Concatenated analysis sheds light on early metazoan evolution and fuels a modern "urmetazoon" hypothesis

Abstract

For more than a century, the origin of metazoan animals has been debated. One aspect of this debate has been centered on what the hypothetical "urmetazoon" bauplan might have been. The morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles an intriguing model for one of several "urmetazoon" hypotheses: the placula hypothesis. Clear support for a basal position of Placozoa would aid in resolving several key issues of metazoan-specific inventions (including, for example, head-foot axis, symmetry, and coelom) and would determine a root for unraveling their evolution. Unfortunately, the phylogenetic relationships at the base of Metazoa have been controversial because of conflicting phylogenetic scenarios generated while addressing the question. Here, we analyze the sum of morphological evidence, the secondary structure of mitochondrial ribosomal genes, and molecular sequence data from mitochondrial and nuclear genes that amass over 9,400 phylogenetically informative characters from 24 to 73 taxa. Together with mitochondrial DNA genome structure and sequence analyses and Hox-like gene expression patterns, these data (1) provide evidence that Placozoa are basal relative to all other diploblast phyla and (2) spark a modernized "urmetazoon" hypothesis.

Figure 1. Discussed Relationships at the Base of the Metazoan Tree

Potential arrangements of five critical taxa (B, Bilateria; Cn, Cnidaria; Ct, Ctenophora; P, Placozoa; and S, Porifera) are shown on the right, and some hypotheses in the literature with only four taxa (Placozoa omitted) on the left. Arrows indicate the root of the networks. The letters at the arrows are for reference to Table S1. The uppercase letters refer to publications in Table S1 that support the indicated root for trees without Placozoa. The lowercase letters refer to publications in Table S1 that support the root for trees with all five taxa.

Figure 2. Maximum Likelihood Phylogenetic Tree of Metazoan Relationships Using the Concatenated Data Matrix

Node support is based on the best ML tree filtered through 1,000 rapid bootstrap replicates. Only support values below 100% are shown. Bayesian inference supported strongly (posterior probability = 1.0) all nodes with the exception of monophyly of Cnidaria. The maximum a posteriori and the Bayesian 50% majority-rule consensus trees disagreed with the best ML tree in supporting a Ctenophora–Anthozoa clade with posterior probability of 0.98. Please note that “Coelenterata” is not a taxonomic unit, but rather it is a traditional grouping for reasons of convenience. The alpha shape parameters of the Gamma distribution were 0.507454 and 0.651659 for the nucleotide and amino acid partitions, respectively. Log-likelihood = −261429.821426.

Figure 3. Phylogeny of Animals and Weighting Schemes

The impact of several weighting schemes on the phylogenetic hypothesis in Figure 2. The values in the table are jackknife values for maximum parsimony, rapid bootstrap for ML, and posterior clade probabilities for Bayesian inference. The color coding for the values is shown at the bottom of the table. The major monophyletic groups examined for jackknife support in Figure 2 are indicated in the top row. See Figure 2 for nodes defined by these groups. Monosiga refers to placing Monosiga as basal to Metazoa, and Placozoa refers to placing Placozoa as basal to diploblasts. Total in the first row refers to the entire dataset analyzed with equal weighting of all characters. The next four rows show results for analyses of partitioned datasets: mtDNA, mitochondrial partition; Nuclear, nuclear; Protein, protein; and rRNA, ribosomal RNAs from both nuclear and mitochondrial genomes. The bottom rows show results for various weighting schemes; 2:rRNA, 10:rRNA, and 100:rRNA refer to weighting schemes in which transversions are weighted 2, 10, and 100 times more than transitions, respectively. Protein weighting schemes are Gonnet weighting matrix, Whelan and Goldman (WAG) matrix, Le and Gascuel (LG) matrix, and genetic identity (GI). For details on weighting matrices, see Figure S4.

Figure 4. Modern Interpretation and Modification of the Placula Hypothesis of Metazoan Origin

Here, a nonsymmetric and axis-lacking bauplan (placula) transforms into a typical symmetric metazoan bauplan with a defined oral–aboral or anterior–posterior body axis. In the placula transformation, a primitive disk consisting of an upper and a lower epithelium (lower row), which can be derived from a flattened multicellular protist, forms an external feeding cavity between its lower epithelium and the substrate (second row from bottom). The latter is achieved by the placula lifting up the center of its body, as this is naturally seen in feeding Trichoplax (i.e., the two Trichoplax images derive from a nonfeeding (first row) and feeding (second row) individual. If this process is continued, the external feeding cavity increases (cross section, third row) while at the same time the outer body shape changes from irregular to more circular (see oral views). Eventually, the process results in a bauplan in which the formerly upper epithelium of the placula remains outside (and forms the ectoderm) and the formerly lower epithelium becomes “inside” (and forms the entoderm; upper row). This is the basic bauplan of Cnidaria and Porifera. Three of the four transformation stages have living counterparts in the form of resting Trichoplax, feeding Trichoplax, and cnidarian polyps and medusae (right column). The above-outlined transformation of a placula into a cnidarian bauplan involves the development of a main body axis and a head region, which allows the invention of new structures and organs for feeding. From a developmental genetics point of view, a single regulatory gene would be required to control separation between the lower and upper epithelium (three lower rows). If the above scenario were correct, the following empirical data would be congruent with it. In the form of the putative ProtoHox/ParaHox gene, Trox-2, in Trichoplax, we find a single regulatory gene, marks the differentiation of an as yet undescribed cell type at the lower–upper epithelium boundary in Trichoplax [46]. More than one regulatory gene would be required to organize new head structures originating from the ectoderm–entoderm boundary of the oral pole (upper row). Quite noteworthy, two putative descendents of the Trox-2 gene, Cnox-1 and Cnox-3, show these hypothesized expression patterns (Diplox expression upper row; for simplicity, only the ring for Cnox-1 expression is shown; see Figure S4 for expression patterns of both genes and Jakob et al. [46,52] for details. Cnox-1 and Cnox-3 expression both mark the ectoderm-entoderm boundary at the oral pole in the hydrozoan Eleutheria dichotoma. Both genes are expressed in parallel in a ring-shaped manner at the tip of the manubrium, with Cnox-3 being expressed more ectodermally and Cnox-1 being expressed more entodermally (unpublished data).