V-shaped Pits in Regions of Ancient Baekje Kingdom Paleoparasitologically Confirmed as Likely Human-Waste Reservoirs

In a paleo-parasitological analysis of soil samples obtained from V-shaped pits dating to the ancient Baekje period in Korean history, we discovered Ascaris lumbricoides, Trichuris trichiura, and Clonorchis sinensis eggs. In light of the samples'' seriously contaminated state, the V-shaped pits might have served as toilets, cesspits, or dung heaps. For a long period of time, researchers scouring archaeological sites in Korea have had difficulties locating such structures. In this context then, the present report is unique because similar kind of the ancient ruins must become an ideal resource for successful sampling in our forthcoming paleoparasitological studies.     

First finds of <Emphasis Type="Italic">Prunus domestica</Emphasis> L. in Italy from the Phoenician and Punic periods (6th–2nd centuries <Emphasis Type="SmallCaps">bc</Emphasis>)

During the archaeological excavations in the Phoenician and Punic settlement of Santa Giusta (Oristano, Sardinia, Italy), dating back to the 6th–2nd centuries bc, several Prunus fruitstones (endocarps) inside amphorae were recovered. The exceptional state of preservation of the waterlogged remains allowed morphometric measurements to be done by image analysis and statistical comparisons made with modern cultivated and wild Prunus samples collected in Sardinia. Digital images of modern and archaeological Prunus fruitstones were acquired with a flatbed scanner and analysed by applying image analysis techniques to measure 26 morphometric features. By applying stepwise linear discriminant analysis, a morphometric comparison was made between the archaeological fruitstones of Prunus and the modern ones collected in Sardinia. These analyses allowed identification of 53 archaeological fruitstones as P. spinosa and 11 as P. domestica. Moreover, the archaeological samples of P. spinosa showed morphometric similarities in 92.5% of the cases with the modern P. spinosa samples currently growing near the Phoenician and Punic site. Likewise, the archaeological fruitstones identified as P. domestica showed similarities with the modern variety of P. domestica called Sanguigna di Bosa which is currently cultivated near the village of Bosa. Currently, these findings represent the first evidence of P. domestica in Italy during the Phoenician and Punic periods.     

Uranium series dating in paleoanthropology

Many hominid and archaeological sites contain materials that were formed or deposited synchronously with the site and can be dated by uranium-series methods, principally ²³⁰Th/²³⁴U dating. The range of the method is approximately 350,000 years. U-series dating yields most accurate results when applied to chemically precipitated calcium carbonate, which occurs in stalagmitic layers, travertines, and lacustrine limestones. Materials such as bone and shell are less reliable. Using mass spectrometry (MS), a precision of ±1% is attainable, whereas conventional alpha counting optimally gives a precision of ±5%. Mass spectrometric dating requires smaller samples, potentially allowing determination of ante quem dates for calcite coatings on hominid fossils and associated faunal skeletons.     

Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenase enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNA gene tree to a considerable degree, which made it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterial communities in sediments of hyper saline and soda lakes. Although diazotrophs were revealed in all environmental samples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophic gammaproteobacteria were predominant in integrated samples. Analysis of the upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria, and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.     

Skeletal chemistry of Nautilus and its taxonomic significance

The strontium (Sr) and magnesium (Mg) chemistry of the shell wall and septum as well as the spherulitic-prismatic and nacreous layers of these structures was determined for Nautilus species: N. belauensis, N. macromphalus, N. pompilius and N. scrobiculatus. Each species of Nautilus exhibits greater variability and higher concentrations of Mg in juvenile portions of the shell than in more mature portions of the shell. This decrease in the variability and amount of Mg in the aragonite lattice suggests a physiochemical system which becomes more efficient with time relative to carbonate production. Statistically significant differences in the Sr and Mg content of spherulitic-prismatic and nacreous layers of the shell and septum indicate that these layers were formed from extracellular fluids of different compositions. Concentrations of Sr and Mg in aragonite of the shell wall are characteristic for each species and sufficiently invariant within species to allow species of Nautilus to be distinguished statistically on the basis of either the Sr or Mg content of the shell wall or the Mg content of septa.     

The degradation of intracrystalline mollusc shell proteins: A proteomics study of Spondylus gaederopus

Mollusc shells represent excellent systems for the preservation and retrieval of genuine biomolecules from archaeological or palaeontological samples. As a consequence, the post-mortem breakdown of intracrystalline mollusc shell proteins has been extensively investigated, particularly with regard to its potential use as a “molecular clock” for geochronological applications. But despite seventy years of ancient protein research, the fundamental aspects of diagenesis-induced changes to protein structures and sequences remain elusive. In this study we investigate the degradation of intracrystalline proteins by performing artificial degradation experiments on the shell of the thorny oyster, Spondylus gaederopus, which is particularly important for archaeological research. We used immunochemistry and tandem mass tag (TMT) quantitative proteomics to simultaneously track patterns of structural loss and of peptide bond hydrolysis.Powdered and bleached shell samples were heated in water at four different temperatures (80, 95, 110, 140 °C) for different time durations. The structural loss of carbohydrate and protein groups was investigated by immunochemical techniques (ELLA and ELISA) and peptide bond hydrolysis was studied by tracking the changes in protein/peptide relative abundances over time using TMT quantitative proteomics. We find that heating does not induce instant organic matrix decay, but first facilitates the uncoiling of cross-linked structures, thus improving matrix detection. We calculated apparent activation energies of structural loss: E_a (carbohydrate groups) = 104.7 kJ/mol, E_a (protein epitopes) = 104.4 kJ/mol, which suggests that secondary matrix structure degradation may proceed simultaneously with protein hydrolysis. While prolonged heating at 110 °C (10 days) results in complete loss of the structural signal, surviving peptide sequences were still observed. Eight hydrolysis-prone peptide bonds were identified in the top scoring shell sequence, the uncharacterised protein LOC117318053 from Pecten maximus. Interestingly, these were not the expected “weak” bonds based on published theoretical stabilities calculated for peptides in solution. This further confirms that intracrystalline protein degradation patterns are complex and that the overall microchemical environment plays an active role in protein stability. Our TMT approach represents a major stepping stone towards developing a model for studying protein diagenesis in biomineralised systems.     

Longer and Less Overlapping Food Webs in Anthropogenically Disturbed Marine Ecosystems: Confirmations from the Past

The human exploitation of marine resources is characterised by the preferential removal of the largest species. Although this is expected to modify the structure of food webs, we have a relatively poor understanding of the potential consequences of such alteration. Here, we take advantage of a collection of ancient consumer tissues, using stable isotope analysis and SIBER to assess changes in the structure of coastal marine food webs in the South-western Atlantic through the second half of the Holocene as a result of the sequential exploitation of marine resources by hunter-gatherers, western sealers and modern fishermen. Samples were collected from shell middens and museums. Shells of both modern and archaeological intertidal herbivorous molluscs were used to reconstruct changes in the stable isotopic baseline, while modern and archaeological bones of the South American sea lion Otaria flavescens, South American fur seal Arctocephalus australis and Magellanic penguin Spheniscus magellanicus were used to analyse changes in the structure of the community of top predators. We found that ancient food webs were shorter, more redundant and more overlapping than current ones, both in northern-central Patagonia and southern Patagonia. These surprising results may be best explained by the huge impact of western sealing on pinnipeds during the fur trade period, rather than the impact of fishing on fish populations. As a consequence, the populations of pinnipeds at the end of the sealing period were likely well below the ecosystem''s carrying capacity, which resulted in a release of intraspecific competition and a shift towards larger and higher trophic level prey. This in turn led to longer and less overlapping food webs.     

First data on the shell structure of the Early Cretaceous (Berriasian) rhynchonellids from Crimea

The shell structure of the Early Cretaceous rhynchonellids from Crimea has not been previously studied. First data on the shell structure of Berriasian rhynchonellids from the family Praecyclothyrididae Makridin, 1964 from southwestern and central Crimea are presented. Sulcirhynchia semenovi (Moisseev, 1939), S. berriasensis (Lobacheva, 1980), S. gracilis (Lobacheva, 1977), Belbekella airgulensis Moisseev, 1939, B. mutabilis Lobacheva, 1983, B. minor Lobacheva, 1983 and partly Lamellaerhynchia rectimarginata (Smirnova, 1972) are studied. The shell wall of most species consists of three layers: external (finely or coarsely crystalline), fibrous, and prismatic layers. The layers are usually strongly recrystallized, especially external and prismatic layers. New fibers were formed by repeated dichotomy of ridges in different areas of the fibrous layer independently of the distance from the anterior margin.     

Food supply and seawater pCO2 impact calcification and internal shell dissolution in the blue mussel Mytilus edulis

Progressive ocean acidification due to anthropogenic CO₂ emissions will alter marine ecosytem processes. Calcifying organisms might be particularly vulnerable to these alterations in the speciation of the marine carbonate system. While previous research efforts have mainly focused on external dissolution of shells in seawater under saturated with respect to calcium carbonate, the internal shell interface might be more vulnerable to acidification. In the case of the blue mussel Mytilus edulis, high body fluid pCO₂ causes low pH and low carbonate concentrations in the extrapallial fluid, which is in direct contact with the inner shell surface. In order to test whether elevated seawater pCO₂ impacts calcification and inner shell surface integrity we exposed Baltic M. edulis to four different seawater pCO₂ (39, 142, 240, 405 Pa) and two food algae (310–350 cells mL⁻¹ vs. 1600–2000 cells mL⁻¹) concentrations for a period of seven weeks during winter (5°C). We found that low food algae concentrations and high pCO₂ values each significantly decreased shell length growth. Internal shell surface corrosion of nacreous ( = aragonite) layers was documented via stereomicroscopy and SEM at the two highest pCO₂ treatments in the high food group, while it was found in all treatments in the low food group. Both factors, food and pCO₂, significantly influenced the magnitude of inner shell surface dissolution. Our findings illustrate for the first time that integrity of inner shell surfaces is tightly coupled to the animals'' energy budget under conditions of CO₂ stress. It is likely that under food limited conditions, energy is allocated to more vital processes (e.g. somatic mass maintenance) instead of shell conservation. It is evident from our results that mussels exert significant biological control over the structural integrity of their inner shell surfaces.     

Inheritance of the background shell color in the snails Littorina obtusata (Gastropoda,Littorinidae)

We investigated in a gastropod mollusk Littorina obtusata (L. obtusata) the inheritance of back-ground shell coloration of the shell, which arises on the basis of three pigments: purple, orange, and yellow. We found that the hypothesis on polyallelic inheritance, as in the genus Cepaea, cannot explain the inheritance of shell color in periwinkles. We demonstrated that a separate genetic system is responsible for incorporation of each pigment into the shell. The composition of these genetic systems includes at least two genes each in the case of the yellow and purple pigments. Our analysis shows that caution should be applied when extending the results obtained in the studies of the Cepaea genus to the other species of gastropods.     

Molecular Paleoparasitological Hybridization Approach as Effective Tool for Diagnosing Human Intestinal Parasites from Scarce Archaeological Remains

Paleoparasitology is the science that uses parasitological techniques for diagnosing parasitic diseases in the past. Advances in molecular biology brought new insights into this field allowing the study of archaeological material. However, due to technical limitations a proper diagnosis and confirmation of the presence of parasites is not always possible, especially in scarce and degraded archaeological remains. In this study, we developed a Molecular Paleoparasitological Hybridization (MPH) approach using ancient DNA (aDNA) hybridization to confirm and complement paleoparasitological diagnosis. Eight molecular targets from four helminth parasites were included: Ascaris sp., Trichuris trichiura, Enterobius vermicularis, and Strongyloides stercoralis. The MPH analysis using 18^th century human remains from Praça XV cemetery (CPXV), Rio de Janeiro, Brazil, revealed for the first time the presence E. vermicularis aDNA (50%) in archaeological sites of Brazil. Besides, the results confirmed T. trichiura and Ascaris sp. infections. The prevalence of infection by Ascaris sp. and E. vermicularis increased considerably when MPH was applied. However, a lower aDNA detection of T. trichiura (40%) was observed when compared to the diagnosis by paleoparasitological analysis (70%). Therefore, based on these data, we suggest a combination of Paleoparasitological and MPH approaches to verify the real panorama of intestinal parasite infection in human archeological samples.     

Paleoparasitological Surveys for Detection of Helminth Eggs in Archaeological Sites of Jeolla-do and Jeju-do

A paleoparasitological survey to detect helminth eggs was performed in archaeological sites of Jeolla-do and Jeju-do, the Republic of Korea. Total 593 soil samples were collected in 12 sites of Jeolla-do and 5 sites of Jeju-do from April to November 2011, and examined by the methods of Pike and coworkers. A total of 4 helminth eggs, 2 eggs each for Trichuris trichiura and Ascaris sp., were found in soil samples from 1 site, in Hyangyang-ri, Jangheung-eup, Jangheung-gun, Jeollanam-do. The egg-recovery layer was presumed to represent a 19th century farm, which fact suggested the use of human manures. This is the third archaeological discovery of parasite eggs in Jeolla-do. Additionally, no helminth eggs in archaeological sites of Jeju-do is an interesting problem to be solved in the further investigations.     

EVIDENCE FROM THE FOSSIL RECORD OF AN ANTIPREDATORY EXAPTATION: CONCHIOLIN LAYERS IN CORBULID BIVALVES

Conchiolin layers, organic-rich laminae, are characteristic of the shells of corbulid bivalves. The retention of these layers, despite their high metabolic cost, throughout the evolutionary history of Corbulidae has prompted the proposal of several adaptive scenarios to explain the origin and maintenance of these layers. The most widely held hypothesis contends that conchiolin layers are an adaptation for inhibiting drilling by predatory naticid gastropods. However, others suggest that the layers are adaptations to retard shell dissolution in waters undersaturated with calcium carbonate or to increase shell strength in the face of durophagous (shell crushing) predators. In this paper, experiments using recent Corbula (Varicorbula) gibba (Olivi) and observations of corbulids' present natural habitat demonstrate the current utility of conchiolin layers for all three functions: retardation of shell dissolution in waters undersaturated in calcium carbonate, increase of mechanical shell strength, and inhibition of drilling by predatory naticid gastropods. Earlier analyses of the extensive history of naticid predator-corbulid prey interactions suggested that conchiolin layers were an adaptation, a feature that promotes fitness and was built by selection for its current role, for deterring naticid predators. Not only are naticid drillholes widespread in fossil and recent corbulid shells, but an unusually large number of incomplete drillholes terminate unsuccessfully at conchiolin layers. In addition, a phylogenetic analysis of the origin of conchiolin layers and its function to deter naticid predators is consistent with a hypothesis of adaptation for this function. However, this hypothesis is rejected by an examination of fossil Jurassic Corbulomima. These oldest corbulids contained conchiolin layers before the evolution of naticid drilling during the Early Cretaceous. Therefore, conchiolin layers appear to be an exaptation, characters evolved for other usages and later “coopted” for their current role, for defense against drilling predators. The layers may in fact be an adaptation to resist durophagous predation.     

Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity

The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second–nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth–nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis.     

Prehistoric salt mining in Hallstatt,Austria. New chronologies out of small wooden fragments

The prehistoric salt mine of Hallstatt together with its burial ground is one of the most prominent archaeological sites in the world, and has given its name to the “Hallstatt period”, an epoch of European prehistory (800 to 400 BCE). Due to the perfect conservation in rock salt a high number of organic materials have been found, including mostly wooden artefacts and structural timber.More than 2000 samples were taken from various archaeological sites in the mines as well as at the surface. It was possible to date 763 samples by the means of dendrochronology and by ¹⁴C wiggle matching. The dendrochronological dating was possible due to crossdating with various available chronologies (like Villingen-Magdalenenberg or Dachstein/Schwarzer See). The fir (Abies alba Mill.) chronologies span the periods: -1232 to -1063; -819 to -425 and -371 to-129. The spruce (Picea abies L. Karst.) chronologies span the periods: -1228 to -1063; -813 to -669 and -342 to -123. The larch (Larix decidua Mill.) chronologies span the periods: -1393 ± 18 to-1286 ± 18 based on wiggle matching data and -252 to -164. A beech (Fagus sylvatica L.) chronology span the time -1182 to -1062.     

Population Genomic Analyses Based on 1 Million SNPs in Commercial Egg Layers

Identifying signatures of selection can provide valuable insight about the genes or genomic regions that are or have been under selective pressure, which can lead to a better understanding of genotype-phenotype relationships. A common strategy for selection signature detection is to compare samples from several populations and search for genomic regions with outstanding genetic differentiation. Wright''s fixation index, F_ST, is a useful index for evaluation of genetic differentiation between populations. The aim of this study was to detect selective signatures between different chicken groups based on SNP-wise F_ST calculation. A total of 96 individuals of three commercial layer breeds and 14 non-commercial fancy breeds were genotyped with three different 600K SNP-chips. After filtering a total of 1 million SNPs were available for F_ST calculation. Averages of F_ST values were calculated for overlapping windows. Comparisons of these were then conducted between commercial egg layers and non-commercial fancy breeds, as well as between white egg layers and brown egg layers. Comparing non-commercial and commercial breeds resulted in the detection of 630 selective signatures, while 656 selective signatures were detected in the comparison between the commercial egg-layer breeds. Annotation of selection signature regions revealed various genes corresponding to productions traits, for which layer breeds were selected. Among them were NCOA1, SREBF2 and RALGAPA1 associated with reproductive traits, broodiness and egg production. Furthermore, several of the detected genes were associated with growth and carcass traits, including POMC, PRKAB2, SPP1, IGF2, CAPN1, TGFb2 and IGFBP2. Our approach demonstrates that including different populations with a specific breeding history can provide a unique opportunity for a better understanding of farm animal selection.