A Concatenated Dataset for Metazoa
Given that both nonphylogenetic interpretation of morphological data as well as molecular analyses of sequence data have failed to resolve the issue, a more comprehensive, systematic analysis of morphological data and new molecular markers are now a requisite for identifying the root of the metazoan tree of life. To approach this goal, we conducted concatenated analyses for 24 metazoan taxa from all of the major organismal lineages in this part of the tree of life that included morphological characters (17 characters), both mitochondrial and nuclear ribosomal gene sequences (five gene partitions for 6,111 nucleotide positions) and molecular morphology [8
] (ten characters), as well as nuclear coding genes (16 gene partitions derived from our database searches and another 18 gene partitions derived from the Dunn et al. (2008) study [25
]; see Materials and Methods
) for 8,307 amino acid positions and protein coding genes (16 gene partitions for 3,004 amino acid characters) to resolve phylogenetic relationships between recent diploblast groups. The total number of characters included was 17,664 from 51 partitions, giving 7,822 phylogenetically informative characters. We also constructed a matrix with a larger number of taxa based on the Dunn et al. (2008) [25
] study with 73 taxa for the same gene partitions (see Materials and Methods
and Tables S2
). This matrix had 17,637 total characters and 9,421 phylogenetically informative characters. In addition, Hox gene expression was compared for a placozoan and a cnidarian bauplan to test predictions from the placula hypothesis [5
Clarity and Confusion at the Root of the Metazoan Tree
Parsimony, likelihood (with morphological characters removed), and mixed Bayesian analysis of the smaller concatenated matrix using a variety of approaches, weighting schemes, and models is generally consistent with the view that Bilateria and diploblasts (Porifera, Ctenophora, Placozoa, and Cnidaria) are sister groups. In addition, Placozoa are robustly observed as the most basal diploblast group ( and ). shows the support for several hypotheses of monophyly obtained from diverse methods of analysis. Porifera, Bilateria, and Fungi all form strong monophyletic groups (). The four cnidarian classes (Anthozoa, Hydrozoa, Scyphozoa, and Cubozoa) together with the Ctenophora form a monophyletic group, the “Coelenterata.” Within the Cnidaria, the generally accepted basal position of the anthozoans is also recovered by this analysis [34
]. Both choanoflagellates and Placozoa are strongly excluded from a Porifera–Coelenterata monophyletic group. The basal position of Placozoa is also strongly supported by comparing the phylogeny in with hypotheses that place it more derived, using the statistical approach of Shimodaira and Hasegawa [36
]. This battery of tests () demonstrates that the basal position of the Placozoa is significantly better than other hypotheses. The 95% confidence tree includes the Maximum Likelihood (ML) and Bayesian trees (both with Placozoa as basal in the diploblasts) with a cumulative expected likelihood weight (ELW) of 0.960763.
Maximum Likelihood Phylogenetic Tree of Metazoan Relationships Using the Concatenated Data Matrix
Phylogeny of Animals and Weighting Schemes
Comparison of Competing Phylogenetic Hypotheses
The tree topology shown in summarizes the best supported phylogenetic hypothesis obtained by using Maximum Parsimony, ML, and Bayesian analyses of the concatenated dataset. Analysis of the larger matrix (Figure S2
) was less well resolved within the Bilateria, but showed the same general topology as the smaller analysis. Specifically, Bilateria are monophyletic and sister to the diploblasts, with the choanoflagellate Monosiga
basal to these taxa with high jackknife values and Bayesian posteriors. Diploblasts are also monophyletic, and Placozoa are the most basal taxon in the diploblasts. In addition, within the diploblasts, Porifera and Coelenterata are monophyletic, and within Bilateria, Ecdysozoa and Deuterostomia are monophyletic; all groupings with high node support.
The topology within the diploblasts is also robust when Bilateria are removed from the analysis. The full analysis seemingly misplaces the Bilateria clade as the sister to all diploblasts. The classical position of the Bilateria is in a highly derived position from within the diploblasts and usually sister to the Cnidaria. The seemingly “weird” prediction of a basal Bilateria from the present analysis has been observed before in other studies (see Table S1
). Several studies have addressed phylogenetic problems specific to this region of the tree of life and have suggested that this region of the tree will be inherently difficult to resolve. These studies suggest that the compression of splitting events in this region renders the resolution of these nodes with high support difficult, if not impossible [38
]. These studies have suggested that even large amounts of data might not resolve the problem. Other studies have pointed to taxon sampling and modeling as a potential problem in resolving this part of the tree of life [25
]. Another problem is that the large number of molecular phylogenetic approaches creates multiple and possibly the most short-lived hypotheses in biology. The large repertoire of algorithms, models, and assumptions sometimes produces a forest of trees from the same dataset (cf. [43
]). Thus, tree-building procedures are highly crucial and deserve particular attention if this region of the tree of life is to be resolved [38
Resurrecting the “Placula”
Although the hypothesis in is in conflict with a recent analysis of coding genes from whole genomes [23
] as well as is in conflict with other studies (Table S1
), the scenario presented here is consistent with another set of studies and also with one of the major urmetazoon hypotheses, the placula hypothesis (). This hypothesis fuels intriguing scenarios for the mechanisms and direction of anagenetic evolution in Metazoa, and in the form presented here, it can illustrate the derivation of Cnidaria and Bilateria from a placozoan-like ancestor. A basal position of Placozoa relative to Cnidaria, and diploblasts sister to Bilateria are cum grano salis
consistent with several recent molecular phylogenetic analyses ([23
] and this study) encouraging us to reconsider the placula hypothesis in a modern light.
Modern Interpretation and Modification of the Placula Hypothesis of Metazoan Origin
The comparison of Hox/ParaHox-like gene expression patterns in Placozoa and Cnidaria creates a new working hypothesis for the origin of the entoderm, a main body axis, and symmetry. Based on the undisputed evidence that Placozoa are basal relative at least to Cnidaria, the Trox-2
gene is likely ancestral to Hox/ParaHox-like genes from Cnidaria (as formerly suggested [44
is expressed at the gastrodermis/epidermis (lower/upper epithelium) boundary in Trichoplax
]. Strikingly, we found similar expression patterns for two putative Trox-2
descendents in the hydrozoan Eleutheria dichotoma
(). These regulatory gene expression data mirror directly the beginning and ending stage of a modern interpretation of the placula hypothesis. The latter explains the origin of a symmetric bauplan with one or two defined body axes and an internal feeding cavity from a simple placuloid (proto-placozoan–like) bauplan that lacked all of the former characteristics. In the most parsimonious scenario, the expression of a single regulatory gene defines polarity in Placozoa, i.e., the differentiation of a lower versus upper epithelium. According to the proposed “new placula hypothesis,” the nonsymmetric placozoan bauplan transforms into a symmetric Cnidaria (or also Bilateria) bauplan by the former ring of epithelia boundary separation transforming into the new “oral” region of the derived symmetric bauplan (). This transformation is simply the result of a placula lifting up its feeding epithelium in order to form an external feeding cavity, keeping function and morphology of the epithelium unchanged. In the final stage, the “oral” pole develops specialized organs, such as a mouth and tentacles for feeding (cf. [47
]). The latter could be driven by duplication of the regulatory gene, which originally defined polarity in the placula (; cf. [48
] for review). Observations on extant Placozoa and Cnidaria mirror this scenario almost perfectly ().
Although prediction and observation match nicely, one has to note, however, that no gene or even gene family, no matter how important, can provide more than just indirect support for a working hypothesis on a hypothetical animal bauplan that can never be observed. It is important to note that multiple topologies can be consistent with the placula hypothesis and that the form of the extant earliest-branching lineage does not necessarily have to represent the form of the ancestor; we consider the latter, however, the more parsimonious alternative. We also point out that the regulatory gene family mentioned here, Hox/ParaHox-like genes, seems to be absent in sponges [49
]. A secondary loss of Hox/ParaHox-like genes in sponges seems plausible, and the work by Peterson and Sperling, 2007 [50
] provides some evidence for this assumption. Whether a possible loss of a Hox/ParaHox gene might be related to the reduction of epithelial organization in Porifera [3
] remains an interesting speculation.
The Hox/ParaHox loss scenario in sponges is just one of several crucial questions raised by the phylogeny in . According to this phylogeny, diploblasts and Bilateria both may have started from a placula-like bauplan as suggested in (“new placula hypothesis”). The shown new placula hypothesis illustrates a potential transition from a nonsymmetric, axis-lacking placula into a radial symmetric and head–foot axis organized cnidarian. In a similar way, the placula could also be transformed into a Bilateria bauplan, i.e., a bilaterally symmetric bauplan with an anterior–posterior body axis. One of the easiest models for adopting a bilateral symmetry suggests that the “urbilaterian” kept the benthic lifestyle of the placula but adopted directional movement. The latter almost automatically leads to an anterior–posterior and ventral–dorsal differentiation. The pole moving forward develops a head and becomes anterior, the body side facing the ground carries the mouth and thus by definition becomes ventral. According to the above scenario, the main body axes of diploblastic animals and Bilateria were independent inventions. Whereas an independent evolution of body axes in diploblastic animals and Bilateria seems easily plausible, the independent evolution of other characters (e.g., the nervous system; see below) seems less plausible given our knowledge of the development and morphology of these characters.
We will never observe the hypothetical placula, but we may draw some conclusions from Placozoa, which seem to have retained many of the characteristics of the placula if our interpretation is valid. This scenario draws into question several aspects of animal evolution that will require reinterpretation if this hypothesis is correct. Most notable of these aspects is the evolution of the nervous system, which in the hypothesis in , can only be explained by convergent evolution of Cnidaria and Bilateria nervous system organization. According to the placula hypothesis, we suggest that the placula already had the genetic capability and basic building blocks to build a nervous system, and that from here, the final build-up of the nervous system developed via independent, but parallel, pathways in diploblasts and Bilateria. The genome of the placozoan Trichoplax adhaerens
indeed delivers some notable evidence that the genetic inventory may precede morphological manifestation of organs [23
]. For example, the placozoan genome harbors representatives of all major genes that are involved in neurogenesis in higher animals, whereas placozoans show not the slightest morphological hint of nerve or sensory cells. Quite noteworthy, however, is that placozoans are quite capable of stimuli reception and perception used to coordinate behavioral responses. In this light, the generally accepted unlikely convergent evolution of a nervous system only looks unlikely from a morphological, but not from a genetic and physiological, point of view.
Regardless of the need for reinterpretation of this and other anatomical characters, the findings presented here provide a viable hypothesis for the major cladogenetic events during the metazoan radiation. Given the basal position of Placozoa, we suggest that at least for diploblastic metazoan life, the body plan started with the following: an asymmetric body plan, a most simple morphology (only two steps above basic definition [51
]), a single ProtoHox gene, a large mitochondrial (mtDNA) genome, an outer feeding epithelium that gave rise to the entoderm, and the smallest of all known (not secondarily reduced) metazoan genomes. If the placula is also the ancestral state for metazoans (i.e., the common ancestor of Bilateria and diploblasts in ), then the same could be said for the urmetazoon.