Marburg, University, Diss., 2004.

Insertions/deletions (indels) are potentially powerful evolutionary markers, but little is known about their evolution and few tools exist to effectively study them. To address this, I developed SeqFIRE, a tool for automated identification and extraction of indels from protein multiple sequence alignments. The program also extracts conserved alignment blocks, thus covering all major steps in preparing multiple sequence alignments for phylogenetic analysis. I then used SeqFIRE to build an indel database, using 299 single copy proteins from a broad taxonomic sampling of mainly multicellular eukaryotes. A total of 4,707 indels were extracted, of which 901 are simple (one genetic event) and 3,806 are complex (multiple events). The most abundant indels are single amino acid simple indels. Indel frequency decreases exponentially with length and shows a linear relationship with host protein size. Singleton indels reveal a strong bias towards insertions (2.31 x deletions on average). These analyses also identify 43 indels marking major clades in Plantae and Fungi (clade defining indels or CDIs), but none for Metazoa. In order to study the 3806 complex indels they were first classified by number of states. Analysis of the 2-state complex and simple indels combined (“bi-state indels”) confirms that insertions are over 2.5 times as frequent as deletions. Three-quarters of the complex indels had three-nine states (“slightly complex indels”). A tree-assisted search method was developed allowing me to identify 1,010 potential CDIs supporting all examined major branches of Plantae and Fungi. Forty-two proteins were also found to host complex indel CDIs for the deepest branches of Metazoa. After expanding the taxon set for these proteins, I identified a total of 49 non-bilaterian specific CDIs. Parsimony analysis of these indels places Ctenophora as sister taxon to all other Metazoa including Porifera. Six CDIs were also found placing Placozoa as sister to Bilateria. I conclude that slightly complex indels are a rich source of CDIs, and my tree-assisted search strategy could be automated and implemented in the program SeqFIRE to facilitate their discovery. This will have important implications for mining the phylogenomic content of the vast resource of protist genome data soon to become available.

S:Als darrers 10 anys, el nostre coneixement de l'evolució dels animals ha estat objecte d'una revolució degut a l'aplicació de la Biologia Molecular al camp de la Filogènia, que ha rebutjat algunes hipòtesis antigues i n'ha proposat de noves. Un dels canvis més importants és el que subdivideix a tots els animals amb simetria bilateral (els Bilateria) en tres grups: els Lophotrochozoa, els Ecdysozoa y els Deuterostomata. Malgrat el gran cabdal d'informació que han suposat, les dades moleculars no han estat capaces de resoldre totes les relacions entre els fílums. Al començament, això era degut al l'ús de pocs marcadors moleculars, que no portaven prou informació i que patien artefactes metodològics com el Long Branch Atraction. L'objectiu d'aquest projecte és obtenir una filogènia molecular dels bilaterals el més resolta possible, mitjançant l'aplicació de dues aproximacions. La primera fa us de la gran quantitat de dades disponibles dels gens nuclears del RNA ribosomal (18S i 28S) a l'hora que es minimitza l'impacte dels artefactes filogenètics; s'ha obtingut una matriu de 104 representants dels bilaterals, pertanyents a 28 fílums i amb 3.700 parells de bases de longitud. Aquesta matriu s'ha analitzat mitjançant mètodes d'inferència filogenètica de tipus probabilístic (Maxima Versemblança i Inferència Bayesiana), tot fent servir models evolutius sofisticats i compartimentant les anàlisis pels grups problemàtics. La segona aproximació pretén construir una matriu dels bilaterals que maximitzi tant el nombre de marcadors com el de fílums representats. S'han seleccionat 13 gens candidat i recollit mostres per un total de 96 espècies de 31 fílums. S'han obtingut un total de 135 seqüències, que junt amb seqüències de les bases de dades, s'han fet servir per construir una matriu de 90 representants de 27 fílums amb 8.880 parells de bases de longitud i un 40% de missing data. Aquesta matriu s'ha analitzat amb mètodes d'inferència probabilístics.Els resultats principals dels dos estudis son: 1) es comprova la monofilia dels tres grans grups de bilaterals, així com la posició d'Acoela i Nemertodermatida com a bilaterals basals, 2) s'obté una filogènia resolta dels deuteròstoms, mentre que la filogènia dels ecdisozous resulta en una tricotomia i 3) s'obté una nova filogènia dels lofotrocozous, a on cal destacar als gnatostomulats i gastrotrics com els fílums més basals, la posició dels platihelmints com a grup germà de la resta d'animals espirals i la parafilia dels espirals degut a la posició de braquiòpodes i foronidis com a grup germà dels mol·luscs, 4) la filogènia obtinguda situa a Xenoturbella dins dels deuteròstoms, tot i que amb un suport molt baix, i no es pot descartar una posició més basal dins dels bilaterals, 5) els Chaetognatha pertanyen als ecdisozous, tot i que la posició exacta dins dels mateixos és dubtosa, i 6) la filogènia obtinguda ens ha permès especular sobre l'evolució de determinades característiques del patró corporal, mostrant la gran plasticitat de la seva aparició i la prevalença de l'adquisició independent d'algunes d'elles. / "Bilaterian molecular phylogeny: a multigenic approach"TEXT:Resolving the relationships among animal phyla is a key biological problem that remains to be solved. Morphology has been unable to determine the relationships among most phyla. During the past decade studies based on a limited number of markers established new hypotheses such as the existence of three bilaterian superclades (Deuterostomia, Ecdysozoa and Lophotrochozoa) but left major questions unresolved.The aim of this project is to obtain a well resolved bilaterian phylogeny applying two approaches. The first uses the great quantity of information available for 18S and 28S genes, while overcoming their pitfalls by combining several methods suggested in previous studies. A matrix was built, comprising 104 bilaterian representatives for 28 phyla and it is 3,700 base pairs long. The methods used include Maximum Likelihood and Bayesian Inference, the application of models with rate-heterogeneity across sites, wide taxon sampling, and compartmentalized analyses for each problematic clade. The second approach is based on maximizing the number of markers and the number of representatives. 13 genes were chosen as candidates and tissues for 96 species belonging to 31 phyla were sampled. 135 sequences were obtained, and used together with databases sequences to build a matrix 8,880 base pairs long, with 90 representatives for 27 bilaterian phyla and bears 40% of missing data.The results obtained show 1) the monophyly of the three superclades and the basal position of a paraphyletic Acoelomorpha, 2) a well resolved deuterostomates phylogeny, while ecdysozoan relationships turn up as a tricotomy, 3) a new lophotrochozoan phylogeny is obtained with gnathostomulids and gastrotrichs as the most basal phyla, flatworms as sistergroup to spiralians animals, and spiralians are paraphyletic due to the position of brachiopods and phoronids as sistergroup to molluscs, 4) Xenoturbella is shown within deuterostomates, though a more basal position cannot be rejected, 5) Chaetognatha belong to Ecdysozoa, though its exact position cannot be solved, and 6) the obtained phylogeny was used for a speculative discussion on morphological traits evolution, showing their great evolving plasticity.

Deciphering the rapid emergence of bilaterian animals around the time of the Cambrian Explosion and reconstructing the interrelationships of animal groups have long been two of the most elusive problems in Zoology. This thesis concerns the phylogenetic interrelationships within and among Acoela and Nemertodermatida, two groups of small worms that are believed to be basal bilaterians and which may provide important clues for understanding the early evolution of animals. In addition to trying to resolve the phylogenetic positions of these groups, major focus is put on inferring how ancestral animals might have looked, given the phylogenetic hypotheses put forward. The data used to infer phylogenies include nuclear ribosomal DNA, the mitochondrial COI gene and microRNAs. Based on phylogenetic analyses of a large number of 18S SSU ribosomal DNA sequences, it is proposed that Cnidaria is the sister taxon to Bilateria. Poor taxon sampling is suggested to be one of the reasons for why earlier assessments of the interrelationships among the most basal animal groups have yielded many conflicting results using the same gene. Analyses of new 18S SSU rDNA and 28S LSU rDNA sequences from six of the nine known species of nemertodermatids corroborate earlier indications that Acoela and Nemertodermatida are not sister taxa, as once thought. Being separate basal bilaterian animal groups, it is suggested that the last common ancestor of all bilaterians shared much of their comparatively simple morphology. Many methods are deployed to assess whether the phylogenetic results are mainly due to long-branch attraction, but no indication of this artifact is detected. The first comprehensive phylogenetic framework of Acoela is reconstructed from the 18S SSU, 28S LSU and COI genes, in combination with morphological data. The ancestral acoel worm is reconstructed using Bayesian methods and morphological observations in extant species. Two indeces, posterior similarity and reconstruction signal, are implemented to assess how similar different species are to the last common ancestor of all acoels and illustrate how clearly different characters or nodes are reconstructed. It is suggested that the ancestral acoel looked much like extant species of Diopisthoporus. The phylogenetic positions of Acoela and Nemertodermatida are assessed using new data on microRNAs in the acoel Hofstenia miamia and the nemertodermatid Meara stichopi. Acoela and Nemertodermatida are again found to be basal bilaterians, in congruence with earlier results. Using the work-flow and indeces developed earlier, it is concluded that the bilaterian ancestral microRNA repertoire can not yet be reconstructed with high confidence. All papers stress the importance of inclusive taxon sampling for making generalized inferences about ancestral features in animals.