De-novo emergence of SINE retroposons during the early evolution of passerine birds.

BACKGROUND: Passeriformes ("perching birds" or passerines) make up more than half of all extant bird species. The genome of the zebra finch, a passerine model organism for vocal learning, was noted previously to contain thousands of short interspersed elements (SINEs), a group of retroposons that is abundant in mammalian genomes but considered largely inactive in avian genomes. RESULTS: Here we resolve the deep phylogenetic relationships of passerines using presence/absence patterns of SINEs. The resultant retroposon-based phylogeny provides a powerful and independent corroboration of previous sequence-based analyses. Notably, SINE activity began in the common ancestor of Eupasseres (passerines excluding the New Zealand wrens Acanthisittidae) and ceased before the rapid diversification of oscine passerines (suborder Passeri - songbirds). Furthermore, we find evidence for very recent SINE activity within suboscine passerines (suborder Tyranni), following the emergence of a SINE via acquisition of a different tRNA head as we suggest through template switching. CONCLUSIONS: We propose that the early evolution of passerines was unusual among birds in that it was accompanied by de-novo emergence and activity of SINEs. Their genomic and transcriptomic impact warrants further study in the light of the massive diversification of passerines.

Morningness-eveningness in a large sample of German adolescents and adults.

People differ in their sleep-wake behavior. This individual difference is conceptualized in different aspects, such as wake up times, bed times, times of peak performance, as well as in morning affect. A total of 14,987 visitors of an exhibition in the LWL State Museum of Natural History, Münster (Germany), did the survey on chronotype and gave their consent that these data can be used for a scientific study. Age groups were coded into 5-year bins. Mean age (mean ± SD) was 28.2 ± 17.5 years. There were 8075 females (54%) and 6912 males in the sample. The German version of the rMEQ (reduced Morningness-Eveningness-Questionnaire) was used for data collection. The data showed clear age effects. Younger children are more morning oriented and become rapidly evening oriented during puberty, while the more attenuated turn towards morningness occurs from the age of 20 years. Then between the ages 25 to 30 morningness-eveningness remained rather stable. Significant gender differences existed in the reproductive age, i.e., the age groups 20 to 50 (corresponding to the age 16-50 years). In other age groups, no gender differences could be detected. Seasonal effects were also found. Chronotype score was lowest during the summer months (and more evening oriented). Based on the single item analysis of the five questions of the rMEQ, we found age group differences in all items. Gender differences occurred in all items except item 1, which deals with the preferred wake-up time. Men always scored significantly lower (i.e. more evening oriented) than women except in item 2 (tiredness after awakening). Seasonal effects were only significant in item 3, which is related to preferred bed times. People showed a later bed time preference during summer. The classification of chronotypes according to the cut-off scores provided by Adan and Almirall (1991) and by using the 20/80 percentile provided identical cut-off scores (values of 11 and below for evening types and 18 and above for morning types).

The genome of a Mesozoic paleovirus reveals the evolution of hepatitis B viruses.

Paleovirology involves the identification of ancient endogenous viral elements within eukaryotic genomes. The evolutionary origins of the reverse-transcribing hepatitis B viruses, however, remain elusive, due to the small number of endogenized sequences present in host genomes. Here we report a comprehensively dated genomic record of hepatitis B virus endogenizations that spans bird evolution from >82 to <12.1 million years ago. The oldest virus relic extends over a 99% complete hepatitis B virus genome sequence and constitutes the first discovery of a Mesozoic paleovirus genome. We show that Hepadnaviridae are >63 million years older than previously known and provide direct evidence for coexistence of hepatitis B viruses and birds during the Mesozoic and Cenozoic Eras. Finally, phylogenetic analyses and distribution of hepatitis B virus relics suggest that birds potentially are the ancestral hosts of Hepadnaviridae and mammalian hepatitis B viruses probably emerged after a bird-mammal host switch. Our study reveals previously undiscovered and multi-faceted insights into prehistoric hepatitis B virus evolution and provides valuable resources for future studies, such as in-vitro resurrection of Mesozoic hepadnaviruses.

A universal method for the study of CR1 retroposons in nonmodel bird genomes.

Presence/absence patterns of retroposon insertions at orthologous genomic loci constitute straightforward markers for phylogenetic or population genetic studies. In birds, the convenient identification and utility of these markers has so far been mainly restricted to the lineages leading to model birds (i.e., chicken and zebra finch). We present an easy-to-use, rapid, and cost-effective method for the experimental isolation of chicken repeat 1 (CR1) insertions from virtually any bird genome and potentially nonavian genomes. The application of our method to the little grebe genome yielded insertions belonging to new CR1 subfamilies that are scattered all across the phylogenetic tree of avian CR1s. Furthermore, presence/absence analysis of these insertions provides the first retroposon evidence grouping flamingos + grebes as Mirandornithes and several markers for all subsequent branching events within grebes (Podicipediformes). Five markers appear to be species-specific insertions, including the hitherto first evidence in birds for biallelic CR1 insertions that could be useful in future population genetic studies.

Retroposon insertion patterns of neoavian birds: strong evidence for an extensive incomplete lineage sorting era.

More than 150 Ma, the avian lineage separated from that of other dinosaurs and later diversified into the more than 10,000 species extant today. The early neoavian bird radiations most likely occurred in the late Cretaceous (more than 65 Ma) but left behind few if any molecular signals of their archaic evolutionary past. Retroposed elements, once established in an ancestral population, are highly valuable, virtually homoplasy-free markers of species evolution; after applying stringent orthology criteria, their phylogenetically informative presence/absence patterns are free of random noise and independent of evolutionary rate or nucleotide composition effects. We screened for early neoavian orthologous retroposon insertions and identified six markers with conflicting presence/absence patterns, whereas six additional retroposons established before or after the presumed major neoavian radiation show consistent phylogenetic patterns. The exceptionally frequent conflicting retroposon presence/absence patterns of neoavian orders are strong indicators of an extensive incomplete lineage sorting era, potentially induced by an early rapid successive speciation of ancestral Neoaves.

Mesozoic retroposons reveal parrots as the closest living relatives of passerine birds.

The relationships of passerines (such as the well-studied zebra finch) with non-passerine birds is one of the great enigmas of avian phylogenetic research, because decades of extensive morphological and molecular studies yielded highly inconsistent results between and within data sets. Here we show the first application of the virtually homoplasy-free retroposon insertions to this controversy. Our study examined ~200,000 retroposon-containing loci from various avian genomes and retrieved 51 markers resolving early bird phylogeny. Among these, we obtained statistically significant evidence that parrots are the closest and falcons the second-closest relatives of passerines, together constituting the Psittacopasserae and the Eufalconimorphae, respectively. Our new and robust phylogenetic framework has substantial implications for the interpretation of various conclusions drawn from passerines as model organisms. This includes insights of relevance to human neuroscience, as vocal learning (that is, birdsong) probably evolved in the psittacopasseran ancestor, >30 million years earlier than previously assumed.

Retroposon insertions and the chronology of avian sex chromosome evolution.

The vast majority of extant birds possess highly differentiated Z and W sex chromosomes. Nucleotide sequence data from gametologs (homologs on opposite sex chromosomes) suggest that this divergence occurred throughout early bird evolution via stepwise cessation of recombination between identical sex chromosomal regions. Here, we investigated avian sex chromosome differentiation from a novel perspective, using retroposon insertions and random insertions/deletions for the reconstruction of gametologous gene trees. Our data confirm that the CHD1Z/CHD1W genes differentiated in the ancestor of the neognaths, whereas the NIPBLZ/NIPBLW genes diverged in the neoavian ancestor and independently within Galloanserae. The divergence of the ATP5A1Z/ATP5A1W genes in galloanserans occurred independently in the chicken, the screamer, and the ancestor of duck-related birds. In Neoaves, this gene pair differentiated in each of the six sampled representatives, respectively. Additionally, three of our investigated loci can be utilized as universal, easy-to-use independent tools for molecular sexing of Neoaves or Neognathae.

Retroposon insertions provide insights into deep lagomorph evolution.

The homogenous mammalian order Lagomorpha comprises about 80 species in two families, Ochotonidae (pikas) and Leporidae (rabbits and hares). However, the phylogenetic relationships among leporids are controversial. Molecular data, particularly from mitochondrial sequences, give highly homoplasious signals. To resolve the controversy between mitochondrial and nuclear data, we analyzed genomic orthologous retroposon insertion sites, a virtually homoplasy-free marker system. From a differential screen of rabbit genomic data for intronic retroposon insertions of CSINE elements, we polymerase chain reaction-amplified and sequenced 11 retroposons in eight representative lagomorphs. We found three retroposons shared among all lagomorphs but absent in outgroups, four confirmed the monophyly of leporids, and three significantly supported Pronolagus as the sister group to all other leporids. One retroposon supported the monophyly of Lepus. The position of Pronolagus outside of the remaining leporids supports the sequence-based signals of nuclear genes and clearly refutes the misleading signals of mitochondrial genes.

Mosaic retroposon insertion patterns in placental mammals.

One and a half centuries after Charles Darwin and Alfred Russel Wallace outlined our current understanding of evolution, a new scientific era is dawning that enables direct observations of genetic variation. However, pure sequence-based molecular attempts to resolve the basal origin of placental mammals have so far resulted only in apparently conflicting hypotheses. By contrast, in the mammalian genomes where they were highly active, the insertion of retroelements and their comparative insertion patterns constitute a neutral, virtually homoplasy-free archive of evolutionary histories. The "presence" of a retroelement at an orthologous genomic position in two species indicates their common ancestry in contrast to its "absence" in more distant species. To resolve the placental origin controversy we extracted approximately 2 million potentially phylogenetically informative, retroposon-containing loci from representatives of the major placental mammalian lineages and found highly significant evidence challenging all current single hypotheses of their basal origin. The Exafroplacentalia hypothesis (Afrotheria as the sister group to all remaining placentals) is significantly supported by five retroposon insertions, the Epitheria hypothesis (Xenarthra as the sister group to all remaining placentals) by nine insertion patterns, and the Atlantogenata hypothesis (a monophyletic clade comprising Xenarthra and Afrotheria as the sister group to Boreotheria comprising all remaining placentals) by eight insertion patterns. These findings provide significant support for a "soft" polytomy of the major mammalian clades. Ancestral successive hybridization events and/or incomplete lineage sorting associated with short speciation intervals are viable explanations for the mosaic retroposon insertion patterns of recent placental mammals and for the futile search for a clear root dichotomy.

Waves of genomic hitchhikers shed light on the evolution of gamebirds (Aves: Galliformes).

BACKGROUND: The phylogenetic tree of Galliformes (gamebirds, including megapodes, currassows, guinea fowl, New and Old World quails, chicken, pheasants, grouse, and turkeys) has been considerably remodeled over the last decades as new data and analytical methods became available. Analyzing presence/absence patterns of retroposed elements avoids the problems of homoplastic characters inherent in other methodologies. In gamebirds, chicken repeats 1 (CR1) are the most prevalent retroposed elements, but little is known about the activity of their various subtypes over time. Ascertaining the fixation patterns of CR1 elements would help unravel the phylogeny of gamebirds and other poorly resolved avian clades. RESULTS: We analyzed 1,978 nested CR1 elements and developed a multidimensional approach taking advantage of their transposition in transposition character (TinT) to characterize the fixation patterns of all 22 known chicken CR1 subtypes. The presence/absence patterns of those elements that were active at different periods of gamebird evolution provided evidence for a clade (Cracidae + (Numididae + (Odontophoridae + Phasianidae))) not including Megapodiidae; and for Rollulus as the sister taxon of the other analyzed Phasianidae. Genomic trace sequences of the turkey genome further demonstrated that the endangered African Congo Peafowl (Afropavo congensis) is the sister taxon of the Asian Peafowl (Pavo), rejecting other predominantly morphology-based groupings, and that phasianids are monophyletic, including the sister taxa Tetraoninae and Meleagridinae. CONCLUSION: The TinT information concerning relative fixation times of CR1 subtypes enabled us to efficiently investigate gamebird phylogeny and to reconstruct an unambiguous tree topology. This method should provide a useful tool for investigations in other taxonomic groups as well.

Identification and subcellular localization of paracellin-1 (claudin-16) in human salivary glands.

Salivary calcium plays an important role in the pathogenesis of dental caries and the bio-mineralization of dental enamel and exposed dentin. The cellular and molecular basis of calcium secretion by the human salivary glands is, however, poorly understood. Recently a transcellular transport of calcium by the acinus cells has been proposed. In this paper we looked for evidence for paracellular calcium transport by investigating the presence and cellular localization of paracellin-1 (claudin-16) that has been implied in paracellular magnesium and calcium transport in the kidney. At the mRNA level, using RT-PCR with primers of appropriate sequence, paracellin-1 mRNA could be found in human Glandula parotis, Glandula submandibularis, Glandula labialis and Glandula sublingualis samples. In addition, a splice variant was detected in three out of 15 glands consisting of exons one and five of the paracellin gene. In immunohistochemical studies paracellin-1 colocalised in the salivary excretory ducts with the tight junction proteins ZO-1 and occludin suggesting a potential role in paracellular calcium and magnesium transport. In the acini no such colocalisation was observed; paracellin was instead detected at the basal poles of the cells, between cells of the same acinus as well as between cells of neighboring acini. At this location paracellin-1 might act as selectivity filter for the paracellular movement of ions and water during stimulated secretion. Thus, both in the ducts and in the acini a paracellular transport of calcium appears possible. Whether it occurs at all and the extent to which it contributes to the overall salivary calcium secretion remains, however, to be determined.

Evolutionary history of 7SL RNA-derived SINEs in Supraprimates.

The evolutionary relationships of 7SL RNA-derived SINEs such as the primate Alu or the rodent B1 elements have hitherto been obscure. We established an unambiguous phylogenetic tree for Supraprimates, and derived intraordinal relationships of the 7SL RNA-derived SINEs. As well as new elements in Tupaia and primates, we also found that the purported ancestral fossil Alu monomer was restricted to Primates, and provide here the first description of a potential chimeric promoter box region in SINEs.

Retroposed elements as archives for the evolutionary history of placental mammals.

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.

Does the AD7c-NTP locus encode a protein?

AD7c-NTP, the only known protein entirely encoded by tandem and nested cassettes of Alu repetitive elements, is reportedly over-expressed in brains of Alzheimer's disease patients [de la Monte et al., J. Clin. Invest. 15 (1997)]. Based on these findings a commercial diagnostic assay ("7c Gold"/"AlzheimAlert" ) has been developed. We analyzed the published cDNA sequence and compared it to corresponding EST clones as well as the genomic sequences of human and chimpanzee. We come to the conclusion that the existence of the gene and in particular the predicted protein is inconsistent with EST and genomic data. Previously published data need to be reassessed.