the hosts from the evolutionary tree of the parasite are very unreliable. For any evolutionary tree we construct for the parasites, there are a great number of host-trees that fit the data. For example, in the case of the study we are considering, we have drawn the conclusion that the parasites have infested other amniotes before they infested the chelonians, because they seem to have diverged along with the amniote/lissamphibian divergence. Another explanation, however, is that this divergence of the parasites had no relationship with the evolution of amniotes, but took place within the lissamphibian host (a socalled duplication event) and that one lineage switched to aquatic amniotes at a certain time, and also went extinct within the lissamphibians. If this is the case, then our conclusion that there must have been another aquatic amniote before the taxa we know of entered the water, is false. The authors reject this possibility because their molecular dating is so close to the palaeontological dating of the origin of amniotes and it is therefore very improbable that the two are unrelated. We have seen, however, that the molecular dating is itself not very reliable. However, it does indeed seem improbable that this fundamental divergence of which the two branches extend to form the amphibian and amniote parasites respectively, would have no relationship with the actual amniote/lissamphibian divergence itself. Even though this study does not give us a very precise time estimate for different divergence events, it does certainly show how valuable cophylogenetic research can be. On the one hand it gives us independent evidence for the basic divergences in the evolution of amphibious tetrapods. On the other hand it gives us supportive, although not conclusive, evidence for the existence of amphibious or aquatic amniotes very early in the paleozoic. 5

References [1] Olivier Verneau, Sophie Bentz, Neeta Devi Sinnappah, Louis du Preez, Ian Whittington and Claude Combes, A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Plystomatidae), 2002: Proc. R. Soc. Lond. B 269, 535-543 [2] R. L. Paton, T. R. Smithson and J. A. Clack, An amniote-like skeleton from the Early Carboniferous of Scotland, 1999: Nature 398, 508-513 [3] Olivier Verneau, Louis du Preez and Mathieu Badets, Lessons from parasitic flatworms about evolution and historical biogeography of their vertebrate hosts, 2009: Comptes Rendus - Biologies 332, Is. 2-3, 149-158 [4] Mathieu Badets, Olivier Verneau, Origin and evolution of alternative developmental strategies in amphibious sarcopterygian parasites (Platyhelminthes, Monogenea, Polystomatidae), 2009: Organisms Diversity and Evolution 9, Is. 3, 155-164 [5] Diego San Mauro, Miguel Vences, Marina Alcobendas, Rafael Zardoya and Axel Meyer, Initial Diversifcation of Living Amphibians Predated the Breakup of Pangaea, 2005: The American Naturalist 165, Is. 5, 590-599 [6] Kim Roelants, David J. Gower, Mark Wilkinson, Simon P. Loader, S. D. Biju, Karen Guillaume, Linde Moriau and Franky Bossuyt, Global patterns of diversification in the history of modern amphibians, 2007: Proc. Natl. Acad. Sci. USA 104, Is. 3, 887-892 [7] Sean Patrick Modesto, The Postcranial Skeleton of the Aquatic Parareptile Mesosaurus tenuidens from the Gondwanan Permian, 2010: Journal of Vertebrate Paleontology 30, Is. 5, 1378-1395 [8] S. P. Modesto, Noteosaurus africanus Broom is a Nomen Dubium, 1996: Journal of Vertebrate Paleontology 16, Is. 1, 172-174 [9] Michael J. Benton, Vertebrate Paleontology, Third edition, 2005: Blackwell Publishing Ltd [10] Damien Germain and Michel Laurin, Microanatomy of the radius and lifestyle in amniotes (Vertebrata, Tetrapoda), 2005: Zoologica Scripta 34, Is. 4, 335350 [11] Aurore Canoville and Michel Laurin, Evolution of humeral microanatomy and lifestyle in amniotes, and some comments on palaeobiological inferences, 2010: Biological Journal of the Linnean Society 100, 384406 6