New phylogenomic data support the monophyly of Lophophorata and an Ectoproct-Phoronid clade and indicate that Polyzoa and Kryptrochozoa are caused by systematic bias.

BACKGROUND: Within the complex metazoan phylogeny, the relationships of the three lophophorate lineages, ectoprocts, brachiopods and phoronids, are particularly elusive. To shed further light on this issue, we present phylogenomic analyses of 196 genes from 58 bilaterian taxa, paying particular attention to the influence of compositional heterogeneity. RESULTS: The phylogenetic analyses strongly support the monophyly of Lophophorata and a sister-group relationship between Ectoprocta and Phoronida. Our results contrast previous findings based on rDNA sequences and phylogenomic datasets which supported monophyletic Polyzoa (= Bryozoa sensu lato) including Ectoprocta, Entoprocta and Cycliophora, Brachiozoa including Brachiopoda and Phoronida as well as Kryptrochozoa including Brachiopoda, Phoronida and Nemertea, thus rendering Lophophorata polyphyletic. Our attempts to identify the causes for the conflicting results revealed that Polyzoa, Brachiozoa and Kryptrochozoa are supported by character subsets with deviating amino acid compositions, whereas there is no indication for compositional heterogeneity in the character subsets supporting the monophyly of Lophophorata. CONCLUSION: Our results indicate that the support for Polyzoa, Brachiozoa and Kryptrochozoa gathered so far is likely an artifact caused by compositional bias. The monophyly of Lophophorata implies that the horseshoe-shaped mesosomal lophophore, the tentacular feeding apparatus of ectoprocts, phoronids and brachiopods is, indeed, a synapomorphy of the lophophorate lineages. The same may apply to radial cleavage. However, among phoronids also spiral cleavage is known. This suggests that the cleavage pattern is highly plastic and has changed several times within lophophorates. The sister group relationship of ectoprocts and phoronids is in accordance with the interpretation of the eversion of a ventral invagination at the beginning of metamorphosis as a common derived feature of these taxa.

Mitochondrial genomes to the rescue--Diurodrilidae in the myzostomid trap.

Diurodrilidae is a taxon of Lophotrochozoa comprising about six, exclusively interstitial species, which are up to 500µm long and dorsoventrally flattened. Traditionally, Diurodrilidae had been regarded as an annelid family. However, recently Diurodrilidae had been excluded from Annelida and been placed in closer relationship to platyzoan taxa based on both morphological and nuclear rRNA data. Since both, Diurodrilidae and platyzoan taxa, exhibit long branches in the molecular analyses, the close relationship might be due to a long branch attraction artifact. The annelid taxon Myzostomida had been trapped in a similar long branch attraction artifact with platyzoan taxa using nuclear rRNA data, but determination of the nearly complete mitochondrial genome of myzostomids revealed their annelid affinity. Therefore, we determined the nearly complete mitochondrial genome of Diurodrilus subterraneus as well as new nuclear rRNA data for D. subterraneus and some platyzoan taxa. All our analyses of nuclear rRNA and mitochondrial sequence and gene order data presented herein clearly place Diurodrilidae within Annelida and with strong nodal support values in some analyses. Therefore, the previously suggested exclusion of Diurodrilidae from Annelida and its close relationship with platyzoan taxa can be attributed to a long branch artifact. Morphological data do not unambiguously support a platyzoan affinity of Diurodrilidae, but instead would also be in line with a progenetic origin of Diurodrilidae within Annelida.

Agent of whirling disease meets orphan worm: phylogenomic analyses firmly place Myxozoa in Cnidaria.

Myxozoa are microscopic obligate endoparasites with complex live cycles. Representatives are Myxobolus cerebralis, the causative agent of whirling disease in salmonids, and the enigmatic "orphan worm" Buddenbrockia plumatellae parasitizing in Bryozoa. Originally, Myxozoa were classified as protists, but later several metazoan characteristics were reported. However, their phylogenetic relationships remained doubtful. Some molecular phylogenetic analyses placed them as sister group to or even within Bilateria, whereas the possession of polar capsules that are similar to nematocysts of Cnidaria and of minicollagen genes suggest a close relationship between Myxozoa and Cnidaria. EST data of Buddenbrockia also indicated a cnidarian origin of Myxozoa, but were not sufficient to reject a closer relationship to bilaterians. Phylogenomic analyses of new genomic sequences of Myxobolus cerebralis firmly place Myxozoa as sister group to Medusozoa within Cnidaria. Based on the new dataset, the alternative hypothesis that Myxozoa form a clade with Bilateria can be rejected using topology tests. Sensitivity analyses indicate that this result is not affected by long branch attraction artifacts or compositional bias.

Detecting the symplesiomorphy trap: a multigene phylogenetic analysis of terebelliform annelids.

BACKGROUND: For phylogenetic reconstructions, conflict in signal is a potential problem for tree reconstruction. For instance, molecular data from different cellular components, such as the mitochondrion and nucleus, may be inconsistent with each other. Mammalian studies provide one such case of conflict where mitochondrial data, which display compositional biases, support the Marsupionta hypothesis, but nuclear data confirm the Theria hypothesis. Most observations of compositional biases in tree reconstruction have focused on lineages with different composition than the majority of the lineages under analysis. However in some situations, the position of taxa that lack compositional bias may be influenced rather than the position of taxa that possess compositional bias. This situation is due to apparent symplesiomorphic characters and known as "the symplesiomorphy trap". RESULTS: Herein, we report an example of the sympleisomorphy trap and how to detect it. Worms within Terebelliformia (sensu Rouse & Pleijel 2001) are mainly tube-dwelling annelids comprising five 'families': Alvinellidae, Ampharetidae, Terebellidae, Trichobranchidae and Pectinariidae. Using mitochondrial genomic data, as well as data from the nuclear 18S, 28S rDNA and elongation factor-1α genes, we revealed incongruence between mitochondrial and nuclear data regarding the placement of Trichobranchidae. Mitochondrial data favored a sister relationship between Terebellidae and Trichobranchidae, but nuclear data placed Trichobranchidae as sister to an Ampharetidae/Alvinellidae clade. Both positions have been proposed based on morphological data. CONCLUSIONS: Our investigation revealed that mitochondrial data of Ampharetidae and Alvinellidae exhibited strong compositional biases. However, these biases resulted in a misplacement of Trichobranchidae, rather than Alvinellidae and Ampharetidae. Herein, we document that Trichobranchidae was apparently caught in the symplesiomorphy trap suggesting that in certain situations even homologies can be misleading.

The complete mitochondrial genome of Flustra foliacea (Ectoprocta, Cheilostomata) - compositional bias affects phylogenetic analyses of lophotrochozoan relationships.

BACKGROUND: The phylogenetic relationships of the lophophorate lineages, ectoprocts, brachiopods and phoronids, within Lophotrochozoa are still controversial. We sequenced an additional mitochondrial genome of the most species-rich lophophorate lineage, the ectoprocts. Although it is known that there are large differences in the nucleotide composition of mitochondrial sequences of different lineages as well as in the amino acid composition of the encoded proteins, this bias is often not considered in phylogenetic analyses. We applied several approaches for reducing compositional bias and saturation in the phylogenetic analyses of the mitochondrial sequences. RESULTS: The complete mitochondrial genome (16,089 bp) of Flustra foliacea (Ectoprocta, Gymnolaemata, Cheilostomata) was sequenced. All protein-encoding, rRNA and tRNA genes are transcribed from the same strand. Flustra shares long intergenic sequences with the cheilostomate ectoproct Bugula, which might be a synapomorphy of these taxa. Further synapomorphies might be the loss of the DHU arm of the tRNA L(UUR), the loss of the DHU arm of the tRNA S(UCN) and the unique anticodon sequence GAG of the tRNA L(CUN). The gene order of the mitochondrial genome of Flustra differs strongly from that of the other known ectoprocts. Phylogenetic analyses of mitochondrial nucleotide and amino acid data sets show that the lophophorate lineages are more closely related to trochozoan phyla than to deuterostomes or ecdysozoans confirming the Lophotrochozoa hypothesis. Furthermore, they support the monophyly of Cheilostomata and Ectoprocta. However, the relationships of the lophophorate lineages within Lophotrochozoa differ strongly depending on the data set and the used method. Different approaches for reducing heterogeneity in nucleotide and amino acid data sets and saturation did not result in a more robust resolution of lophotrochozoan relationships. CONCLUSION: The contradictory and usually weakly supported phylogenetic reconstructions of the relationships among lophotrochozoan phyla based on mitochondrial sequences indicate that these alone do not contain enough information for a robust resolution of the relations of the lophotrochozoan phyla. The mitochondrial gene order is also not useful for inferring their phylogenetic relationships, because it is highly variable in ectoprocts, brachiopods and some other lophotrochozoan phyla. However, our study revealed several rare genomic changes like the evolution of long intergenic sequences and changes in the structure of tRNAs, which may be helpful for reconstructing ectoproct phylogeny.

Compositional heterogeneity and phylogenomic inference of metazoan relationships.

Compositional heterogeneity of sequences between taxa may cause systematic error in phylogenetic inference. The potential influence of such bias might be mitigated by strategies to reduce compositional heterogeneity in the data set or by phylogeny reconstruction methods that account for compositional heterogeneity. We adopted several of these strategies to analyze a large ribosomal protein data set representing all major metazoan taxa. Posterior predictive tests revealed that there is compositional bias in this data set. Only a few taxa with strongly deviating amino acid composition had to be excluded to reduce this bias. Thus, this is a good solution, if these taxa are not central to the phylogenetic question at hand. Deleting individual proteins from the data matrix may be an appropriate method, if compositional heterogeneity among taxa is concentrated in a few proteins. However, half of the ribosomal proteins had to be excluded to reduce the compositional heterogeneity to a degree that the CAT model was no longer significantly violated. Recoding of amino acids into groups is another alternative but causes a loss of information and may result in badly resolved trees as demonstrated by the present data set. Bayesian inference with the CAT-BP model directly accounts for compositional heterogeneity between lineages by introducing breakpoints along the branches of the phylogeny at which the amino acid composition is allowed to change but is computationally expensive. Finally, a neighbor joining tree based on equal input distances that consider pattern and rate heterogeneity showed several unusual groupings, which are most likely artifacts, probably caused by the loss of information resulting from the transformation of the sequence data into distances. As long as no more efficient phylogenetic inference methods are available that can directly account for compositional heterogeneity in large data sets, using methods for reducing compositional heterogeneity in the data in combination with methods that assume a stationary amino acid composition remains an option for controlling systematic errors in tree reconstruction that result from compositional bias. Our analyses indicated that the paraphyly of Deuterostomia in some analyses is the result of systematic errors that also affected the relationships of Entoprocta and Ectoprocta.

Phylogenetic relationships within the lophophorate lineages (Ectoprocta, Brachiopoda and Phoronida).

We produced two new EST datasets of so far uncovered clades of ectoprocts to investigate the phylogenetic relationships within the lophophorate lineages, Ectoprocta, Brachiopoda and Phoronida. Maximum-likelihood analyses based on 78 ribosomal proteins of 62 metazoan taxa support the monophyly of Ectoprocta and a sister group relationship of Phylactolaemata living in freshwater and the mainly marine Gymnolaemata. Hypotheses suggesting that Ectoprocta is diphyletic with phylactolaemates forming a clade with phoronids or paraphyletic with respect to Entoprocta could be rejected by topology tests. The hypotheses that Stenolaemata are the sister group of all other ectoprocts, that Stenolaemata constitutes a monophyletic group with Cheilostomata, and that Phylactolaemata have been derived from Ctenostomata could also be excluded. However, the hypothesis that Phylactolaemata and Stenolaemata form a monophyletic group could not be rejected. Brachiopoda and Phoronida constitute a monophylum, Brachiozoa. The hypotheses that phoronids are the sister group of articulate or inarticulate brachiopods could be rejected by topology tests, thus confirming the monophyly of Brachiopoda.

Detecting possibly saturated positions in 18S and 28S sequences and their influence on phylogenetic reconstruction of Annelida (Lophotrochozoa).

Phylogenetic reconstructions may be hampered by multiple substitutions in nucleotide positions obliterating signal, a phenomenon called saturation. Traditionally, plotting ti/tv ratios against genetic distances has been used to reveal saturation by assessing when ti/tv stabilizes at 1. However, interpretation of results and assessment of comparability between different data sets or partitions are rather subjective. Herein, we present the new C factor, which quantifies convergence of ti/tv ratios, thus allowing comparability. Furthermore, we introduce a comparative value for homoplasy, the O/E ratio, based on alterations of tree length. Simulation studies and an empirical example, based on annelid rRNA-gene sequences, show that the C factor correlates with noise, tree length and genetic distance and therefore is a proxy for saturation. The O/E ratio correlates with the C factor, which does not provide an intrinsic threshold of exclusion, and thus both together can objectively guide decisions to exclude saturated nucleotide positions. However, analyses also showed that, for reconstructing annelid phylogeny using Maximum Likelihood, an increase in numbers of positions improves tree reconstruction more than does the exclusion of saturated positions.