Xenoturbella and Xenacoelomorpha

Xenoturbella, Acoela, and Nemertodermatida are worm-like simple bilateral animals, which might give insights into the earliest steps in the evolution of Bilateria. Phylogenomic analyses (Philippe et al. 2011; Cannon et al. 2016) and some ultrastructural characters (Franzén & Afzelius 1987; Lundin 1998) group them into a monophyletic group, which has been named Xenacoelomorpha (Philippe et al. 2011; Tyler & Schilling 2011).

Xenoturbella has been kind of a puzzle for zoologists since it was described in 1949. First considered to be a primitive turbellarian flatworm, occasionally affiliated with Hemichordata, suggested to be a sister group to other bilaterians, for a moment mistaken for a mollusc, then suggested to be a deuterostome, then again together with Acoelomorpha (Acoela and Nemertodermatida) as a sister group to Bilateria, and then back to deuterostome affinity, as a sister group to Ambulacraria (Echinodermata and Hemichordata) (Telford 2008; Hejnol et al. 2009; Philippe et al. 2011). It is exciting that four new species of Xenoturbella have now been found in the Pacific ocean (Rouse et al. 2016). Before, only one small (up to 4 cm) species (X. bocki) was known from the west coast of Sweden (the second, X. westbladi, from the same place is probably the same species). Three of the new species are much bigger than X. bocki, some of them 20 cm or longer, but all are otherwise anatomically and in their habits (feeding on bivalves on the muddy ocean floor) quite similar to each other, which is also consistent with the genetic data. I heard about a new large Xenoturbella species found in the Pacific in 2010 while at EDIT taxonomy course in Kristineberg, Sweden. Sounded pretty unbelievable. I was hoping that this amazing new discovery will help to solve the phylogenetic position of Xenoturbella and Xenacoelomorpha more reliably, but it seems that the debate will continue for years to come.

Rouse et al. (2016) gathered also phylogenomic data for one of the new species and sequenced complete mitochondrial genomes of all the new species. Their phylogenetic analyses were incongruent to each other. Phylogenomic analyses based on nearly 1200 nuclear protein coding genes found Xenacoelomorpha (composed in this dataset of one Acoela and two Xenoturbella species) as sister group to other Bilateria (Nephrozoa) (using site homogeneous models) or as a sister group to Protostomia (using site heterogeneous CAT model). Analyses based on mitochondrial proteins grouped Xenacoelomorpha (4 acoels and 4 xenoturbellid species) with deuterostomes. In the same issue of Nature where Rouse et al's paper was published, Cannon et al. (2016) investigated the placement of Xenacoelomorpha more thoroughly, although using only one Xenoturbella species, but with many more Acoela species (7) and including Nemertodermatida (4 species). Cannon et al. (2016) added impressive amount of new transcriptome data (including for Xenoturbella), which enabled them to have pretty complete datasets for phylogenetic analyses (phylogenomic datasets tend to be gappy if not based on fully sequenced genomes). Cannon et al. (2016) results were much more consistent compared to Rouse et al. (2016), finding support only for sister-group relationship between Xenacoelomorpha and Nephrozoa (all other Bilateria, hypothetically having ancestrally nephridia in contrast to Xenacoelomorpha, which lack them). However, when fast-evolving acoelomorphs where excluded (retaining only clearly slower evolving Xenoturbella), statistical support for Nephrozoa and Deuterostomia decreased (from 99–100% to 81% in both cases). Unfortunately, Cannon et al. (2016) did not analyse this dataset with more complex CAT model and I suspect if they had, support for Nephrozoa and Deuterostomia would have disappeared entirely and Xenoturbella might have jumped next to Ambulacraria (as in Philippe et al. 2011). Cannon et al. (2016) did analyze their main dataset (which included Acoelomorpha) also with CAT model fully supporting Nephrozoa and Deuterostomia, but the internal branches leading to Nephrozoa and Deuterostomia of the resulting phylogenetic tree were clearly shorter (barely visible in the published figure) than in the analyses using simpler phylogenetic models. Simpler site homogeneous models underestimate the amount of sequence evolution compared to site heterogeneous models (like CAT) and can therefore produce artefactual groupings (grouping of fast-evolving, long-branch taxa with each other or with a distant out-group, regardless of their actual phylogenetic affinities). It could be that the fast-evolving Acoelomorpha (which is clearly evident in the long branches they display in phylogenetic analyses, the shortest ones perhaps still about twice as long as the Xenoturbella branch) caused the whole Xenacoelomorpha to shift artefactually at the base of Bilateria, away from more slowly evolving Deuterostomia and this way creating the Nephrozoa. CAT model was able to shorten the branches supporting monophyly of Nephrozoa and Deuterostomia, but not entirely to overcome possible non-phylogenetic signal present in the fast-evolving Acoelomorpha.

In conclusion, I think it is too early to exclude deuterostome affinity of Xenacoelomorpha. Curiously, while looking at the pictures of the new large species of Xenoturbella, I thought that they looked a bit like enteropneusts (Hemichordata). Could it be that the ring furrow of Xenoturbella, which divides the body into the head and tail region, is homologous to the collar (or to part of it) of Hemichordata (Deuterostomia: Ambulacraria)?

References

Cannon JT, Vellutini BC, Smith III J, Ronquist F, Jondelius U, Hejnol A (2016) Xenacoelomorpha is the sister group to Nephrozoa. Nature 530: 89–93. doi: 10.1038/nature16520

Franzen A, Afzelius BA (1987) The ciliated epidermis of Xenoturbella bocki (Platyhelminthes, Xenoturbellida) with some phylogenetic considerations. Zoologica Scripta 16: 9–17. doi: 10.1111/j.1463-6409.1987.tb00046.x

Hejnol A, Obst M, Stamatakis A, Ott M, Rouse GW, Edgecombe GD, Martinez P, Baguñà J, Bailly X, Jondelius U, Wiens M, Müller WEG, Seaver E, Wheeler WC, Martindale MQ, Giribet G, Dunn CW (2009) Assessing the root of bilaterian animals with scalable phylogenomic methods. Proceedings. Biological sciences / The Royal Society 276: 4261–4270. doi: 10.1098/rspb.2009.0896

Lundin K (1998) The epidermal ciliary rootlets of Xenoturbella bocki (Xenoturbellida) revisited: new support for a possible kinship with the Acoelomorpha (Platyhelminthes). Zoologica Scripta 27: 263–270. doi: 10.1111/j.1463-6409.1998.tb00440.x

Philippe H, Brinkmann H, Copley RR, Moroz LL, Nakano H, Poustka AJ, Wallberg A, Peterson KJ, Telford MJ (2011) Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470: 255–258. doi: 10.1038/nature09676

Rouse GW, Wilson NG, Carvajal JI, Vrijenhoek RC (2016) New deep-sea species of Xenoturbella and the position of Xenacoelomorpha. Nature 530: 94–97. doi: 10.1038/nature16545

Telford MJ (2008) Xenoturbellida: the fourth deuterostome phylum and the diet of worms. Genesis 46: 580–586. doi: 10.1002/dvg.20414

Tyler S, Schilling S (2011) Phylum Xenacoelomorpha Philippe, et al., 2011. In: Zhang, Z.-Q. (Ed.) Animal biodiversity: An outline of higher-level classification and survey of taxonomic richness. Zootaxa 3148: 24–25.

Ctenophora is not a sister group to other animals after all

Since 2008 there have been a debate whether the phylum Ctenophora is a phylogenetic sister group to other animals or not (Dunn et al. 2008; Philippe et al. 2009). There were some who claimed to have resolved the debate (that Ctenophora really is a sister group to other animals: Ryan et al. 2013), but the presented evidence was not at all convincing, as I discussed some time ago. Recently, another paper (Whelan et al. 2015) came out that favoured Ctenophora as the sister group to other animals (hereafter referred to as Ctenophora-basal tree), but this time the results were much more convincing and I was prepared to believe the Ctenophora-basal tree.

But not so fast. It turns out I was fooled. Only half a year after the Whelan et al. (2015) publication, Pisani et al. (2015) showed that the analyses by Whelan et al., as well as all previous analyses favouring Ctenophora-basal tree, were still plagued by systematic errors. Pisani et al. (2015) found that the sponges (Porifera) are probably the sister group to other animals (as is usually assumed) after all, although exact position of Ctenophora relative to the remaining animals still needs additional research. For me, one of the most striking results from Pisani et al. (2015) was the re-analysis of genome content data (presence/absence of protein coding genes) from Ryan et al. (2013). The genome content tree that Ryan et al. (2013) recovered, contained several phylogenetic relationships that are highly suspect (e.g. non-monophyly of Annelida and a group containing a mollusc, an annelid and a chordate Branchiostoma). Remarkably, when Pisani et al. (2015) used a less biased model of gene content evolution, which does not underestimate gene losses as much as did the model used by Ryan et al. (2013), they recovered an animal tree of life that fully agreed with independent phylogenomic analyses based gene sequences. There was not a single nonsense clade left. And in that tree, Porifera replaced Ctenophora as the sister group to other animals.

As for the Whelan et al. (2015) study that fooled me, the main issue comes down to the choice of an out-group. Whelan et al. (2015) analysed some of their datasets also with more realistic, but computationally more demanding CAT model, but recovered nevertheless Ctenophora-basal tree. It turns out, though, that when more distant out-groups to animals (Fungi or Fungi plus non-choanoflagllate members of Holozoa) are excluded from the analysis (retaining only the members of Holozoa or only the choanoflagllates), Whelan et al. (2015) datasets do not support Ctenophora-basal tree anymore or even favour Porifera-basal tree. Whelan et al. (2015) actually did analyse their datasets also with alternative out-group compositions, but only with simpler evolutionary models and therefore it was not possible to discover that different out-group compositions affect results when using more complex CAT model! Such a simple trick which the authors could have done to test their results...

Another recent study also found Ctenophora-basal tree (Chang et al. 2015) using the CAT model. Although Chang et al. (2015) analyses did not include Fungi, it was not tested if excluding more distant non-choanoflagllate Holozoa taxa could affect the results. But this Ctenophora question was not the topic of this paper (it was about Myxozoa, highly reduced parasitic Cnidaria), although one of the co-authors was (Hervé Philippe) also a co-author in Pisani et al. (2015) paper, which studied the effect of out-group composition.

Still, it is disconcerting that depending on the composition of the out-group, even if the closest relatives are included, the results can change that dramatically: using CAT model and including Fungi, the Ctenophora-basal tree can be recovered with maximal statistical support, but using CAT model and excluding all out-groups except choanoflagllates (the closest relatives of animals), Porifera-basal tree can be recovered with maximal support instead. I would have thought that as long as the closest out-groups are also included (choanoflagllates) in the analyses, the more distant out-groups (Fungi) should not influence the results that much. This tells me again (as I discussed before) that whatever phylogenetic signal there is for the relationships between Porifera, Ctenophora, Placozoa, Cnidaria, and Bilateria, it is quite tiny. These groups separated from each other in the Precambrian (>540 Ma) probably rather rapidly, perhaps within a few tens of millions of years or even within a shorter time period. Because it starts to look like the nervous systems and perhaps muscles might have evolved independently even three times in Ctenophora, Cnidaria, and Bilateria (Liebeskind et al. 2015; Moroz et al. 2014), it seems to be of less importance what are the exact phylogenetic relationships between Porifera, Ctenophora, Placozoa, Cnidaria, and Bilateria. Ctenophora, Cnidaria, and Bilateria might have evolved their morphological complexity independently from Porifera or Placozoa-like ancestors.

References

Chang ES, Neuhof M, Rubinstein ND, Diamant A, Philippe H, Huchon D, Cartwright P (2015) Genomic insights into the evolutionary origin of Myxozoa within Cnidaria. Proceedings of the National Academy of Sciences 112: 14912–14917. doi: 10.1073/pnas.1511468112

Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith S a, Seaver E, Rouse GW, Obst M, Edgecombe GD, Sørensen M V, Haddock SHD, Schmidt-Rhaesa A, Okusu A, Kristensen RM, Wheeler WC, Martindale MQ, Giribet G (2008) Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452: 745–749. doi: 10.1038/nature06614

Liebeskind BJ, Hillis DM, Zakon HH (2015) Convergence of ion channel genome content in early animal evolution. Proceedings of the National Academy of Sciences 112: E846–E851. doi: 10.1073/pnas.1501195112

Moroz LL, Kocot KM, Citarella MR, Dosung S, Norekian TP, Povolotskaya IS, Grigorenko AP, Dailey C, Berezikov E, Buckley KM, Ptitsyn A, Reshetov D, Mukherjee K, Moroz TP, Bobkova Y, Yu F, Kapitonov V V, Jurka J, Bobkov Y V, Swore JJ, Girardo DO, Fodor A, Gusev F, Sanford R, Bruders R, Kittler E, Mills CE, Rast JP, Derelle R, Solovyev V V, Kondrashov F a, Swalla BJ, Sweedler J V, Rogaev EI, Halanych KM, Kohn AB (2014) The ctenophore genome and the evolutionary origins of neural systems. Nature 510: 109–114. doi: 10.1038/nature13400

Philippe H, Derelle R, Lopez P, Pick K, Borchiellini C, Boury-Esnault N, Vacelet J, Renard E, Houliston E, Quéinnec E, Da Silva C, Wincker P, Le Guyader H, Leys S, Jackson DJ, Schreiber F, Erpenbeck D, Morgenstern B, Wörheide G, Manuel M (2009) Phylogenomics revives traditional views on deep animal relationships. Current Biology 19: 706–712. doi: 10.1016/j.cub.2009.02.052

Pisani D, Pett W, Dohrmann M, Feuda R, Rota-Stabelli O, Philippe H, Lartillot N, Wörheide G (2015) Genomic data do not support comb jellies as the sister group to all other animals. Proceedings of the National Academy of Sciences 112: 15402–15407. doi: 10.1073/pnas.1518127112

Ryan JF, Pang K, Schnitzler CE, Nguyen A-D, Moreland RT, Simmons DK, Koch BJ, Francis WR, Havlak P, Smith S a, Putnam NH, Haddock SHD, Dunn CW, Wolfsberg TG, Mullikin JC, Martindale MQ, Baxevanis AD (2013) The Genome of the Ctenophore Mnemiopsis leidyi and Its Implications for Cell Type Evolution. Science 342: 1242592–1242592. doi: 10.1126/science.1242592

Whelan N V., Kocot KM, Moroz LL, Halanych KM (2015) Error, signal, and the placement of Ctenophora sister to all other animals. Proceedings of the National Academy of Sciences 112: 5773–5778. doi: 10.1073/pnas.1503453112

Root of the Eukaryotic Tree of Life

While it has become quite clear that the last eukaryotic common ancestor (LECA) was a bikont, i.e. had two anterior cilia to move around (as I wrote previously), it now seems that the LECA wasn't only a bikont, but also an excavate. Excavates are one of the five main groups of eukaryotes (Adl et al.2012) having ancestrally two cilia and a ventral feeding groove. Excavates include for example Trichomonas (Parabasalia), Giardia (Fornicata), Euglenozoa (e.g. Euglena, Trypanosoma), Heterolobosea, Jakobida (latter three belonging to Discoba), and Malawimonas, but it has turned out that they might not form a monophyletic group that seemed quite likely in 2009 (Hampl et al. 2009). In my previous post I treated excavates as monophyletic and avoided discussing them, although I was aware of some problems. Particularly, Malawimonas, which is structurally a typical excavate (possessing two cilia with a characteristic ciliary apparatus and a ventral feeding groove; Simpson, 2003) did not want to group very well with other excavates in phylogenomic analyses (Rodríguez-Ezpeletaet al. 2007; Derelle & Lang, 2012; Zhao et al. 2013; Brown et al. 2013). I thought perhaps the data was incomplete to make a big deal about this. Now it looks pretty clear (Cavalier-Smith et al. 2014; 2015; Derelle et al. 2015) that Malawimonas is more closely related to unikont/Opimoda branch (amoebae, animals, fungi and others; Fig. 1) than to (most) other excavates. Interestingly, phylogenomic analyses by Cavalier-Smith et al. (2014; 2015) reveal that excavate groups Parabasalia, Fornicata, and Preaxostyla might also be more closely related to unikonts than to Discoba (Euglenozoa, Heterolobosoa, and Jakobida). Parabasalia, Fornicata, and Preaxostyla are classified under Metamonada, who are all anaerobic or microaerophilic and lack typical respiratory mitochondria. Many of them are parasites or symbionts of animals. Unfortunately most of them are fast evolving, making it difficult to place them in phylogenetic analyses, particularly Trichomonas and Giardia for which full genomes are available. It would be necessary to obtain additional genome scale data for more slowly evolving free-living species from groups like Dysnectes and Carpediemonas (Takishita et al. 2012) to place representatives of Metamonada among eukaryotes more reliably.

Figure 1. Phylogeny of eukaryotes updated from my previous post mainly on the basis of Cavalier-Smith et al. (2014; 2015) and Derelle et al. (2015) papers. Although since 2010, Cavalier-Smith prefers to root the tree between Euglenozoa (member of Discoba) and other eukaryotes, the rooting found by Derelle et al. (2015) is more reliable, because it is based on large number of mitochondrial and other bacterial genes for which there are closer out-groups available than for genes of archaeal origin (see for example the rooting found by Lasek-Nesselquist& Gogarten, 2013 which fits Cavalier-Smith's scenario). Cavalier-Smith (2010; 2013) lists some genomic characters that appear to be ancestral (i.e. shared with prokaryotes) in Euglenozoa but derived in other eukaryotes. The problem is that full genome sequences are available only for few fast evolving and mostly parasitic Euglenozoa and other excavates, which makes these kinds of lists highly speculative.

If the rooting of the eukoryote tree (Fig. 1) is correct, then it really seems that the LECA might have been quite similar to a typical excavate like Malawimonas or a jakobid (e.g. Jakoba, Reclinomonas, Andalucia). This scenario finds support also from some alveolates (e.g. Colponema) that are structurally quite similar to excavates (Tikhonenkov et al. 2014; Cavalier-Smith et al. 2014). Apparently, Colponema, Acavomonas and many other similar undescribed groups (Janouškovecet al. 2013; Tikhonenkov et al. 2014) are phylogenetically diverse bunch that are variously related to, but clearly outside of the three main groups of alveolates (ciliates, apicomplexans, and dinoflagellates).

References

Adl SM, Simpson AGB, Lane CE, Lukeš J, Bass D, Bowser SS, Brown MW, Burki F, Dunthorn M, Hampl V, Heiss A, Hoppenrath M, Lara E, Gall L le, Lynn DH, McManus H, Mitchell E a D, Mozley-Stanridge SE, Parfrey LW, Pawlowski J, Rueckert S, Shadwick L, Schoch CL, Smirnov A, Spiegel FW (2012) The revised classification of eukaryotes. The Journal of eukaryotic microbiology 59 (5): 429–514. doi: 10.1111/j.1550-7408.2012.00644.x

Brown MW, Sharpe SC, Silberman JD, Heiss AA, Lang BF, Simpson AGB, Roger AJ (2013) Phylogenomics demonstrates that breviate flagellates are related to opisthokonts and apusomonads. Proceedings. Biological sciences / The Royal Society 280 (1769): 20131755. doi: 10.1098/rspb.2013.1755

Cavalier-Smith T (2010) Kingdoms Protozoa and Chromista and the eozoan root of the eukaryotic tree. Biology letters 6: 342–345. doi: 10.1098/rsbl.2009.0948

Cavalier-Smith T (2013) Early evolution of eukaryote feeding modes, cell structural diversity, and classification of the protozoan phyla Loukozoa, Sulcozoa, and Choanozoa. European journal of protistology 49 (2): 115–178. doi: 10.1016/j.ejop.2012.06.001

Cavalier-Smith T, Chao EE, Snell E a, Berney C, Fiore-Donno AM, Lewis R (2014) Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa. Molecular phylogenetics and evolution 81: 71–85. doi: 10.1016/j.ympev.2014.08.012

Cavalier-Smith T, Chao EE, Lewis R (2015) Multiple origins of Heliozoa from flagellate ancestors: New cryptist subphylum Corbihelia, superclass Corbistoma, and monophyly of Haptista, Cryptista, Hacrobia and Chromista. Molecular phylogenetics and evolution. doi: 10.1016/j.ympev.2015.07.004

Derelle R, Lang BF (2012) Rooting the Eukaryotic Tree with Mitochondrial and Bacterial Proteins. Molecular biology and evolution 29: 1277–1289. doi: 10.1093/molbev/msr295

Derelle R, Torruella G, Klimeš V, Brinkmann H, Kim E, Vlček Č, Lang BF, Eliáš M (2015) Bacterial proteins pinpoint a single eukaryotic root. Proceedings of the National Academy of Sciences 112: E693–E699. doi: 10.1073/pnas.1420657112

Hampl V, Hug L, Leigh JW, Dacks JB, Lang BF, Simpson AGB, Roger AJ (2009) Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic “supergroups”. Proceedings of the National Academy of Sciences of the United States of America 106: 3859–3864. doi: 10.1073/pnas.0807880106

Janouškovec J, Tikhonenkov D V, Mikhailov K V, Simdyanov TG, Aleoshin V V, Mylnikov AP, Keeling PJ (2013) Colponemids represent multiple ancient alveolate lineages. Current biology 23: 2546–2552. doi: 10.1016/j.cub.2013.10.062

Lasek-Nesselquist E, Gogarten JP (2013) The effects of model choice and mitigating bias on the ribosomal tree of life. Molecular phylogenetics and evolution 69: 17–38. doi: 10.1016/j.ympev.2013.05.006

Rodríguez-Ezpeleta N, Brinkmann H, Burger G, Roger AJ, Gray MW, Philippe H, Lang BF (2007) Toward resolving the eukaryotic tree: the phylogenetic positions of jakobids and cercozoans. Current Biology 17: 1420–1425. doi: 10.1016/j.cub.2007.07.036

Simpson AGB (2003) Cytoskeletal organization, phylogenetic affinities and systematics in the contentious taxon Excavata (Eukaryota). International Journal of Systematic and Evolutionary Microbiology 53: 1759–1777. doi: 10.1099/ijs.0.02578-0

Takishita K, Kolisko M, Komatsuzaki H, Yabuki A, Inagaki Y, Cepicka I, Smejkalová P, Silberman JD, Hashimoto T, Roger AJ, Simpson AGB (2012) Multigene Phylogenies of Diverse Carpediemonas-like Organisms Identify the Closest Relatives of ‘Amitochondriate’ Diplomonads and Retortamonads. Protist 163: 344–355. doi: 10.1016/j.protis.2011.12.007

Tikhonenkov DV, Janouškovec J, Mylnikov AP, Mikhailov KV, Simdyanov TG, Aleoshin VV, Keeling PJ (2014) Description of Colponema vietnamica sp.n. and Acavomonas peruviana n. gen. n. sp., Two New Alveolate Phyla (Colponemidia nom. nov. and Acavomonidia nom. nov.) and Their Contributions to Reconstructing the Ancestral State of Alveolates and Eukaryotes. PloS one 9: e95467. doi: 10.1371/journal.pone.0095467

Zhao S, Shalchian-Tabrizi K, Klaveness D (2013) Sulcozoa revealed as a paraphyletic group in mitochondrial phylogenomics. Molecular phylogenetics and evolution 69 (3): 462–468. doi: 10.1016/j.ympev.2013.08.005

Phylogenetic position of Ctenophora. A follow-up.

In a previous post I nagged about a paper reporting a genome of a ctenophore Mnemiopsis leidyi (Ryan et al. 2013), where it was suggested that ctenophores are the sister group to all other animals. Now another genome from this animal phylum, that of Pleurobrachia bachei, has been sequenced along with transcriptomes of additional 10 ctenophore species (Moroz et al. 2014)! Regarding the phylogenetic position of Ctenophora, the analysis by Moroz et al. are even less convincing than those by Ryan et al. (2013). Apparently only maximum likelihood analyses with RAxML were done (or at least reported), which either support Ctenophora as sister group to other animals or are inconclusive. But this is not the main point of the article. Moroz et al. suggest that nervous system and possibly muscles of ctenophores evolved independently from other animals (Cnidaria and Bilateria). They showed not only that ctenophores lack many Cnidaria+Bilateria specific genes associated with muscles, nervous system etc, but that ctenophores have recruited for those purposes nearly entirely different set of genes. So it seems unlikely that Ctenophora lost muscles and nervous system specified by genetic toolkit found in Cnidaria and Bilateria and then invented everything from scratch once again. More likely, Ctenophora and Cnidaria+Bilateria evolved phenotypic complexity independently from simpler ancestors. However, this does not mean that Ctenophora has to be a sister group to other animals, but a phylogenetic arrangement where Ctenophora is sister to Cnidaria and these together (called Coelenterata) form a sister group of Bilateria (as found by Philippe et al. 2009), seems less likely now, because it might exactly mean the loss and re-evolution of nerves and muscles in Ctenophora. Then again, compared to Bilateria, Cnidaria, and Porifera, contemporary ctenophores are genetically very closely related to each other (see Podar et al. 2001 and Extended Data Figure 3d in Moroz et al. 2014), meaning that they diverged from each other relatively recently, leaving a long stem going probably back to Precambrian. A lot can happen along this long stem, so who knows...

Regardless of the deepest phylogenetic relationships between animals, independent evolution of muscles and nervous system in ctenophores seems quite likely based on the results by Moroz et al. 2014. This led me to a realization that maybe the phylogenetic relationships between Porifera, Placozoa, Ctenophora, Cnidaria, and Bilateria actually do not matter much. These relationships tend to vary from study to study and the internal branches uniting these groups in different combinations tend to be very short. As short branches suggest little amount of evolution, maybe nothing remarkable happened along those branches anyway and most of the possible relationships between those main animal lineages are more or less equivalent? Then again, considering that these lineages diverged from each other more than 540 million years ago, difficulties in reconstructing their relationships can be expected. Too early to give up. More sophisticated analyses might help to figure out what are the likely causes for conflicting results (systematic errors or true lack of phylogenetic signal).

References

Moroz LL, Kocot KM, Citarella MR, Dosung S, Norekian TP, Povolotskaya IS, Grigorenko AP, Dailey C, Berezikov E, Buckley KM, Ptitsyn A, Reshetov D, Mukherjee K, Moroz TP, Bobkova Y, Yu F, Kapitonov V V, Jurka J, Bobkov Y V, Swore JJ, Girardo DO, Fodor A, Gusev F, Sanford R, Bruders R, Kittler E, Mills CE, Rast JP, Derelle R, Solovyev V V, Kondrashov F a, Swalla BJ, Sweedler J V, Rogaev EI, Halanych KM, Kohn AB (2014) The ctenophore genome and the evolutionary origins of neural systems. Nature 510: 109–114. doi: 10.1038/nature13400 

Philippe H, Derelle R, Lopez P, Pick K, Borchiellini C, Boury-Esnault N, Vacelet J, Renard E, Houliston E, Quéinnec E, Da Silva C, Wincker P, Le Guyader H, Leys S, Jackson DJ, Schreiber F, Erpenbeck D, Morgenstern B, Wörheide G, Manuel M (2009) Phylogenomics revives traditional views on deep animal relationships. Current biology 19: 706–712. doi: 10.1016/j.cub.2009.02.052

Podar M, Haddock SH, Sogin ML, Harbison GR (2001) A molecular phylogenetic framework for the phylum Ctenophora using 18S rRNA genes. Molecular phylogenetics and evolution 21: 218–230. doi: 10.1006/mpev.2001.1036

Ryan JF, Pang K, Schnitzler CE, Nguyen A-D, Moreland RT, Simmons DK, Koch BJ, Francis WR, Havlak P, Smith S a, Putnam NH, Haddock SHD, Dunn CW, Wolfsberg TG, Mullikin JC, Martindale MQ, Baxevanis AD (2013) The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science 342: 1242592. doi: 10.1126/science.1242592

Phylogenetic position of Ctenophora

The first genome of Ctenophora has now been sequenced (Ryan et al. 2013), specifically that of Mnemiopsis leidyi. Genomes of most of the 30 or so animal phyla are still unavailable, but this might change in the coming years (Bracken-Grissomet al. 2014). I had great hopes of getting new insights about pinpointing the phylogenetic position of ctenophores. Unfortunately, the Science paper was disappointing and mainly not because the phylogenetic position of Ctenophora remains unsolved, but because the authors want to give the impression that it is. The abstract of the paper is terribly misleading. Of course, it is more attractive to present the results in a way that one of the long standing questions in animal evolution has been solved, rather than admitting that even after sequencing the whole genome, it's still not sure.

Ryan et al. want to make the case, that ctenophores are the sister group to all other animals. This was suggested for the first time in 2008 (Dunn et al.), but it wasn't taken very seriously even by the authors and a year later (Philippe et al. 2009) it was shown that the likely problem causing such a strange placement of ctenophores was their fast molecular evolution. Taking this into account, Philippe et al. found that ctenophores most likely belong to Eumetazoa (animals with true tissues, muscles and nervous system) and the sister group to all other animals is Porifera (sponges, who do not have muscles and nervous system), as always suspected. According to Philippe et al. ctenophores are most closely related to Cnidaria, with whom they superficially resemble, and were traditionally classified together as Coelenterata. The results of Philippe et al. can by no means be considered final, however, and reliably deciphering the relationships between the main lineages of animals (Porifera, Placozoa, Ctenophora, Cnidaria, and Bilateria) is still difficult (Nosenko et al. 2013).

So what evidence Ryan et al. provide for such a surprising phylogenetic placement of ctenophores? The two main lines of evidence were phylogenetic analyses of protein sequences and of genome gene contents (presence/absence of genes).

Despite the impression the authors are giving, their phylogenetic analyses are far from conclusive. They used two methods, maximum likelihood (ML) and Bayesian inference, to construct phylogenies based on protein sequences. These methods gave different results, but most likely not because of the methodological differences, but because of different evolutionary models employed.

In the ML framework, they used the standard GTR model, which is a site homogeneous model. This means, that probabilities describing different amino acid (or nucleotide) replacements (termed replacement matrix) do not vary along the sequence. Although the overall rate of replacement among sites can change when gamma rate parameter is introduced in the model, the relative probabilities of amino acid replacements remain the same. In reality, however, different regions of proteins do not only evolve faster or slower, but also qualitatively differently because of various constraints. This means that depending on the position in the protein, only some types of amino acids tend to be allowed (e.g. hydrophobic or aromatic etc). This fact makes it necessary to consider different amino acid replacement probabilities for different positions even when the rate of change is the same (these kinds of models are called site heterogeneous). Fortunately, there is no need to assign to every position in the sequence its own replacement matrix (which would make the analyses computationally intractable), but they can be grouped into fewer categories. The Bayesian CAT model (Lartillot & Philippe 2004) estimates from the data the number of different categories and which kind of amino acid replacements describe these categories the best. As with the GTR model, the site heterogeneous models can be combined with gamma rate parameter to vary the overall rate at sites, adding an additional layer of complexity (but also making analysis computationally more demanding).

The ML analysis using GTR+gamma model favored a tree where ctenophores were the sister group to all other animals. The Bayesian analyses with CAT model favored either a tree were Ctenophores were the sister group to Porifera (105 000 site dataset with little missing data, but small taxon sampling) or positioned within Eumetazoa (88 000 site dataset with lot of missing data, but large taxon sampling).

The GTR model is less realistic and clearly more prone to long-branch attraction artefacts than CAT (Lartillot et al. 2007). Long-branch attraction causes fast evolving (long-branch) taxa to group together regardless of their phylogenetic affinities or pull them towards distant out-group taxa. As ctenophores appear to be at least at molecular level fast evolving (Philippe et al. 2009; Pett et al. 2011; Kohn et al. 2012), it cannot be excluded that the position of ctenophores in the ML analysis is caused by long-branch attraction artefact. Ryan et al. results also show that the ctenophores are among the faster evolving taxa in their dataset, but because the ctenophores were not extremely fast evolving, the authors thought that it is not a problem (unclear to me what gave them this confidence).

Unfortunately their Bayesian analyses are not without problems either. The small taxon analyses (where Mnemiopsis+sponge clade was sister to other animals) were problematic precisely because of poor taxon sampling (15–19 taxa depending on the outgroup size). Large number of taxa are required (Lartillot & Philippe 2004) to reliably estimate parameters of CAT model and decide between ancestral and derived character states. For the large taxon datasets the problem appeared to be the opposite – they were too big to get reliable results even after running analyses on average 200 days. This could perhaps have been solved by excluding some of the taxa (especially among well sampled Bilateria) and analyzing datasets containing for example random 50% of the original positions (44 000 instead of 88 000). PhyloBayes-MPI manual mentions that getting consistent results becomes challenging already beyond 20 000 positions.

Although CAT model is not available in the ML framework, nevertheless there are similar alternatives for ML. For example, structural and empirical mixture models containing 2–6 matrices (instead of just one) implemented in PhyML programs (Le& Gascuel 2010; Le et al. 2012). Some of these models have already been used in studying ancient phylogenetic relationships and shown to affect the results (Lasek-Nesselquist & Gogarten 2013). Pity that Ryan et al. did not explore these models.

The second main evidence Ryan et al. gave regarding phylogenetic position of ctenophores was gene content analyses. It appears that Mnemiopsis lacks many genes that are present in all other animals (including sponges) but not in outgroup species. Although the list of these missing genes for ctenophores as a whole is somewhat smaller (already authors found that few genes that were missing in Mnemiopsis were in fact present in some other ctenophore species), it probably remains quite large as all ctenophores appear to be rather closely related to each other (Podar et al. 2001). As ctenophores evolve fast and the genome of Mnemiopsis is compact and among the smallest in animals (Ryan et al. 2013), it seems likely that the missing genes have been lost secondarily. Two features of ctenophore reproductive biology might explain their fast evolution: inbreeding caused by self-fertilization (almost all ctenophores are hermaphrodites) and capability for rapid and massive reproduction. This can lead to frequent massive die-offs creating genetic bottlenecks, which facilitates the accumulation of deleterious mutations (Pett et al. 2011). It is also evident from Ryan et al's results of ML phylogenetic analyses of gene content that nonsense phylogenetic relationships can be produced: for example Annelida was not monophyletic, because one species was together with a mollusk as a sister group to a cephalochordate.

In summary, Ryan et al's phylogenetic analyses aren’t particularly convincing. Claiming that phylogenetic position of ctenophores is now resolved is annoying. Before we rearrange the animal tree of life, let's wait for more thorough analyses. And more data wouldn't hurt either.

References

Bracken-Grissom H, Collins AG, Collins T, Crandall K, Distel D, Dunn C, Giribet G, Haddock S, Knowlton N, Martindale M, Medina M, Messing C, O’Brien SJ, Paulay G, Putnam N, Ravasi T, Rouse GW, Ryan JF, Schulze A, Wörheide G, Adamska M, Bailly X, Breinholt J, Browne WE, Diaz MC, Evans N, Flot J-F, Fogarty N, Johnston M, Kamel B, Kawahara AY, Laberge T, Lavrov D, Michonneau F, Moroz LL, Oakley T, Osborne K, Pomponi SA, Rhodes A, Santos SR, Satoh N, Thacker RW, Van de Peer Y, Voolstra CR, Welch DM, Winston J, Zhou X (2014) The Global Invertebrate Genomics Alliance (GIGA): developing community resources to study diverse invertebrate genomes. The Journal of heredity 105: 1–18. doi: 10.1093/jhered/est084

Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith S a, Seaver E, Rouse GW, Obst M, Edgecombe GD, Sørensen M V, Haddock SHD, Schmidt-Rhaesa A, Okusu A, Kristensen RM, Wheeler WC, Martindale MQ, Giribet G (2008) Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452: 745–749. doi: 10.1038/nature06614

Kohn AB, Citarella MR, Kocot KM, Bobkova Y V, Halanych KM, Moroz LL (2012) Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei. Molecular phylogenetics and evolution 63: 203–207. doi: 10.1016/j.ympev.2011.12.009

Lartillot N, Philippe H (2004) A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Molecular biology and evolution 21: 1095–109. doi: 10.1093/molbev/msh112

Lartillot N, Brinkmann H, Philippe H (2007) Suppression of long-branch attraction artefacts in the animal phylogeny using a site-heterogeneous model. BMC evolutionary biology 7 Suppl 1: S4. doi: 10.1186/1471-2148-7-S1-S4

Le SQ, Gascuel O (2010) Accounting for solvent accessibility and secondary structure in protein phylogenetics is clearly beneficial. Systematic biology 59: 277–87. doi: 10.1093/sysbio/syq002

Le SQ, Dang CC, Gascuel O (2012) Modeling protein evolution with several amino Acid replacement matrices depending on site rates. Molecular biology and evolution 29: 2921–36. doi: 10.1093/molbev/mss112

Lasek-Nesselquist E, Gogarten JP (2013) The effects of model choice and mitigating bias on the ribosomal tree of life. Molecular phylogenetics and evolution 69: 17–38. doi: 10.1016/j.ympev.2013.05.006

Nosenko T, Schreiber F, Adamska M, Adamski M, Eitel M, Hammel J, Maldonado M, Müller WEG, Nickel M, Schierwater B, Vacelet J, Wiens M, Wörheide G (2013) Deep metazoan phylogeny: When different genes tell different stories. Molecular phylogenetics and evolution 67: 223–233. doi: 10.1016/j.ympev.2013.01.010

Pett W, Ryan JF, Pang K, Mullikin JC, Martindale MQ, Baxevanis AD, Lavrov D V (2011) Extreme mitochondrial evolution in the ctenophore Mnemiopsis leidyi: Insight from mtDNA and the nuclear genome. Mitochondrial DNA 22: 130–142. doi: 10.3109/19401736.2011.624611; alternateive link

Philippe H, Derelle R, Lopez P, Pick K, Borchiellini C, Boury-Esnault N, Vacelet J, Renard E, Houliston E, Quéinnec E, Da Silva C, Wincker P, Le Guyader H, Leys S, Jackson DJ, Schreiber F, Erpenbeck D, Morgenstern B, Wörheide G, Manuel M (2009) Phylogenomics revives traditional views on deep animal relationships. Current biology 19: 706–712. doi: 10.1016/j.cub.2009.02.052

Podar M, Haddock SH, Sogin ML, Harbison GR (2001) A molecular phylogenetic framework for the phylum Ctenophora using 18S rRNA genes. Molecular phylogenetics and evolution 21: 218–230. doi: 10.1006/mpev.2001.1036

Ryan JF, Pang K, Schnitzler CE, Nguyen A-D, Moreland RT, Simmons DK, Koch BJ, Francis WR, Havlak P, Smith S a, Putnam NH, Haddock SHD, Dunn CW, Wolfsberg TG, Mullikin JC, Martindale MQ, Baxevanis AD (2013) The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science 342: 1242592. doi: 10.1126/science.1242592

Phylogeny of eukaryotes

There have been some interesting developments regarding eukaryote phylogenetics recently. Particularly, it has become quite obvious that some biokonts are more closely related to unikonts than to the other bikonts, and it now seems likely that the last common ancestor of extant eukaryotes was a bikont with two anterior (part of the cell that faces the direction of movement) cilia arising from two basal bodies, one cilium directed forward, the other one backwards (Paps et al. 2013). A few years ago it was unclear if the last common ancestor of eukaryotes was uni- or bikont. Apusomonadida, Ancyromonadida, Mantamonadida, Breviatea, Diphyllatea (Fig. 1), and some other groups were  on morphological grounds regarded as bikonts (although some species among them are with only one or more than two cilia) and therefore more closely related to other bikonts (Diaphoretickes and Excavata) than to unikonts (Amoebozoa and Opisthokonta), but no phylogenomic data were available from these enigmatic lineages. Some limited molecular data supported that (Stechmann & Cavalier-Smith 2002) and some not (Kim et al. 2006). Now, in short succession, several interesting papers have been published analysing phylogenomic datasets including Apusomonadida, Breviatea, and Diphyllatea (Derelle & Lang 2012; Brown et al. 2013; Zhao et al. 2013). It is quite clear now that bikonts, Apusomonadida and Breviatea, are more closely related to Opisthokonta (one of the unikont lineages with two posterior basal bodies, but only one posterior flagellum) than to any other eukaryote group. Phylogenetic position of Diphyllatea is at the moment less certain, but it appears also to be more closely related to unikonts than to other bikonts (Zhao et al. 2013). There is a nice figure in Paps et al. (2013) summarizing evolution of ciliary apparatus in eukaryotes. In short, last common ancestor of extant eukaryotes may have possessed two anterior cilia arising from two basal bodies. In majority of Amoebozoa the cilia were lost (nevertheless there are amoebozoans with one or two anterior cilia) and in Opisthokonta ciliary apparatus with two basal bodies and one cilium moved to posterior part of the cell. Some Diphyllatea have more than two cilia and some Breviatea only one. Diverse group of Bikonta (Diaphoretickes + Excavata) includes many groups with more than two cilia or with none at all, but ancestral condition of two anterior cilia seems quite likely.

Fig. 1. Phylogeny of Eukaryota based on recent phylogenetic analyses (Hampl et al. 2009; Katz et al. 2011; Burki et al. 2012; Laurin-Lemay et al. 2012; Price et al. 2012; Timme et al. 2012; Zhao etal. 2012, 2013; Brown et al. 2013; Paps et al. 2013; Seenivasan et al. 2013; Yabuki et al. 2010; 2013). Amoeboid intracellular algal parasite group Aphelidea, which were thought to belong to Holozoa (Adl et al. 2012), were actually found to group with Fungi (Karpovet al. 2013). An intracellular parasite of oysters, Mikrocytos, has been confirmed to be a rhizarian (Burki et al. 2013). Consulted classifications: Adl et al. 2012; Cavalier-Smith (2013). Cavalier-Smith's classifications are quite idiosyncratic, super complicated, and change rather often (he's a very prolific author). At least this is the impression I'm getting. He also uses many (often novel) paraphyletic taxa.

The other interesting development is the rooting of the eukaryote tree, which is a difficult problem to solve because of the lack of a close out-group (all the prokaryotes are too distant) and rapid radiation of lineages at the base of extant eukaryotic tree of life (it is more like a bush than a tree in this case) (Brinkmann & Philippe 2007). Derelle & Lang (2012) have compiled an interesting dataset of 42 mitochondrial proteins (encoded by mitochondrial or nuclear genome) of alpha-proteobacterial origin to root the eukaryote tree. All known eukaryotes have mitochondria or their derivatives (mitosomes or hydrogenosomes; Hjort etal. 2010; Shiflett & Johnson 2010) and thanks to this, genes of mitochondrial origin can be used to reconstruct eukaryote phylogeny (Brinkmann & Philippe 2007). And the other good news is that there is a relatively close outgroup available - Alphaproteobacteria. Previous analyses relying heavily on nuclear informational genes (replication, transcription, translation) of archaeal origin (closest prokaryotic group to eukaryote nuclear lineage) were plagued by long-branch attraction (LBA) artefacts. LBA is an artefact of phylogenetic analyses where long branches (measured in the amount of estimated character change, e.g. nucleotide or amino acid substitutions) tend to group together regardless of evolutionary relationships, if the model or method used is not adequate to correct for multiple substitutions at the same site. For example, the fast evolving lineages (evident as long branches in the phylogenetic trees) of eukaryotes (often parasites) tend to be attracted towards the outgroup represented by long branch leading to Archaea, and in consequence distorting true relationships between eukaryotes. Derelle & Lang (2012) and now also Zhao et al. (2013) found that the root of the eukaryote tree falls between bikonts and unikonts, as suggested about 10 years ago (Stechmann & Cavalier-Smith 2002, 2003; Richards & Cavalier-Smith 2005), with the caveat that some bikonts, like Apusomonadida and Diphyllatea, are 'unikonts'. Standard phylogenomic analyses of eukaryotes (based on usually 100­­–200 highly expressed genes; e.g. many references in the caption of Fig. 1) without prokaryote outgroups are consistent with this kind of rooting (it is possible to root the resulting unrooted trees so that unikonts and bikonts are monophyletic). It is remarkable, apart from the rooting, that most of the well supported clades found in standard phylogenomic analyses are upheld by Derelle & Lang (2012) and Zhao et al. (2013), because most of the 42 proteins used in the mitochondrial dataset are different from the other analyses. For example, only 3 genes are in common with a dataset of 143 genes used by Hampl et al. (2009) (Derelle & Lang 2012). This gives some confidence that we are not completely lost in deciphering these ancient phylogenies.

Another fascinating aspect of eukaryote evolution is how they came to be in the first place. A straw man of a traditional view is that eukaryote gradually evolved from prokaryotic ancestor, acquiring cytoskeleton, phagocytosis, internal membranes (nuclear envelope, endoplasmic reticulum, golgi apparatus) and then engulfing bacteria which later became mitochondria and plastids. But now, more and more viable alternative seems to be that the origin of mitochondria marks the origin of eukaryotes. Martin & Müller (1998) proposed the hydrogen hypothesis for the first eukaryote, where symbiotic association between an Archaea and Alphaproteobacteria finally lead to Alphaproteobacteria living inside the Archaea. Nick Lane popularized this hypotheses in a book "Power, Sex, Suicide: Mitochondria and the Meaning of Life" (2005). Although the title might suggest some kind of a new age nonsense, it is in reality amazingly insightful and wonderfully written book about fundamentals of biology. I wish I read it long time ago (what other similar gems I might be missing?). Lane makes a convincing case why prokaryotes could not simply evolve to eukaryotes and that mitochondria were needed to give a possibility to evolve large and dynamic cells with large genomes, which could then lead to multicellularity. This idea has been elaborated and supported with numbers by Lane & Martin (2010). According to this view, most of the defining features of eukaryotes, like dynamic cytoskeleton, phagocytosis, nucleus, endomembrane trafficking, and sex, all evolved because of mitochondria! The arguments are related to energy generation and requirement for mitochondria to retain control over respiration. Internalization of energy production by mitochondria inside the host cell made it more efficient (by overcoming surface-to-volume constraints) and freed its cell membrane for other tasks (prokaryotes do not have this kind of luxury). Because oxygen is very reactive and easily damages cell constituents, it was necessary to tightly regulate oxygen level inside the cell. The most efficient way seems to be that mitochondria retain part of their genome so that they can quickly respond to varying levels of oxygen by controlling synthesis of respiratory complexes. There is the same core set of about 10–30 genes, which all free living aerobic eukaryotes have retained in their mitochondrial genomes (some have additional genes, which others have lost). Granted with specialized energy producing organelles inside the cell, nuclear genome could freely acquire more and more different protein coding genes and regulatory sequences without high energetic costs to express them. This opened the road to higher complexity and eventually to multicellularity. In line with the view that mitochondria started eukaryogenesis are recent (e.g. Cox et al. 2008; Guy & Ettema 2011; Lasek-Nesselquist & Gogarten 2013; Williams et al. 2013) and not so recent (e.g. Lake et al.1984; Rivera & Lake 1992) findings that nuclear lineage of eukaryotes probably originates within Archaea (it appears that Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota are more closely related to eukaryotes than they are to other Archaea, Euryarchaeota and Nanoarchaeota). Nevertheless, these findings themselves do not exclude the possibility that the eukaryotic lineage within Archaea evolved (some) eukaryotic characters before acquiring mitochondria.