Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

Analyses of the evolution and function of voltage-gated sodium channel proteins (Navs) have largely been limited to mutations from individual people with diagnosed neuromuscular disease. This project investigates the carboxyl-terminus of the Nav paralog (locus scn4aa 3’) that is preferentially expressed in electric organs of Neotropical weakly-electric fishes (Order Gymnotiformes). As a model system, I used the genus Gymnotus, a diverse clade of fishes that produce species-specific electric organ discharges (EODs). I clarified evolutionary relationships among Gymnotus species using mitochondrial (cytochrome b, and 16S ribosome) and nuclear (rag2, and scn4aa) gene sequences (3739 nucleotide positions from 28 Gymnotus species). I analyzed the molecular evolution of scn4aa 3’, and detected evidence for positive selection at eight amino acid sites in seven Gymnotus lineages. These eight amino acid sites are located in motifs that may be important for modulation of EOD frequencies.

Analyses of the evolution and function of voltage-gated sodium channel proteins (Navs) have largely been limited to mutations from individual people with diagnosed neuromuscular disease. This project investigates the carboxyl-terminus of the Nav paralog (locus scn4aa 3’) that is preferentially expressed in electric organs of Neotropical weakly-electric fishes (Order Gymnotiformes). As a model system, I used the genus Gymnotus, a diverse clade of fishes that produce species-specific electric organ discharges (EODs). I clarified evolutionary relationships among Gymnotus species using mitochondrial (cytochrome b, and 16S ribosome) and nuclear (rag2, and scn4aa) gene sequences (3739 nucleotide positions from 28 Gymnotus species). I analyzed the molecular evolution of scn4aa 3’, and detected evidence for positive selection at eight amino acid sites in seven Gymnotus lineages. These eight amino acid sites are located in motifs that may be important for modulation of EOD frequencies.

Une réconciliation entre un arbre de gènes et un arbre d’espèces décrit une histoire d’évolution des gènes homologues en termes de duplications et pertes de gènes. Pour inférer une réconciliation pour un arbre de gènes et un arbre d’espèces, la parcimonie est généralement utilisée selon le nombre de duplications et/ou de pertes. Les modèles de réconciliation sont basés sur des critères probabilistes ou combinatoires. Le premier article définit un modèle combinatoire simple et général où les duplications et les pertes sont clairement identifiées et la réconciliation parcimonieuse n’est pas la seule considérée. Une architecture de toutes les réconciliations est définie et des algorithmes efficaces (soit de dénombrement, de génération aléatoire et d’exploration) sont développés pour étudier les propriétés combinatoires de l’espace de toutes les réconciliations ou seulement les plus parcimonieuses. Basée sur le processus classique nommé naissance-et-mort, un algorithme qui calcule la vraisemblance d’une réconciliation a récemment été proposé. Le deuxième article utilise cet algorithme avec les outils combinatoires décrits ci-haut pour calculer efficacement (soit approximativement ou exactement) les probabilités postérieures des réconciliations localisées dans le sous-espace considéré. Basé sur des taux réalistes (selon un modèle probabiliste) de duplication et de perte et sur des données réelles/simulées de familles de champignons, nos résultats suggèrent que la masse probabiliste de toute l’espace des réconciliations est principalement localisée autour des réconciliations parcimonieuses. Dans un contexte d’approximation de la probabilité d’une réconciliation, notre approche est une alternative intéressante face aux méthodes MCMC et peut être meilleure qu’une approche sophistiquée, efficace et exacte pour calculer la probabilité d’une réconciliation donnée. Le problème nommé Gene Tree Parsimony (GTP) est d’inférer un arbre d’espèces qui minimise le nombre de duplications et/ou de pertes pour un ensemble d’arbres de gènes. Basé sur une approche qui explore tout l’espace des arbres d’espèces pour les génomes considérés et un calcul efficace des coûts de réconciliation, le troisième article décrit un algorithme de Branch-and-Bound pour résoudre de façon exacte le problème GTP. Lorsque le nombre de taxa est trop grand, notre algorithme peut facilement considérer des relations prédéfinies entre ensembles de taxa. Nous avons testé notre algorithme sur des familles de gènes de 29 eucaryotes. / A reconciliation between a gene tree and a species tree depicts an evolutionary scenario of the homologous genes in terms of gene duplications and gene losses. To infer such a reconciliation given a gene tree and a species tree, parsimony is generally used according to the number of gene duplications and/or losses. The combinatorial models of reconciliation are based on probabilistic or combinatorial criteria. The first paper defines a simple and more general combinatorial model of reconciliation which clearly identifies duplication and loss events and does not only induce the most parsimonious reconciliation. An architecture of all possible reconciliations is developed together with efficient algorithms (that is counting, randomization, and exploration) to study combinatorial properties of the space of all reconciliations or only the most parsimonious ones. Based on the classical birth-death process, an algorithm that computes the likelihood of a reconciliation has recently been proposed. The second paper uses this algorithm together with the combinatorial tools described above to compute efficiently, either exactly or approximately, the posterior probability of the reconciliations located in the considered subspace. Based on realistic gene duplication and loss rates and on real/simulated datasets of fungal gene families, our results suggest that the probability mass of the whole space of reconciliations is mostly located around the most parsimonious ones. In the context of posterior probability approximation, our approach is a valuable alternative to a MCMC method and can competes against a sophisticated, efficient, and exact computation of the probability of a given reconciliation. The Gene Tree Parsimony (GTP) problem is to infer a species tree that minimizes the number of duplications and/or losses over a set of gene family trees. Based on a new approch that explores the whole species tree space for the considered taxa and an efficient computation of the reconciliation cost, the third paper describes a Branch-and- Bound algorithm that solves exactly the GTP problem. When the considered number of taxa is too large, our algorithm can naturally take into account predefined relationships between sets of taxa. We test our algorithm on a dataset of eukaryotic gene families spanning 29 taxa.

Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.