Tracking the origins of Yakutian horses and the genetic basis for their fast adaptation to subarctic environments.

Yakutia, Sakha Republic, in the Siberian Far East, represents one of the coldest places on Earth, with winter record temperatures dropping below -70 °C. Nevertheless, Yakutian horses survive all year round in the open air due to striking phenotypic adaptations, including compact body conformations, extremely hairy winter coats, and acute seasonal differences in metabolic activities. The evolutionary origins of Yakutian horses and the genetic basis of their adaptations remain, however, contentious. Here, we present the complete genomes of nine present-day Yakutian horses and two ancient specimens dating from the early 19th century and ∼5,200 y ago. By comparing these genomes with the genomes of two Late Pleistocene, 27 domesticated, and three wild Przewalski's horses, we find that contemporary Yakutian horses do not descend from the native horses that populated the region until the mid-Holocene, but were most likely introduced following the migration of the Yakut people a few centuries ago. Thus, they represent one of the fastest cases of adaptation to the extreme temperatures of the Arctic. We find cis-regulatory mutations to have contributed more than nonsynonymous changes to their adaptation, likely due to the comparatively limited standing variation within gene bodies at the time the population was founded. Genes involved in hair development, body size, and metabolic and hormone signaling pathways represent an essential part of the Yakutian horse adaptive genetic toolkit. Finally, we find evidence for convergent evolution with native human populations and woolly mammoths, suggesting that only a few evolutionary strategies are compatible with survival in extremely cold environments.

Improving the extraction of ancient Yersinia pestis genomes from the dental pulp.

Ancient DNA preserved in the dental pulp offers the opportunity to characterize the genome of some of the deadliest pathogens in human history. However, while DNA capture technologies help, focus sequencing efforts, and therefore, reduce experimental costs, the recovery of ancient pathogen DNA remains challenging. Here, we tracked the kinetics of ancient Yersinia pestis DNA release in solution during a pre-digestion of the dental pulp. We found that most of the ancient Y. pestis DNA is released within 60 min at 37°C in our experimental conditions. We recommend a simple pre-digestion as an economical procedure to obtain extracts enriched in ancient pathogen DNA, as longer digestion times release other types of templates, including host DNA. Combining this procedure with DNA capture, we characterized the genome sequences of 12 ancient Y. pestis bacteria from France dating to the second pandemic outbreaks of the 17th and 18th centuries Common Era.

Detection of age-related duplications in mtDNA from human muscles and bones.

Several studies have demonstrated the age-related accumulation of duplications in the D-loop of mitochondrial DNA (mtDNA) extracted from skeletal muscle. This kind of mutation had not yet been studied in bone. The detection of age-related mutations in bone tissue could help to estimate age at death within the context of legal medicine or/and anthropological identification procedures, when traditional osteological markers studied are absent or inefficient. As we detected an accumulation of a point mutation in mtDNA from an older individual's bones in a previous study, we tried here to identify if three reported duplications (150, 190, 260 bp) accumulate in this type of tissue. We developed a sensitive method which consists in the use of back-to-back primers during amplification followed by an electrophoresis capillary analysis. The aim of this study was to confirm that at least one duplication appears systematically in muscle tissue after the age of 20 and to evaluate the duplication age appearance in bones extracted from the same individuals. We found that the number of duplications increase from 38 years and that at least one duplicated fragment is present in 50% of cases after 70 years in this tissue. These results confirm that several age-related mutations can be detected in the D-loop of mtDNA and open the way for the use of molecular markers for age estimation in forensic and/or anthropological identification.

No particular genomic features underpin the dramatic economic consequences of 17th century plague epidemics in Italy.

The 17th century plague epidemic had a particularly strong demographic toll in Southern Europe, especially Italy, where it caused long-lasting economical damage. Whether this resulted from ineffective sanitation measures or more pathogenic Yersinia pestis strains remains unknown. DNA screening of 26 skeletons from the 1629-1630 plague cemetery of Lariey (French Alps) identified two teeth rich in plague genetic material. Further sequencing revealed two Y. pestis genomes phylogenetically closest to those from the 1636 outbreak of San Procolo a Naturno, Italy. They both belonged to a cluster extending from the Alps to Northern Germany that probably propagated during the Thirty Years war. Sequence variation did not support faster evolutionary rates in the Italian genomes and revealed only rare private non-synonymous mutations not affecting virulence genes. This, and the more heterogeneous spatial diffusion of the epidemic outside Italy, suggests environmental or social rather than biological causes for the severe Italian epidemic trajectory.

Heterogeneous Hunter-Gatherer and Steppe-Related Ancestries in Late Neolithic and Bell Beaker Genomes from Present-Day France.

The transition from the Late Neolithic to the Bronze Age has witnessed important population and societal changes in western Europe.1 These include massive genomic contributions of pastoralist herders originating from the Pontic-Caspian steppes2,3 into local populations, resulting from complex interactions between collapsing hunter-gatherers and expanding farmers of Anatolian ancestry.4-8 This transition is documented through extensive ancient genomic data from present-day Britain,9,10 Ireland,11,12 Iberia,13 Mediterranean islands,14,15 and Germany.8 It remains, however, largely overlooked in France, where most focus has been on the Middle Neolithic (n = 63),8,9,16 with the exception of one Late Neolithic genome sequenced at 0.05× coverage.16 This leaves the key transitional period covering ∼3,400-2,700 cal. years (calibrated years) BCE genetically unsampled and thus the exact time frame of hunter-gatherer persistence and arrival of steppe migrations unknown. To remediate this, we sequenced 24 ancient human genomes from France spanning ∼3,400-1,600 cal. years BCE. This reveals Late Neolithic populations that are genetically diverse and include individuals with dark skin, hair, and eyes. We detect heterogeneous hunter-gatherer ancestries within Late Neolithic communities, reaching up to ∼63.3% in some individuals, and variable genetic contributions of steppe herders in Bell Beaker populations. We provide an estimate as late as ∼3,800 years BCE for the admixture between Neolithic and Mesolithic populations and as early as ∼2,650 years BCE for the arrival of steppe-related ancestry. The genomic heterogeneity characterized underlines the complex history of human interactions even at the local scale.

Analysis of 5000 year-old human teeth using optimized large-scale and targeted proteomics approaches for detection of sex-specific peptides.

The study demonstrates the high potential of MS-based proteomics coupled to an iterative database search strategy for the in-depth investigation of ancient proteomes. An efficient targeted PRM MS-based approach, although limited to the detection of a single pair of sex-specific amelogenin peptides, allowed confirming the sex of individuals in ancient dental remains, an essential information for paleoanthropologists facing the issue of sex determination and dimorphism.

Predicting haplogroups using a versatile machine learning program (PredYMaLe) on a new mutationally balanced 32 Y-STR multiplex (CombYplex): Unlocking the full potential of the human STR mutation rate spectrum to estimate forensic parameters.

We developed a new mutationally well-balanced 32 Y-STR multiplex (CombYplex) together with a machine learning (ML) program PredYMaLe to assess the impact of STR mutability on haplogourp prediction, while respecting forensic community criteria (high DC/HD). We designed CombYplex around two sub-panels M1 and M2 characterized by average and high-mutation STR panels. Using these two sub-panels, we tested how our program PredYmale reacts to mutability when considering basal branches and, moving down, terminal branches. We tested first the discrimination capacity of CombYplex on 996 human samples using various forensic and statistical parameters and showed that its resolution is sufficient to separate haplogroup classes. In parallel, PredYMaLe was designed and used to test whether a ML approach can predict haplogroup classes from Y-STR profiles. Applied to our kit, SVM and Random Forest classifiers perform very well (average 97 %), better than Neural Network (average 91 %) and Bayesian methods (< 90 %). We observe heterogeneity in haplogroup assignation accuracy among classes, with most haplogroups having high prediction scores (99-100 %) and two (E1b1b and G) having lower scores (67 %). The small sample sizes of these classes explain the high tendency to misclassify the Y-profiles of these haplogroups; results were measurably improved as soon as more training data were added. We provide evidence that our ML approach is a robust method to accurately predict haplogroups when it is combined with a sufficient number of markers, well-balanced mutation rate Y-STR panels, and large ML training sets. Further research on confounding factors (such as CNV-STR or gene conversion) and ideal STR panels in regard to the branches analysed can be developed to help classifiers further optimize prediction scores.

Detection of the A189G mtDNA heteroplasmic mutation in relation to age in modern and ancient bones.

The aim of this study was to demonstrate the presence of the A189G age-related point mutation on DNA extracted from bone. For this, a peptide nucleic acid (PNA)/DNA sequencing method which can determine an age threshold for the appearance of the mutation was used. Initially, work was done in muscle tissue in order to evaluate the sensitivity of the technique and afterwards in bone samples from the same individuals. This method was also applied to ancient bones from six well-preserved skeletal remains. The mutation was invariably found in muscle, and at a rate of up to 20% in individuals over 60 years old. In modern bones, the mutation was detected in individuals aged 38 years old or more, at a rate of up to 1%, but its occurrence was not systematic (only four out of ten of the individuals over 50 years old carried the heteroplasmy). For ancient bones, the mutation was also found in the oldest individuals according to osteologic markers. The study of this type of age-related mutation and a more complete understanding of its manifestation has potentially useful applications. Combined with traditional age markers, it could improve identification accuracy in forensic cases or in anthropological studies of ancient populations.

Oral health status in historic population: Macroscopic and metagenomic evidence.

Recent developments in High-Throughput DNA sequencing (HTS) technologies and ancient DNA (aDNA) research have opened access to the characterization of the microbial communities within past populations. Most studies have, however, relied on the analysis of dental calculus as one particular material type particularly prone to the molecular preservation of ancient microbial biofilms and potential of entire teeth for microbial characterization, both of healthy communities and pathogens in ancient individuals, remains overlooked. In this study, we used shotgun sequencing to characterize the bacterial composition from historical subjects showing macroscopic evidence of oral pathologies. We first carried out a macroscopic analysis aimed at identifying carious or periodontal diseases in subjects belonging to a French rural population of the 18th century AD. We next examined radiographically six subjects showing specific, characteristic dental pathologies and applied HTS shotgun sequencing to characterize the microbial communities present in and on the dental material. The presence of Streptococcus mutans and also Rothia dentocariosa, Actinomyces viscosus, Porphyromonas gingivalis, Tannerella forsythia, Pseudoramibacter alactolyticus, Olsenella uli and Parvimonas micra was confirmed through the presence of typical signatures of post-mortem DNA damage at an average depth-of-coverage ranging from 0.5 to 7X, with a minimum of 35% (from 35 to 93%) of the positions in the genome covered at least once. Each sampled tooth showed a specific bacterial signature associated with carious or periodontal pathologies. This work demonstrates that from a healthy independent tooth, without visible macroscopic pathology, we can identify a signature of specific pathogens and deduce the oral health status of an individual.

Ancient genomic changes associated with domestication of the horse.

The genomic changes underlying both early and late stages of horse domestication remain largely unknown. We examined the genomes of 14 early domestic horses from the Bronze and Iron Ages, dating to between ~4.1 and 2.3 thousand years before present. We find early domestication selection patterns supporting the neural crest hypothesis, which provides a unified developmental origin for common domestic traits. Within the past 2.3 thousand years, horses lost genetic diversity and archaic DNA tracts introgressed from a now-extinct lineage. They accumulated deleterious mutations later than expected under the cost-of-domestication hypothesis, probably because of breeding from limited numbers of stallions. We also reveal that Iron Age Scythian steppe nomads implemented breeding strategies involving no detectable inbreeding and selection for coat-color variation and robust forelimbs.

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA.

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.

History of Smallpox and Its Spread in Human Populations.

Smallpox is considered among the most devastating of human diseases. Its spread in populations, initiated for thousands of years following a probable transmission from an animal host, was concomitant with movements of people across regions and continents, trade and wars. Literature permitted to retrace the occurrence of epidemics from ancient times to recent human history, smallpox having affected all levels of past society including famous monarchs. The disease was officially declared eradicated in 1979 following intensive vaccination campaigns.Paleomicrobiology dedicated to variola virus is restricted to few studies, most unsuccessful, involving ancient material. Only one recent approach allowed the identification of viral DNA fragments from lung tissue of a 300-year-old body excavated from permafrost in Eastern Siberia; phylogenetic analysis revealed that this ancient strain was distinct from those described during the 20th century.

About 42% of 154 remains from the "Battle of Le Mans", France (1793) belong to women and children: Morphological and genetic evidence.

Mass graves were discovered in Le Mans and 154 skeletons were exhumed, representing a remarkable historical series of human remains from western France. We aimed to characterise the age-class and sex of these subjects, and to determine whether their profile fits with that of the Catholic and Royal Army of Vendée, who fought against the Republican Army during the Battle of Le Mans (12th-13th December, 1793). This atypical army was composed of male soldiers, but also of civilian people who followed the troops, including the elderly, children and women. In total 154 skeletons from nine mass graves were exhumed from 2009 to 2010. Two morphological methods were used to determine the sex of the subjects: the Probabilist Sexual Diagnosis (DSP) and Secondary Sexual Diagnosis (DSS) methods. Samples were handled cautiously to avoid any pre-laboratory contamination. Molecular genetic sex-typing using a recently developed assay was used to maximise sex-diagnosis of the ancient DNA samples, and 97 successful profiles including immatures were generated. Using morphological and genetic data combined, we successfully determined the sex of 93% of the subjects; 62% were male and 31% female. About 87% of subjects could be considered adults (>18 years old), 6% adolescents (15-19 years old) and 7% infants (<15 years old). Our results of an unexpected population profile for an armed conflict (42% were women and children), in addition to traumatological and historical elements, tend to confirm that these subjects were involved in the Battle of Le Mans, either actively (Republican Army, the Catholic and Royal Army) or passively (collateral victims from the civilian population of Le Mans). They represent 5-6% of the estimated 2500-3000 victims.

Shotgun sequencing of the mitochondrial genome of the Aldabra giant tortoise (Aldabrachelys gigantea).

The complete mitochondrial genome of the Aldabra giant tortoise [Aldabrachelys gigantea (Schweigger, 1812): Reptilia, Testudines, Testudinidae] was sequenced using a shotgun approach on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA). This genome was 16 467 bp long and presents the typical organization found in vertebrates. The mean coverage of sequencing was 116×. A phylogenetic analysis of the Testudinidae confirms the placement of Aldabrachelys in an Indian Ocean group (including Madagascar). This mitogenome constitutes a reference for ancient DNA analyses of the extinct Madagascan lineages of Aldabrachelys.

The genetics of kinship in remote human groups.

For fifteen years, part of the work of our research team has been focused on the study of parental links between individuals living hundreds or thousands of years ago, whose remains have been found in single graves or large funerary complexes. These studies have been undertaken using methods developed by forensic genetics to identify individuals, mainly based on the genotyping of autosomal STR (Short Tandem Repeats). Issues arose from this work, namely the limits of studying small numbers of subjects, originating from groups of finite sizes where kinships cannot be inferred a priori and for which reference allelic frequencies do not exist. Although ideal human populations are rare when undertaking such studies, the Yakuts of Eastern Siberia constitute a very advantageous model, with large numbers of small pastoral communities and well-preserved archaeological material. The study of kinship in the ancient Yakuts allowed us to highlight the difficulties in analysing genetic data from small ancient human groups and to develop a strategy to improve the accuracy of statistical computations. This work describes this strategy and possible solutions to the study of populations outside of the frame of reference of global meta-populations, due either to isolation, remoteness or antiquity.

Tuberculosis epidemiology and selection in an autochthonous Siberian population from the 16th-19th century.

Tuberculosis is one of most ancient diseases affecting human populations. Although numerous studies have tried to detect pathogenic DNA in ancient skeletons, the successful identification of ancient tuberculosis strains remains rare. Here, we describe a study of 140 ancient subjects inhumed in Yakutia (Eastern Siberia) during a tuberculosis outbreak, dating from the 16(th)-19(th) century. For a long time, Yakut populations had remained isolated from European populations, and it was not until the beginning of the 17(th) century that first contacts were made with European settlers. Subsequently, tuberculosis spread throughout Yakutia, and the evolution of tuberculosis frequencies can be tracked until the 19(th) century. This study took a multidisciplinary approach, examining historical and paleo-epidemiological data to understand the impact of tuberculosis on ancient Yakut population. In addition, molecular identification of the ancient tuberculosis strain was realized to elucidate the natural history and host-pathogen co-evolution of human tuberculosis that was present in this population. This was achieved by the molecular detection of the IS6110 sequence and SNP genotyping by the SNaPshot technique. Results demonstrated that the strain belongs to cluster PGG2-SCG-5, evocating a European origin. Our study suggests that the Yakut population may have been shaped by selection pressures, exerted by several illnesses, including tuberculosis, over several centuries. This confirms the validity and necessity of using a multidisciplinary approach to understand the natural history of Mycobacterium tuberculosis infection and disease.

Close genetic relationships in vast territories: autosomal and X chromosome Alu diversity in Yakuts from Siberia.

Twelve autosomal and 8 X chromosome Alu markers were genotyped for the first time in 161 Central and West Yakuts to test their ability to reconstruct the genetic history of these populations, the northernmost Turkic-speaker ethnic group living in Siberia. Autosomal data revealed that both groups showed extremely close genetic distances to other populations of Siberian origins that occupied areas from Lake Baikal, the ancestral place of origin of Yakuts, to North Siberia, their current territories. Autosomal and X chromosome data revealed some discrepancies on the genetic differentiation and the effective sizes of Central and West Yakuts. Such discrepancies could be related to the patrilineal and occasionally polygamous structure of these populations. Autosomal and X Alu markers are informative markers to reconstruct population past demography and history, but their utility is limited by the available data. This study represents a contribution for further investigations on these populations.

Molecular identification of bacteria by total sequence screening: determining the cause of death in ancient human subjects.

Research of ancient pathogens in ancient human skeletons has been mainly carried out on the basis of one essential historical or archaeological observation, permitting specific pathogens to be targeted. Detection of ancient human pathogens without such evidence is more difficult, since the quantity and quality of ancient DNA, as well as the environmental bacteria potentially present in the sample, limit the analyses possible. Using human lung tissue and/or teeth samples from burials in eastern Siberia, dating from the end of 17(th) to the 19(th) century, we propose a methodology that includes the: 1) amplification of all 16S rDNA gene sequences present in each sample; 2) identification of all bacterial DNA sequences with a degree of identity ≥ 95%, according to quality criteria; 3) identification and confirmation of bacterial pathogens by the amplification of the rpoB gene; and 4) establishment of authenticity criteria for ancient DNA. This study demonstrates that from teeth samples originating from ancient human subjects, we can realise: 1) the correct identification of bacterial molecular sequence signatures by quality criteria; 2) the separation of environmental and pathogenic bacterial 16S rDNA sequences; 3) the distribution of bacterial species for each subject and for each burial; and 4) the characterisation of bacteria specific to the permafrost. Moreover, we identified three pathogens in different teeth samples by 16S rDNA sequence amplification: Bordetella sp., Streptococcus pneumoniae and Shigella dysenteriae. We tested for the presence of these pathogens by amplifying the rpoB gene. For the first time, we confirmed sequences from Bordetella pertussis in the lungs of an ancient male Siberian subject, whose grave dated from the end of the 17(th) century to the early 18(th) century.

Population genetics of 17 Y-chromosomal STR loci in Yakutia.

Haplotype and allele frequencies of 17 Y-chromosomal short tandem repeat (Y-STR) markers in a population sample of 133 Yakut male volunteers from two regions: Central (n=41) and Western Yakutia (n=92) were determined using the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems). A total of 65 haplotypes were identified in the Yakut population, with 15 haplotypes in Central sample and 54 haplotypes in Western sample. Haplotype diversity values of 0.79 and 0.96, and average gene diversity values of 0.14 and 0.41 were calculated for Central and Western samples, respectively. The Fst distances between both our Yakut populations with other Russian, Siberian and Chinese populations were represented by MDS plot. The graphical view demonstrated close distances between most Yakut populations and differences with other Siberian populations.