Phylogenomische Untersuchungen zur Aufklärung des Metazoa-Stammbaums

Zentrales Thema der Arbeit war die Aufklärung von Verwandtschaftsverhältnissen im „Tree of Life“ der vielzelligen Tiere (Metazoa) unter Einsatz großer DNA-Sequenzdatensätze und phylogenomischer Methoden. Zur Untersuchung der internen Phylogenie der Syndermata (= meist freilebende Rädertiere („Rotifera“) + endoparasitische Kratzwürmer (Acanthocephala)) sowie ihrer Position im Metazoen-Stammbaum wurden insgesamt sieben neue mitochondriale (mt) Genome sowie neue Transkriptom-Sequenzdaten von sieben verschiedenen Syndermata-Spezies generiert und/oder analysiert. Die Stammbaumrekonstruktionen auf Grundlage dieser sowie orthologer Sequenzen anderer Spezies in Form von phylogenomischen Datensätzen mit bis zu 82.000 Aminosäurepositionen ergaben folgende Aussagen zur Evolution: (i) Innerhalb der Acanthocephala bilden monophyletische Palaeacanthocephala das Schwestertaxon zu den Eoacanthocephala. Die Archiacanthocephala sind Schwestertaxon zu allen vorgenannten. (ii) Innerhalb der Syndermata bilden die epizoisch lebenden Seisonidea das Schwestertaxon zu den endoparasitischen Acanthocephala (= Pararotatoria), die Bdelloidea sind das Schwestertaxon zu den Pararotatoria (= Hemirotifera) und die Monogononta das Schwestertaxon zu den Hemirotifera. Die klassischen Eurotatoria (= Bdelloidea + Monogononta) sind demnach paraphyletisch. (iii) Innerhalb der Metazoa bilden die Syndermata gemeinsam mit den Gnathostomulida die Gnathifera. Diese sind die Schwestergruppe zu allen anderen Spiralia-Taxa, welche sich in Rouphozoa (= Platyhelminthes + Gastrotricha) sowie die Lophotrochozoa aufspalten. Die Platyzoa (= Gnathifera + Platyhelminthes + Gastrotricha) sind demnach paraphyletisch. Diese phylogenetischen Hypothesen wurden im Hinblick auf ihre Implikationen für die Evolution morphologischer und ökologischer Merkmale interpretiert. Demnach sind während der Evolution dieser Tiergruppen mehrfach sekundäre Verlustereignisse von komplexen morphologischen Merkmalen aufgetreten (laterale sensorische Organe innerhalb der Acanthocephala und das Räderorgan (Corona) innerhalb der Syndermata), was die Verwendung dieser Merkmale im Sinne einer klassisch-morphologischen Phylogenetik kritisch erscheinen lässt. Der Endoparasitismus der Acanthocephala hat sich wahrscheinlich über ein epizoisches Zwischenstadium, wie man es heute noch bei den Seisonidea findet, entwickelt. Der letzte gemeinsame Vorfahre der Spiralia war vermutlich klein und unsegmentiert und besaß keine echte Leibeshöhle (Coelom). Demnach hätten sich Segmentierung und Coelome innerhalb der Metazoa mehrfach unabhängig voneinander (konvergent) entwickelt. Die Arbeit beinhaltete folgende weitere, zum Teil methodische Aspekte: (i) die Analyse der Architektur der mt Genome der Monogononta bestätigte die aberrante Organisation in zwei Subgenomen für die Brachionidae. (ii) Eine Prüfung der Tauglichkeit ribosomaler Proteine für molekular-phylogenetische Arbeiten ergab das Vorhandensein widersprüchlicher phylogenetischer Signale in diesen speziellen Proteinsequenzen. (iii) Es konnte nachgewiesen werden, dass systematische Fehler wie „long-branch attraction“ bei der Positionierung der Syndermata im Stammbaum der Metazoa eine große Rolle spielen und adressiert werden müssen.Main subject of the thesis was the clarification of relationships in the “Tree of Life” of multicellular animals (Metazoa) by using large DNA-sequence datasets and phylogenomic methods. To investigate the internal phylogeny of Syndermata (= mostly free-living wheel animals (“Rotifera”) + endoparasitic thorny-headed worms (Acanthocephala)) and their position in the metazoan tree, a total of seven new mitochondrial (mt) genomes and new transcriptomic data of seven different Syndermata species were generated and/or analyzed. Tree reconstructions were based on these as well as orthologous sequences of other species in the form of phylogenomic datasets comprising up to 82,000 amino acid positions and resulted in the following statements concerning evolution: (i) Within Acanthocephala, monophyletic Palaeacanthocephala are sister taxon to Eoacanthocephala. Archiacanthocephala are sister taxon to all former mentioned taxa. (ii) Within Syndermata, epizoic Seisonidea are sister taxon to endoparasitic Acanthocephala (= Pararotatoria), Bdelloidea are sister taxon to Pararotatoria (= Hemirotifera) and Monogononta are sister taxon to Hemirotifera. Hence, classical Eurotatoria (= Bdelloidea + Monogononta) are paraphyletic. (iii) Within Metazoa, Syndermata and Gnathostomulida constitute Gnathifera. This taxon is sister taxon to all other taxa of the Spiralia, which split up into Rouphozoa (= Platyhelminthes + Gastrotricha) and Lophotrochozoa. Hence, Platyzoa (= Gnathifera + Platyhelminthes + Gastrotricha) are paraphyletic. These phylogenetic hypotheses were interpreted concerning their implications for the evolution of morphological and ecological features. Accordingly, several secondary loss events of complex morphological traits (lateral sensory organs within Acanthocephala and the wheel organ (corona) within Syndermata) occurred during the evolution of these animal groups. Therefore the usability of these traits in terms of classic morphological phylogenetic analyses appears to be critical. Acanthocephalan endoparasitism probably evolved via an epizoic intermediate stage, a lifestyle that can still be observed today in Seisonidea. The last common ancestor of Spiralia presumably was small and unsegmented and was lacking a true body cavity (coelom). Thus, segmentation and coeloms evolved several times independently (convergent) within Metazoa. The thesis furthermore included the following, partly methodological aspects: (i) analysis of the architecture of monogonont mt genomes confirmed the aberrant organization in two sub-genomes for Brachionidae. (ii) The suitability of ribosomal proteins for molecular-phylogenetic analyses was tested and the existence of conflicting phylogenetic signals in these special protein sequences was observed. (iii) It could be demonstrated that systematic errors like “long-branch attraction” play a major role in placing Syndermata in the metazoan tree and have to be addressed

Transcriptome data reveal syndermatan relationships and suggest the evolution of endoparasitism in Acanthocephala via an epizoic stage

The taxon Syndermata comprises the biologically interesting wheel animals (“Rotifera”: Bdelloidea + Monogononta + Seisonidea) and thorny-headed worms (Acanthocephala), and is central for testing superordinate phylogenetic hypotheses (Platyzoa, Gnathifera) in the metazoan tree of life. Recent analyses of syndermatan phylogeny suggested paraphyly of Eurotatoria (free-living bdelloids and monogononts) with respect to endoparasitic acanthocephalans. Data of epizoic seisonids, however, were absent, which may have affected the branching order within the syndermatan clade. Moreover, the position of Seisonidea within Syndermata should help in understanding the evolution of acanthocephalan endoparasitism. Here, we report the first phylogenomic analysis that includes all four higher-ranked groups of Syndermata. The analyzed data sets comprise new transcriptome data for Seison spec. (Seisonidea), Brachionus manjavacas (Monogononta), Adineta vaga (Bdelloidea), and Paratenuisentis ambiguus (Acanthocephala). Maximum likelihood and Bayesian trees for a total of 19 metazoan species were reconstructed from up to 410 functionally diverse proteins. The results unanimously place Monogononta basally within Syndermata, and Bdelloidea appear as the sister group to a clade comprising epizoic Seisonidea and endoparasitic Acanthocephala. Our results support monophyly of Syndermata, Hemirotifera (Bdelloidea + Seisonidea + Acanthocephala), and Pararotatoria (Seisonidea + Acanthocephala), rejecting monophyly of traditional Rotifera and Eurotatoria. This serves as an indication that early acanthocephalans lived epizoically or as ectoparasites on arthropods, before their complex lifecycle with arthropod intermediate and vertebrate definite hosts evolved

Data from: Platyzoan paraphyly based on phylogenomic data supports a non-coelomate ancestry of Spiralia

Based on molecular data three major clades have been recognized within Bilateria: Deuterostomia, Ecdysozoa and Spiralia. Within Spiralia, small-sized and simply organized animals such as flatworms, gastrotrichs and gnathostomulids have recently been grouped together as Platyzoa. However, the representation of putative platyzoans was low in the respective molecular phylogenetic studies, in terms of both, taxon number and sequence data. Furthermore, increased substitution rates in platyzoan taxa raised the possibility that monophyletic Platyzoa represents an artefact due to long-branch attraction. In order to overcome such problems, we employed a phylogenomic approach, thereby substantially increasing i) the number of sampled species within Platyzoa and ii) species-specific sequence coverage in datasets of up to 82,162 amino acid positions. Using established and new measures (long-branch score) we disentangled phylogenetic signal from misleading effects such as long-branch attraction. In doing so, our phylogenomic analyses did not recover a monophyletic origin of platyzoan taxa that, instead, appeared paraphyletic with respect to the other spiralians. Platyhelminthes and Gastrotricha formed a monophylum, which we name Rouphozoa. To the exclusion of Gnathifera, Rouphozoa and all other spiralians represent a monophyletic group, which we name Platytrochozoa. Platyzoan paraphyly suggests that the last common ancestor of Spiralia was a simple-bodied organism lacking coelomic cavities, segmentation and complex brain structures, and that more complex animals such as annelids evolved from such a simply organized ancestor. This conclusion contradicts alternative evolutionary scenarios proposing an annelid-like ancestor of Bilateria and Spiralia and several independent events of secondary reduction

Implications for character evolution in Syndermata.

<p>Morphological and biological features of representatives of Syndermata and Gnathostomulida were projected on our phylogenetic tree (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone-0088618-g002" target="_blank">Figs. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone-0088618-g003" target="_blank">3</a>). Present features are denoted with a plus, absent features are denoted with a minus. The corona in Seisonidea is reduced (denoted with plus/minus). The pictogram of Gnathostomulida has been modified from biodidac.bio.uottawa.ca. Pictograms of other representatives: courtesy of Bernd Baumgart (Göttingen, Germany).</p

Phylogenetic relationships obtained with different phylogenomic data sets.

<p>Tree and branch lengths represent results of the TreeFinder analysis of the partitioned mintax4 data set. Node labels represent minimum and maximum support values of analyses of the phylogenomic data sets (mintax4, mintax8; upper row), phylogenomic data sets without RPs (mintax4_noRPs, mintax8_noRPs; middle row), and most purposive subset (MPS) data sets (with or without RPs; lower row). For further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone.0088618.s005" target="_blank">Table S4</a>. ML bootstrap and ELW support (RAxML, TreeFinder) | PP values (MrBayes, PhyloBayes). A star denotes maximum support, a double star denotes maximum support in all analyses.</p

Comparison of branch lengths obtained with different data sets.

<p>Results of RAxML analyses based on [A] mintax4 data set, [B] mintax4_slow data set, [C] mintax4 data set with reduced taxon sampling within Syndermata (mintax4_4Synd), and [D] mintax4_4Synd data set excluding “singletons” and “dingletons” (mintax4_4Synd-DS).</p

Two alternative hypotheses for the internal syndermatan phylogeny.

<p>Alternative hypotheses for the internal syndermatan relationships are (A) the morphology-based Lemniscea hypothesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone.0088618-Lorenzen1" target="_blank">[45]</a>, and (B) the Hemirotifera hypothesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone.0088618-Srensen1" target="_blank">[35]</a>, which is mainly based on molecular data. The latter did not specify the sister group of Acanthocephala. Support for a potential sister group relationship of Acanthocephala and Seisonidea (Pararotatoria) comes from morphological data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone.0088618-Ahlrichs1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone.0088618-Ahlrichs2" target="_blank">[23]</a> and analyses of partial 18S rRNA sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088618#pone.0088618-Herlyn1" target="_blank">[26]</a>.</p

Clinically relevant variants in a large cohort of Indian patients with Marfan syndrome and related disorders identified by next-generation sequencing

Abstract Marfan syndrome and related disorders are a group of heritable connective tissue disorders and share many clinical features that involve cardiovascular, skeletal, craniofacial, ocular, and cutaneous abnormalities. The majority of affected individuals have aortopathies associated with early mortality and morbidity. Implementation of targeted gene panel next-generation sequencing in these individuals is a powerful tool to obtain a genetic diagnosis. Here, we report on clinical and genetic spectrum of 53 families from India with a total of 83 patients who had a clinical diagnosis suggestive of Marfan syndrome or related disorders. We obtained a molecular diagnosis in 45/53 (85%) index patients, in which 36/53 (68%) had rare variants in FBN1 (Marfan syndrome; 63 patients in total), seven (13.3%) in TGFBR1/TGFBR2 (Loeys–Dietz syndrome; nine patients in total) and two patients (3.7%) in SKI (Shprintzen–Goldberg syndrome). 21 of 41 rare variants (51.2%) were novel. We did not detect a disease-associated variant in 8 (15%) index patients, and none of them met the Ghent Marfan diagnostic criteria. We found the homozygous FBN1 variant p.(Arg954His) in a boy with typical features of Marfan syndrome. Our study is the first reporting on the spectrum of variants in FBN1, TGFBR1, TGFBR2, and SKI in Indian individuals