Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry combined with multidimensional scaling, binary hierarchical cluster tree and selected diagnostic masses improves species identification of Neolithic keratin sequences from furs of the Tyrolean Iceman Oetzi.

The identification of fur origins from the 5300-year-old Tyrolean Iceman's accoutrement is not yet complete, although definite identification is essential for the socio-cultural context of his epoch. Neither have all potential samples been identified so far, nor there has a consensus been reached on the species identified using the classical methods. Archaeological hair often lacks analyzable hair scale patterns in microscopic analyses and polymer chain reaction (PCR)-based techniques are often inapplicable due to the lack of amplifiable ancient DNA. To overcome these drawbacks, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was used exclusively based on hair keratins. Thirteen fur specimens from his accoutrement were analyzed after tryptic digest of native hair. Peptide mass fingerprints (pmfs) from ancient samples and from reference species mostly occurring in the Alpine surroundings at his lifetime were compared to each other using multidimensional scaling and binary hierarchical cluster tree analysis. Both statistical methods highly reflect spectral similarities among pmfs as close zoological relationships. While multidimensional scaling was useful to discriminate specimens on the zoological order level, binary hierarchical cluster tree reached the family or subfamily level. Additionally, the presence and/or absence of order, family and/or species-specific diagnostic masses in their pmfs allowed the identification of mammals mostly down to single species level. Red deer was found in his shoe vamp, goat in the leggings, cattle in his shoe sole and at his quiver's closing flap as well as sheep and chamois in his coat. Canid species, like grey wolf, domestic dog or European red fox, were discovered in his leggings for the first time, but could not be differentiated to species level. This is widening the spectrum of processed fur-bearing species to at least one member of the Canidae family. His fur cap was allocated to a carnivore species, but differentiation between brown bear and a canid species could not be made with certainty.

Genetic differentiation among migrant and resident populations of the threatened Asian houbara bustard.

The Asian houbara bustard Chlamydotis macqueenii is a partial migrant of conservation concern found in deserts of central Asia and the Middle East. In the southern part of the species range, resident populations have been greatly fragmented and reduced by sustained human pressure. In the north, birds migrate from breeding grounds between West Kazakhstan and Mongolia to wintering areas in the Middle East and south central Asia. Extensive satellite tracking has shown substantial partitioning in migration routes and wintering grounds, suggesting a longitudinal barrier to present-day gene flow among migrants. In this context, we explored genetic population structure using 17 microsatellite loci and sampling 108 individuals across the range. We identified limited but significant overall differentiation (F(CT) = 0.045), which was overwhelmingly due to the differentiation of resident Arabian populations, particularly the one from Yemen, relative to the central Asian populations. Population structure within the central Asian group was not detectable with the exception of subtle differentiation of West Kazakh birds on the western flyway, relative to eastern populations. We interpret these patterns as evidence of recent common ancestry in Asia, coupled with a longitudinal barrier to present-day gene flow along the migratory divide, which has yet to translate into genetic divergence. These results provide key parameters for a coherent conservation strategy aimed at preserving genetic diversity and migration routes.

Species identification of Oetzi's clothing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on peptide pattern similarities of hair digests.

Identification of ancient biological samples from the 1991-discovered and more than 5300-year-old Tyrolean mummy, also called iceman or Oetzi, is very difficult. The species of origins of four animal-hair-bearing samples of the accoutrement of the mummy not yet diagnosed were identified by a special proteomics method. Ha 43/91/130 and Ha 6/91, two samples from his coat, and Ha 5/91, a sample from his leggings, were assigned to sheep. The upper leather of his moccasins, Ha 2/91, was made from cattle. Despite the enormous age of these samples with partial (bio)chemical alterations, reliable identification was possible using a recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometric ((MALDI-TOF MS)-based analytical method. The method is exclusively based on the analysis of proteins and uses minute amounts of peptides directly derived from tryptic hair digests without any separation or enrichment steps. Unknown species are identified by comparison of their peptide ion patterns with known spectra stored in existing databases. Hereby, the correlation distance, a form of Euclidean distance, and deduced parameters are used to measure similarities. If more than one potential hit remains, specific diagnostic peptide ions are used to stepwise exclude incorrect matches. These ions are specific for orders, families, subfamilies/genera and/or even species. Peptide mass fingerprinting data combined with those from collision-induced dissociation spectra (combined MS & MS/MS) were used for interpretation with the MASCOT search engine and the NCBI database to find the potential parentage of hair proteins. For this technique, selected precursor ions were identified as specific diagnostic peptide ions.

Evolutionary aspects in evaluating mutations in the melanocortin 4 receptor.

More than 70 missense mutations have been identified in the human melanocortin 4 receptor (MC4R), and many of them have been associated with obesity. In a number of cases, the causal link between mutations in MC4R and obesity is controversially discussed. Here, we mined evolution as an additional source of structural information that may help to evaluate the functional relevance of naturally occurring variations in MC4R. The sequence information of more than 60 MC4R orthologs enabled us to identify residues that are important for maintaining receptor function. More than 90% of all inactivating mutations found in obese patients were located at amino acid positions that are highly conserved during 450 million years of MC4R evolution in vertebrates. However, for a reasonable number of MC4R variants, we found no correlation between structural conservation of the mutated position and the reported functional consequence. By re-evaluating selected mutations in the MC4R, we demonstrate the usefulness of combining functional and evolutionary approaches.

Amelogenin cross-amplification in the family Bovidae and its application for sex determination.

Sex-specific sequence variability of the amelogenin gene had been observed in a variety of mammalian species. In our study, the suitability of the amelogenin gene for sex determination in different species of the family Bovidae was examined. Based on a sequence insertion/deletion characteristic for X- and Y-specific amelogenin (AMELX and AMELY), PCR amplification on male and female genomic DNA from domestic and wild bovine species, sheep and goat, consistently displayed a sex-specific pattern. Thus, the amelogenin amplification by PCR proved to be a reliable method for sex determination not only in domestic and wild species of the tribe Bovini, but also in the related species sheep and goat. Sex determination using the amelogenin-based assay can be performed with at least 40 pg of genomic DNA. The assay enables the investigation of small amounts of DNA from meat, hair, bones, and embryo biopsies to identify species and sex for a number of applications in animal production, forensics, population research, and monitoring within the family Bovidae. Sequence comparison of the amplified amelogenin gene region specific for male and female animals from domestic and wild bovide species revealed further sequence variations within and between sexes as well as between species. Sequence variations in the AMELX gene can be applied to discriminate Bos and Bison individuals from other bovine species, and also from sheep and goat.

Phylogenetic position of the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminichthys (Teleostei: Cyprinidae).

The Dalmatian cyprinid genus Phoxinellus is characterized by reductive characters most likely associated with the environmental conditions of small karstic streams, where all species of this genus occur. Based on 33 morphological traits, nuclear and mtDNA sequences Phoxinellus was found to be paraphyletic and included three not closely related monophyletic units. The scientific name Phoxinellus should therefore be restricted to species having plain coloration, small or absent postcleithrum, no genital papilla and an almost entirely naked body such as P. alepidotus, P. dalmaticus, and P. pseudalepidotus. Species that also have a small or absent postcleithrum and no genital papilla but display a dark stripe from the head to the caudal peduncle, and are entirely covered by distinct, not overlapping scales should be positioned closely to Telestes. Thus, we suggest inclusion of Phoxinellus croaticus, P. fontinalis and Paraphoxinus metohiensis in the genus Telestes. The Phoxinellus species that have a irregularly spotted color pattern, a large postcleithrum, an increased number of precaudal anal-fin pterygiophores, and a large genital papilla in females represent its own evolutionary line closely related to the Balkan species of Pseudophoxinus. For this monophyletic group, we propose to introduce a new genus: Delminichthys. This genus includes the species D. adspersus, D. ghetaldii, D. krbavensis and D. jadovensis.

Molecules and morphology: evidence for introgression of mitochondrial DNA in Dalmatian cyprinids.

In one population of Scardinius dergle, mitochondrial DNA was observed originating from Squalius tenellus. Scardinius dergle shared all diagnostic morphological characters and similarities at a highly variable nuclear region with the genus Scardinius. While crosses and backcrosses most likely resulted in the loss of morphologically diagnostic Squalius-features, maternal inheritance of mtDNA fixed their diagnostic substitutions. Anthropogenic influences resulting from dam building in 1962 are suspected to be the initial force for the hybridization events. However, because hybridization took place despite both lineages being only very distantly related (p=11.2%), we conclude that introgressive hybridization events can be seen as generally possible among leuciscine cyprinids.

The rise and fall of the chemoattractant receptor GPR33.

Chemokine and chemoattractant receptors are members of the large superfamily of G protein-coupled receptors (GPCR), which control leukocyte chemotaxis. In addition to their physiological role, several chemokine and chemoattractant receptors, such as CCR5 and Duffy, have been directly associated with pathogen entry. GPR33 is an orphan chemoattractant GPCR that was previously identified as a pseudogene in humans. GPR33 evolved in mammals about 125-190 million years ago. The cloning and analysis of more than 120 mammalian GPR33 orthologs from 16 of 18 eutherian orders revealed an inactivation of this chemoattractant GPCR not only in humans, but also in several great ape and rodent species. Intriguingly, in all ape and some rodent species where the inactivation occurred, samples harbored both pseudogene and intact gene variants. The analysis of over 1200 human individuals representing all major linguistic groups revealed that the intact allele of GPR33 is still present in the human population. Estimates of the age of the human alleles suggest inactivation in the past 1 million years. Similarly, analysis of more than 120 wild-caught gray rats (Rattus norvegicus), revealed that inactivation of gpr33 is worldwide fixed and occurred in less than 0.7 million years ago. The coincidental inactivation and its fixation in several species of distantly related mammalian orders suggest a selective pressure on this chemoattractant receptor gene.

Nucleocapsid protein of cell culture-adapted Seoul virus strain 80-39: analysis of its encoding sequence, expression in yeast and immuno-reactivity.

Seoul virus (SEOV) is a hantavirus causing a mild to moderate form of hemorrhagic fever with renal syndrome that is distributed mainly in Asia. The nucleocapsid (N) protein-encoding sequence of SEOV (strain 80-39) was RT-PCR-amplified and cloned into a yeast expression vector containing a galactose-inducible promoter. A survey of the pattern of synonymous codon preferences for a total of 22 N protein-encoding hantavirus genes including 13 of SEOV strains revealed that there is minor variation in codon usage by the same gene in different viral genomes. Introduction of the expression plasmid into yeast Saccharomyces cerevisiae resulted in the high-level expression of a hexahistidine-tagged N protein derivative. The nickel-chelation chromatography purified, yeast-expressed SEOV N protein reacted in the immunoblot with a SEOV-specific monoclonal antibody and certain HTNV- and PUUV-cross-reactive monoclonal antibodies. The immunization of a rabbit with the recombinant N protein resulted in the induction of a high-titered antibody response. In ELISA studies, the N protein was able to detect antibodies in sera of experimentally infected laboratory rats and in human anti-hantavirus-positive sera or serum pools of patients from different geographical origin. The yeast-expressed SEOV N protein represents a promising antigen for development of diagnostic tools in serology, sero prevalence studies and vaccine development.

Evolution and phylogeny of old world deer.

The phylogenetic pattern and timing of the radiation of Old World deer was determined based on the complete mitochondrial cytochrome b gene from 33 Cervinae taxa. Using rooted and unrooted phylogenies derived from distinct theoretical approaches, strong support was achieved for monophyly of the Old World deer with muntjacs as sister group as well as for the divergence of at least three distinct genera: Rucervus, Dama, and Cervus. The latter clade comprises what have previously been regarded as the genera or subgenera Panolia, Rusa, Cervus, Sika, and probably Przewalskium. Our data also consistently confirmed paraphyly of nominate C. elaphus and did not support the monophyly of Axis. We used these molecular phylogenies to assess the homoplastic evolution of morphological, geographical, ecological, and selected behavioural character state differences within the Cervinae. Reliable fossil calibrations, large molecular data sets, and improved dating methods are shaping a molecular time scale for the evolutionary radiation of Old World deer that occurred at the Miocene/Pliocene transition and is largely compatible with existing palaeontological evidence. Using node ages estimated from sequence data, we estimated an average per-lineage diversification rate of 0.51+/-0.1 species per million years (my) over roughly the last 6 mya.

Coronavirus infection of spotted hyenas in the Serengeti ecosystem.

Sera from 38 free-ranging spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, were screened for exposure to coronavirus of antigenic group 1. An immunofluorescence assay indicated high levels of exposure to coronavirus among Serengeti hyenas: 95% when considering sera with titer levels of > or = 1:10 and 74% when considering sera with titer levels of > or = 1:40. Cubs had generally lower mean titer levels than adults. Exposure among Serengeti hyenas to coronavirus was also confirmed by a serum neutralisation assay and an ELISA. Application of RT-PCR to 27 fecal samples revealed viral RNA in three samples (11%). All three positive fecal samples were from the 15 juvenile animals (<24 months of age) sampled, and none from the 12 adults sampled. No viral RNA was detected in tissue samples (lymph node, intestine, lung) from 11 individuals. Sequencing of two amplified products from the S protein gene of a positive sample revealed the presence of coronavirus specific RNA with a sequence homology to canine coronavirus of 76 and 78% and to feline coronavirus type II of 80 and 84%, respectively. Estimation of the phylogenetic relationship among coronavirus isolates indicated considerable divergence of the hyena variant from those in European, American and Japanese domestic cats and dogs. From long-term observations of several hundred known individuals, the only clinical sign in hyenas consistent with those described for coronavirus infections in dogs and cats was diarrhea. There was no evidence that coronavirus infection in hyenas caused clinical signs similar to feline infectious peritonitis in domestic cats or was a direct cause of mortality in hyenas. To our knowledge, this is the first report of coronavirus infection in Hyaenidae.

A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.

When the American sea sturgeon swam east.

The two species of Atlantic sea sturgeon on either shore of the North Atlantic, Acipenser sturio in Europe and A. oxyrinchus in North America, probably diverged with the closure of the Tethys Sea and the onset of the North Atlantic Gyre 15-20 million years ago, and contact between them was then presumably precluded by geographic distance. Here we present genetic, morphological and archaeological evidence indicating that the North American sturgeon colonized the Baltic during the Middle Ages and replaced the native sturgeon there, before recently becoming extinct itself in Europe as a result of human activities. In addition to representing a unique transatlantic colonization event by a fish that swims upriver to spawn, our findings have important implications for projects aimed at restocking Baltic waters with the European sturgeon.

Phylogenetic relationships and ancestral areas of the bustards (Gruiformes: Otididae), inferred from mitochondrial DNA and nuclear intron sequences.

The taxonomy of the bustards is poorly understood phylogenetically and has not been extensively evaluated using molecular methods. We sequenced part of the mitochondrial cytochrome b gene, the control region (central domain II), and an intron-exon crossing fragment of the nuclear chromo-helicase-DNA binding gene (CHD1) in 27 bustard taxa (including multiple subspecies) representing 11 genera and four gruiform outgroup species. Molecular datings suggest a Miocene origin for the family. Inferred phylogenetic relationships include the following: (i) the basal polytomy consists of 10 branches (mostly consistent with traditional genera), suggesting a rapid early radiation; (ii) sister relationships between several couplets of genera include Ardeotis with Neotis, Afrotis with Eupodotis (excluding E. rueppellii), Otis with Chlamydotis, and Houbaropsis with Sypheotides; (iii) the genus Eupodotis may be polyphyletic; and (iv) the currently delimited genera Ardeotis and Neotis do not form independent monophyletic lineages. Molecular evidence for the Afro-tropical origin of the Otididae is provided.

An exceptional case of historical outbreeding in African sable antelope populations.

Empirical investigations of intraspecific outbreeding and subsequent introgressive hybridization in natural populations are rare, particularly among conspecific populations of large mammals. Using mitochondrial DNA data [partial control region (496 basepairs - bp) and cytochrome b gene (343 bp) sequences analysed from 95 individuals representing 17 sampling locations scattered through the African miombo (Brachystegia) woodland ecosystem] and phylogeographical statistical procedures (gene genealogy, nested cladistic and admixture proportion analyses), we (i) give a detailed dissection of the geographical genetic structure of Hippotragus niger; (ii) infer the processes and events potentially involved in the population history; and (iii) trace extensive introgressive hybridization in the species. The present-day sable antelope population shows a tripartite pattern of genetic subdivision representing West Tanzanian, Kenya/East Tanzanian and Southern Africa locations. Nested clade analysis revealed that past allopatric fragmentation, caused probably by habitat discontinuities associated with the East African Rift Valley system, together with intermediary episodic long-distance colonization and restricted, recurrent gene flow have played an predominant role in shaping the extent of maternal genetic diversity (10.4%) and population structure. An extensive (average rate of admixture = 20.0%), but geographically circumscribed and unidirectional hybridization event in the past was inferred, resulting in an extreme (the highest discovered so far in mammals) intraspecific difference of 18.2% among morphologically monotypic sable antelopes from West Tanzania. The results are used to provide an evolutionary framework within which taxonomic implications and conservation decisions can be evaluated.

Great bustard population structure in central Spain: concordant results from genetic analysis and dispersal study.

We found significant sex differences in the mtDNA genetic structure and dispersal patterns of great bustards in a population of 11 breeding groups, "leks", in central Spain. The analysis of genetic distances showed that the female population was divided into three groups of leks separated by ca. 50 km, whereas male haplotypes were randomly distributed among leks. Genetic distances among pairs of leks were positively correlated with geographical distances in females but not in males. While female haplotype distributions were homogeneous among leks at close distances, differences in male genetic structure were highly variable even between two close leks. These results from genetic analyses were concordant with those from a radiotracking study on natal dispersal. Natal dispersal distances were higher in males than in females. Also, the frequency of movement of a female between two leks was positively correlated with their genetic affinity and geographical proximity. In males, the frequency of movement was correlated with geographical proximity but not with genetic affinity. Males dispersed among genetically unrelated leks, contributing to keep nuclear genetic diversity in the population, whereas females tended to be philopatric. These results suggest that isolation-by-distance influences the distribution of maternal lineages at a regional level.

Emerging viruses: the case 'hantavirus'.

This review briefly summarises the recent knowledge about hantavirus infections and raises particular problems in hantavirus research that need further investigation. The following questions are addressed: (i) are hantaviruses distributed worldwide and what leads to new outbreaks, (ii) what is known about hantavirus evolution, (iii) how can hantavirus species be defined, (iv) what are the determinants of hantavirus pathogenesis in humans, and (v) what problems are associated with the development of new vaccines and antiviral therapeutics.