New insights into the long-term evolutionary history of the avian MHC

SummaryGene duplication and neofunctidnalization are important processes in the evolution of phenotypic complexity. They account for important evolutionary novelties that confer ecological adaptation, such as the major histocompatibility complex (MHC), a multigene family with a central role in vertebrates' adaptive immune system. Multigene families, which evolved in large part through duplication, represent promising systems to study the still strongly depbated relative roles of neutral and adaptive processes in the evolution of phenotypic complexity. Detailed knowledge on ecological function and a well-characterized evolutionary history place the mammals' MHC amongst ideal study systems. However mammalian MHCs usually encompass several million base pairs and hold a large number of functional and non-functional duplicate genes, which makes their study complex. Avian MHCs on the other hand are usually way more compact, but the reconstruction of. their evolutionary history has proven notoriously difficult. However, no focused attempt has been undertaken so far to study the avian MHC evolutionary history in a broad phylogenetic context and using adequate gene regions.In the present PhD, we were able to make important contributions to the understanding of the long-term evolution of the avian MHC class II Β (MHCI1B). First, we isolated and characterized MHCIIB genes in barn owl (Tyto alba?, Strigiformes, Tytonidae), a species from an avian lineage in which MHC has not been studied so far. Our results revealed that with only two functional MHCIIB genes the MHC organization of barn owl may be similar to the 'minimal essential' MHC of chicken (Gallus gallus), indicating that simple MHC organization may be ancestral to birds. Taking advantage of the sequence information from barn owl, we studied the evolution of MHCIIB genes in 13 additional species of 'typical' owls (Strigiformes, Strigidae). Phylogenetic analyses revealed that according to their function, in owls the peptide-binding region (PBR) encoding exon 2 and the non-PBR encoding exon 3 evolve by different patterns. Exon 2 exhibited an evolutionary history of positive selection and recombination, while exon 3 traced duplication history and revealed two paralogs evolving divergently from each other in owls, and in a shorebird, the great snipe {Gallinago media). The results from exon 3 were the first ever from birds to demonstrate gene orthology in species that diverged tens of millions of years ago, and strongly questioned whether the taxa studied before provided an adequate picture of avian MHC evolution. In a follow-up study, we aimed at explaining a striking pattern revealed by phylogenetic trees analyzing the owl sequences along with MHCIIB sequences from other birds: One owl paralog (termed DAB1) grouped with sequences of passerines and falcons, while the other (DAB2) grouped with wildfowl, penguins and birds of prey. This could be explained by either a duplication event preceding the evolution of these bird orders, or by convergent evolution of similar sequences in a number of orders. With extensive phylogenetic analyses we were able to show, that indeed a duplication event preceeded the major avian radiation -100 my ago, and that following this duplication, the paralogs evolved under positive selection. Furthermore, we showed that the divergently evolving amino acid residues in the MHCIIB-encoded β-chain potentially interact with the MHCI I α-chain, and that molecular coevolution of the interacting residues may have been involved in the divergent evolution of the MHCIIB paralogs.The findings of this PhD are of particular interest to the understanding of the evolutionary history of the avian MHC and, by providing essential information on long-term gene history in the avian MHC, open promising perspectives for advances in the understanding of the evolution of multigene families in general, and for avian MHC organization in particular. Amongst others I discuss the importance of including protein structure in the phylogenetic study of multigene families, and the roles of ecological versus molecular selection pressures. I conclude by providing a population genomic perspective on avian MHC, which may serve as a basis for future research to investigate the relative roles of neutral processes involving effective population size effects and of adaptation in the evolution of avian MHC diversity and organization.RésuméLa duplication de gènes et leur néo-fonctionnalisation sont des processus importants dans l'évolution de la complexité phénotypique. Ils sont impliqués dans l'apparition d'importantes nouveautés évolutives favorisant l'adaptation écologique, comme c'est le cas pour le complexe majeur d'histocompatibilité ancestrale chez les oiseaux. En se basant sur l'information obtenue pour cette espèce, nous avons ensuite étudié l'évolution des gènes CMHIIB chez 13 espèces de chouettes "typiques" (Strigiformes, Strigidae). Les analyses phylogénétiques ont révélé que chez les chouettes, selon leur fonction, l'exon 2 codant pour la 'peptide-binding region' (PBR) et l'exon 3 ne codant pas pour la région PBR évoluent de manière distincte. L'exon 2 montre une histoire évolutive de sélection positive et recombinaison, tandis que l'exon 3 retrace l'histoire de duplication et révèle deux paralogues évoluant de façon divergente chez les chouettes, ainsi que chez un limicole, la bécassine double (Gallinago media). Les résultats découlant de l'analyse de l'exon 3 ont montré pour la première fois chez les oiseaux une orthologie de gènes chez des espèces ayant divergé il y a des dizaines de millions d'années. Dans une deuxième étude, nous avons voulu expliquer le pattern surprenant révélé par les arbres phylogénétiques lorsque les séquences CMHIIB des chouettes sont analysées avec les séquences d'autres oiseaux: un paralogue de chouette (appelé DABI) forme un groupe avec les séquences de passereaux et faucons, tandis que l'autre (DAB2) se regroupe avec les gallo-ansériformes, les manchots et les rapaces diurnes. Ceci pourrait être expliqué soit par une duplication qui a précédé l'évolution de ces ordres aviaires soit par évolution convergente dans un certain nombre d'ordres. Grâce à des analyses phylogénétiques approfondies, nous avons pu montrer qu'un événement de duplication a effectivement précédé la radiation aviaire principale d'il y a environ 100 millions d'années, et qu'après cette duplication les paralogues ont évolué sous sélection positive. De plus, les résidus d'acides aminés évoluant de façon divergente dans la chaîne β codée par le CMHIIB interagissent potentiellement avec la chaîne α codée par le CMHII, et que la coévolution moléculaire de ces résidus pourrait être à la base de l'évolution divergente des paralogues CMHIIB.Dans cette thèse sont discutés entre autres l'importance d'inclure la structure des protéines dans l'étude phylogénétique des familles multigéniques, ainsi que les rôles respectifs des pressions sélectives écologique et moléculaire. La conclusion comporte une perspective sur la génomique des populations du CMH aviaire, qui pourrait servir de base pour de futures recherches sur les rôles relatifs joués par Îes processus neutres comprenant les effets de la taille efficace des populations et l'adaptation dans l'évolution de la diversité et l'organisation du MHC aviaire.

Chromosomal gene movements reflect the recent origin and biology of therian sex chromosomes

Mammalian sex chromosomes stem from ancestral autosomes and have substantially differentiated. It was shown that X-linked genes have generated duplicate intronless gene copies (retrogenes) on autosomes due to this differentiation. However, the precise driving forces for this out-of-X gene "movement" and its evolutionary onset are not known. Based on expression analyses of male germ-cell populations, we here substantiate and extend the hypothesis that autosomal retrogenes functionally compensate for the silencing of their X-linked housekeeping parental genes during, but also after, male meiotic sex chromosome inactivation (MSCI). Thus, sexually antagonistic forces have not played a major role for the selective fixation of X-derived gene copies in mammals. Our dating analyses reveal that although retrogenes were produced ever since the common mammalian ancestor, selectively driven retrogene export from the X only started later, on the placental mammal (eutherian) and marsupial (metatherian) lineages, respectively. Together, these observations suggest that chromosome-wide MSCI emerged close to the eutherian-marsupial split approximately 180 million years ago. Given that MSCI probably reflects the spread of the recombination barrier between the X and Y, crucial for their differentiation, our data imply that these chromosomes became more widely differentiated only late in the therian ancestor, well after the divergence of the monotreme lineage. Thus, our study also provides strong independent support for the recent notion that our sex chromosomes emerged, not in the common ancestor of all mammals, but rather in the therian ancestor, and therefore are much younger than previously thought

Evolution of vitellogenin genes: comparative analysis of the nucleotide sequences downstream of the transcription initiation site of four Xenopus laevis and one chicken gene.

Electron microscopic analysis of heteroduplexes between the most distantly related Xenopus vitellogenin genes (A genes X B genes) has revealed the distribution of homologous regions that have been preferentially conserved after the duplication events that gave rise to the multigene family in Xenopus laevis. DNA sequence analysis was limited to the region downstream of the transcription initiation site of the Xenopus genes A1, B1 and B2 and a comparison with the Xenopus A2 and the major chicken vitellogenin gene is presented. Within the coding regions of the first three exons, nucleotide substitutions resulting in amino acid changes accumulate at a rate similar to that observed in globin genes. This suggests that the duplication event which led to the formation of the A and B ancestral genes in Xenopus laevis occurred about 150 million years ago. Homologous exons of the A1-A2 and B1-B2 gene pairs, which formed about 30 million years ago, show a quite similar sequence divergence. In contrast, A1-A2 homologous introns seem to have evolved much faster than their B1-B2 counterparts.

Variation and evolution of herkogamy in Exochaenium (Gentianaceae): implications for the evolution of distyly.

Backgrounds and Aims The spatial separation of stigmas and anthers (herkogamy) in flowering plants functions to reduce self-pollination and avoid interference between pollen dispersal and receipt. Little is known about the evolutionary relationships among the three main forms of herkogamy - approach, reverse and reciprocal herkogamy (distyly) - or about transitions to and from a non-herkogamous condition. This problem was examined in Exochaenium (Gentianaceae), a genus of African herbs that exhibits considerable variation in floral morphology, including the three forms of herkogamy. Methods Using maximum parsimony and maximum likelihood methods, the evolutionary history of herkogamic and non-herkogamic conditions was reconstructed from a molecular phylogeny of 15 species of Exochaenium and four outgroup taxa, based on three chloroplast regions, the nuclear ribosomal internal transcribed spacer (ITS1 and 2) and the 5·8S gene. Ancestral character states were determined and the reconstructions were used to evaluate competing models for the origin of reciprocal herkogamy. Key results Reciprocal herkogamy originated once in Exochaenium from an ancestor with approach herkogamy. Reverse herkogamy and the non-herkogamic condition homostyly were derived from heterostyly. Distylous species possessed pendent, slightly zygomorphic flowers, and the single transition to reverse herkogamy was associated with the hawkmoth pollination syndrome. Reductions in flower size characterized three of four independent transitions from reciprocal herkogamy to homostyly. Conclusions The results support Lloyd and Webb's model in which distyly originated from an ancestor with approach herkogamy. They also demonstrate the lability of sex organ deployment and implicate pollinators, or their absence, as playing an important role in driving transitions among herkogamic and non-herkogamic conditions.

Genome size rather than content might affect call properties in toads of three ploidy levels (Anura: Bufonidae: Bufo viridis subgroup).

In vertebrates, genome size has been shown to correlate with nuclear and cell sizes, and influences phenotypic features, such as brain complexity. In three different anuran families, advertisement calls of polyploids exhibit longer notes and intervals than diploids, and difference in cellular dimensions have been hypothesized to cause these modifications. We investigated this phenomenon in green toads (Bufo viridis subgroup) of three ploidy levels, in a different call type (release calls) that may evolve independently from advertisement calls, examining 1205 calls, from ten species, subspecies, and hybrid forms. Significant differences between pulse rates of six diploid and four polyploid (3n, 4n) green toad forms across a range of temperatures from 7 to 27 °C were found. Laboratory data supported differences in pulse rates of triploids vs. tetraploids, but failed to reach significance when including field recordings. This study supports the idea that genome size, irrespective of call type, phylogenetic context, and geographical background, might affect call properties in anurans and suggests a common principle governing this relationship. The nuclear-cell size ratio, affected by genome size, seems the most plausible explanation. However, we cannot rule out hypotheses under which call-influencing genes from an unexamined diploid ancestral species might also affect call properties in the hybrid-origin polyploids.

A key transcription cofactor on the nascent sex chromosomes of European tree frogs (Hyla arborea)

We show that MED15, a key component of the transcription complex Mediator, lies within the nonrecombining segment of nascent sex chromosomes in the male-heterogametic Hyla arborea. Both X and Y alleles are expressed during embryonic development and differ by three frame-preserving indels (eight amino acids in total) within their glutamine-rich central part. These changes have the potential to affect the conformation of the Mediator complex and to activate genes in a sex-specific way and might thus represent the first steps toward the acquisition of a male-specific function. Alternatively, they might result from an ancestral neutral polymorphism, with different alleles picked by chance on the X and Y chromosomes when MED15 was trapped in the nonrecombining segment.

Forensic science - A true science ?

While the US jurisprudence of the 1993 Daubert requires judges to question not only the methodology behind, but also the principles governing, a body of knowledge to qualify it as scientific, can forensic science, based on Locard's and Kirk's Principles, pretend to this higher status in the courtroom ? Moving away from the disputable American legal debate, this historical and philosophical study will screen the relevance of the different logical epistemologies to recognize the scientific status of forensic science. As a consequence, the authors are supporting a call for its recognition as a science of its own, defined as the science of identifying and associating traces for investigative and security purposes, based o its fundamental principles and the case assesment and interpretation process that follows with its specific and relevant mode of inference.

The balanced lethal system of crested newts: a ghost of sex chromosomes past?

Balanced lethal systems are more than biological curiosities: as theory predicts, they should quickly be eliminated through the joint forces of recombination and selection. That such systems might become fixed in natural populations poses a challenge to evolutionary theory. Here we address the case of a balanced lethal system fixed in crested newts and related species, which makes 50% of offspring die early in development. All adults are heteromorphic for chromosome pair 1. The two homologues (1A and 1B) have different recessive deleterious alleles fixed on a nonrecombining segment, so that heterozygotes are viable, while homozygotes are lethal. Given such a strong segregation load, how could autosomes stop recombining? We propose a role for a sex-chromosome turnover from pair 1 (putative ancestral sex chromosome) to pair 4 (currently active sex chromosome). Accordingly, 1A and 1B represent two variants (Y(A) and Y(B)) of the Y chromosome from an ancestral male-heterogametic system. We formalize a scenario in which turnovers are driven by sex ratio selection stemming from gene-environment interactions on sex determination. Individual-based simulations show that a balanced lethal system can be fixed with significant likelihood, provided the masculinizing allele on chromosome 4 appears after the elimination of the feminizing allele on chromosome 1. Our study illustrates how strikingly maladaptive traits might evolve through natural selection.

An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

Total hemoglobin mass--a new parameter to detect blood doping?

PURPOSE: All kinds of blood manipulations aim to increase the total hemoglobin mass (tHb-mass). To establish tHb-mass as an effective screening parameter for detecting blood doping, the knowledge of its normal variation over time is necessary. The aim of the present study, therefore, was to determine the intraindividual variance of tHb-mass in elite athletes during a training year emphasizing off, training, and race seasons at sea level. METHODS: tHb-mass and hemoglobin concentration ([Hb]) were determined in 24 endurance athletes five times during a year and were compared with a control group (n = 6). An analysis of covariance was used to test the effects of training phases, age, gender, competition level, body mass, and training volume. Three error models, based on 1) a total percentage error of measurement, 2) the combination of a typical percentage error (TE) of analytical origin with an absolute SD of biological origin, and 3) between-subject and within-subject variance components as obtained by an analysis of variance, were tested. RESULTS: In addition to the expected influence of performance status, the main results were that the effects of training volume (P = 0.20) and training phases (P = 0.81) on tHb-mass were not significant. We found that within-subject variations mainly have an analytical origin (TE approximately 1.4%) and a very small SD (7.5 g) of biological origin. CONCLUSION: tHb-mass shows very low individual oscillations during a training year (<6%), and these oscillations are below the expected changes in tHb-mass due to Herythropoetin (EPO) application or blood infusion (approximately 10%). The high stability of tHb-mass over a period of 1 year suggests that it should be included in an athlete's biological passport and analyzed by recently developed probabilistic inference techniques that define subject-based reference ranges.

The emergence of new genes on the young therian X.

Recent studies have revealed that our sex chromosomes differentiated relatively recently from ancestral autosomes in the common ancestor of placental and marsupial mammals (therians). Here, we show that the therian X started to accumulate new retroduplicate genes with overall sex-biased expression upon therian sex chromosome differentiation. This process reached its peak within the first approximately 90 million years of sex chromosome evolution and then leveled off. Taken together, our observations suggest that the major sex-related functional remodeling of the X was completed relatively soon after the origination of therian sex chromosomes.

Evolution of asexuality via different mechanisms in grass thrips (thysanoptera: Aptinothrips).

Asexual lineages can derive from sexual ancestors via different mechanisms and at variable rates, which affects the diversity of the asexual population and thereby its ecological success. We investigated the variation and evolution of reproductive systems in Aptinothrips, a genus of grass thrips comprising four species. Extensive population surveys and breeding experiments indicated sexual reproduction in A. elegans, asexuality in A. stylifer and A. karnyi, and both sexual and asexual lineages in A. rufus. Asexuality in A. stylifer and A. rufus coincides with a worldwide distribution, with sexual A. rufus lineages confined to a limited area. Inference of molecular phylogenies and antibiotic treatment revealed different causes of asexuality in different species. Asexuality in A. stylifer and A. karnyi has most likely genetic causes, while it is induced by endosymbionts in A. rufus. Endosymbiont-community characterization revealed presence of Wolbachia, and lack of other bacteria known to manipulate host reproduction. However, only 69% asexual A. rufus females are Wolbachia-infected, indicating that either an undescribed endosymbiont causes asexuality in this species or that Wolbachia was lost in several lineages that remained asexual. These results open new perspectives for studies on the maintenance of mixed sexual and asexual reproduction in natural populations.

The hypogene iron oxide copper-gold mineralization in the Mantoverde District, Northern Chile

The Mantoverde iron oxide copper-gold (IOCC) district, northern Chile, is known for its Cu production from supergene ores. Recently, exploration outlined an additional hypogene ore resource of 440 Mt with 0.56 percent Cu, and 0.12 g/t An. The hypogene sulfide mineralization occurs mainly as chalcopyrite and pyrite, typically in specularite or magnetite-cemented breccias and associated stockworks. The host rocks underwent variably intense K feldspar alteration, chloritization, sericitization, silicification, and/or carbonatization. A district scale Na(-Ca) alteration is absent. The IOCC mineralization in the district shows a strong tectonic control by northwest- to north-northwest-trending brittle structures. Large Cu sulfide-rich veins or Cu sulfide-cemented breccias are absent. Therefore, head grades of 4 percent Cu are an exception. There is a positive correlation between Cu and An grades. Gold is probably contained mostly in chalcopyrite and pyrite. Elevated concentrations of light rare-earth elements (LREE) occur locally but are attributed to redistribution of LREE within the deposits rather than to derivation from external sources. The Cu-Au ores in the Mantoverde district are low in and have relatively low contents in heavy metals that are potentially hazardous to the environment, such as As (avg 14 ppm), Hg (<5 ppm), or Cd (<0.2 ppm). The sulfur isotope ratios of chalcopyrite from the IOCC deposits lie between -5.6 and 8.9 per mil delta(34)S(VCDT). They show systematic variations within the district, which are interpreted to reflect relative distance to inferred fluid conduits and the level of deposition within the hydrothermal system. Most initial (87)Sr/(86)Sr values of altered volcanic rocks and hydrothermal calcite from the Mantoverde district are between 0.7031 and 0.7060 and are similar to those of the igneous rocks of the region. Lead isotope ratios of chalcopyrite are consistent with Pb (and by inference Cu) derived from Early Cretaceous magmatism. The sulfur, strontium, and lead isotope data of chalcopyrite, calcite gangue, or altered host rocks, respectively, are compatible with a genetic model that involves cooling of metal and sulfur-bearing magmatic-hydrothermal fluids that mix with meteoric waters or seawater at relatively shallow crustal levels. An additional exotic sulfur input is likely, though not required, for the copper mineralization. Apart from the IOCC. deposits, there are a number of smaller magnetite(-apatite) bodies in the district. These are geologically similar to the Cu-Au-bearing magnetite bodies, but are related to splays of the north-south-trending Atacama fault zone and differ in alteration and texture.