Evaluation of whole-genome enrichment and sequencing of T. pallidum from FFPE samples after 75 years.

The recent developments in genomic sequencing have permitted the publication of many new complete genome sequences of Treponema pallidum pallidum, the bacterium responsible for syphilis, which has led to a new understanding of its phylogeny and diversity. However, few archived samples are available, because of the degradability of the bacterium and the difficulties in preservation. We present a complete genome obtained from a Formalin-Fixed Paraffin-Embedded (FFPE) organ sample from 1947, kept at the Strasbourg Faculty of Medicine. This is the preliminary, proof-of concept study of this collection/biobank of more than 1.5 million FFPE samples and the evaluation of the feasibility of genomic analyses. We demonstrate here that even degraded DNA from fragile bacteria can be recovered from 75-year-old FFPE samples and therefore propose that such collections as this one can function as sources of biological material for genetic studies of pathogens, cancer, or even the historical human population itself.

Genetic evidence suggests a sense of family, parity and conquest in the Xiongnu Iron Age nomads of Mongolia.

In an effort to characterize the people who composed the groups known as the Xiongnu, nuclear and whole mitochondrial DNA data were generated from the skeletal remains of 52 individuals excavated from the Tamir Ulaan Khoshuu (TUK) cemetery in Central Mongolia. This burial site, attributed to the Xiongnu period, was used from the first century BC to the first century AD. Kinship analyses were conducted using autosomal and Y-chromosomal DNA markers along with complete sequences of the mitochondrial genome. These analyses suggested close kin relationships between many individuals. Nineteen such individuals composed a large family spanning five generations. Within this family, we determined that a woman was of especially high status; this is a novel insight into the structure and hierarchy of societies from the Xiongnu period. Moreover, our findings confirmed that the Xiongnu had a strongly admixed mitochondrial and Y-chromosome gene pools and revealed a significant western component in the Xiongnu group studied. Using a fine-scale approach (haplotype instead of haplogroup-level information), we propose Scytho-Siberians as ancestors of the Xiongnu and Huns as their descendants.

The genetic legacy of legendary and historical Siberian chieftains.

Seventeen years of archaeological and anthropological expeditions in North-Eastern Siberia (in the Sakha Republic, Yakutia) have permitted the genetic analysis of 150 ancient (15th-19th century) and 510 modern individuals. Almost all males were successfully analysed (Y-STR) and this allowed us to identify paternal lineages and their geographical expansion through time. This genetic data was confronted with mythological, historical and material evidence to establish the sequence of events that built the modern Yakut genetic diversity. We show that the ancient Yakuts recovered from this large collection of graves are not representative of an ancient population. Uncommonly, we were also able to demonstrate that the funerary preference observed here involved three specific male lineages, especially in the 18th century. Moreover, this dominance was likely caused by the Russian conquest of Siberia which allowed some male clans to rise to new levels of power. Finally, we give indications that some mythical and historical figures might have been the actors of those genetic changes. These results help us reconsider the genetic dynamics of colonization in some regions, question the distinction between fact and myth in national histories and provide a rare insight into a funerary ensemble by revealing the biased process of its composition.

The limitations of kinship determinations using STR data in ill-defined populations.

The likelihood ratio (LR) method is commonly used to determine kinship in civil, criminal, or forensic cases. For the past 15 years, our research group has also applied LR to ancient STR data and obtained kinship results for collections of graves or necropolises. Although we were able to reconstruct large genealogies, some pairs of individuals showed ambiguous results. Second-degree relationships, half-sibling pairs for example, were often inconsistent with detected first-degree relationships, such as parent/child or brother/sister pairs. We therefore set about providing empirical estimations of the error rates for the LR method in living populations with STR allelic diversities comparable to that of the ancient populations we had previously studied. We collected biological samples in the field in North-Eastern Siberia and West Africa and studied more than 800 pairs of STR profiles from individuals with known relationships. Because commercial STR panels were constructed for specific regions (namely Europe and North America), their allelic makeup showed a significant deficit in diversity when compared to European populations, replicating a situation often faced in ancient DNA studies. We assessed the capacity of the LR method to confirm known relationships (effectiveness) and its capacity to detect those relationships (reliability). Concerns over the effectiveness of LR determinations are mostly an issue in forensic studies, while the reliability of the detection of kinship is an issue for the study of necropolises or other large gatherings of unidentified individuals, such as disaster victims or mass graves. We show that the application of LR to both test populations highlights specific issues (both false positives and false negatives) that prevent the confirmation of second-degree kinship or even full siblingship in small populations. Up to 29% of detected full sibling relationships were either overestimated half-sibling relationships or underestimated parent-offspring relationships. The error rate for detected half-sibling relationships was even higher, reaching 41%. Only parent-offspring pairs were reliably detected or confirmed. This implies that, in populations that are small, ill-defined, or for which the STR loci analyzed are inappropriate, an examiner might not be able to distinguish a pair of full siblings from a pair of half-siblings. Furthermore, half-sibling pairs might be overlooked altogether, an issue that is exacerbated by the common confusion, in many languages and cultures, between half-siblings and full siblings. Consequently, in the study of ancient populations, human remains of unknown origins, or poorly surveyed modern populations, we recommend a conservative approach to kinship determined by LR. Next-generation sequencing data should be used when possible, but the costs and technology involved might be prohibitive. Therefore, in potentially contentious situations or cases lacking sufficient external information, uniparental markers should be analyzed: ideally, complete mitochondrial genomes and Y-chromosome haplotypes (STR, SNP, and/or sequencing).

Genetic kinship and admixture in Iron Age Scytho-Siberians.

Scythians are known from written sources as horse-riding nomadic peoples who dominated the Eurasian steppe throughout the Iron Age. However, their origins and the exact nature of their social organization remain debated. Three hypotheses prevail regarding their origins that can be summarized as a "western origin", an "eastern origin" and a "multi-regional origin". In this work, we first aimed to address the question of the familial and social organization of some Scythian groups (Scytho-Siberians) by testing genetic kinship and, second, to add new elements on their origins through phylogeographical analyses. Twenty-eight Scythian individuals from 5 archeological sites in the Tuva Republic (Russia) were analyzed using autosomal Short Tandem Repeats (STR), Y-STR and Y-SNP typing as well as whole mitochondrial (mtDNA) genome sequencing. Familial relationships were assessed using the Likelihood Ratio (LR) method. Thirteen of the 28 individuals tested were linked by first-degree relationships. When related, the individuals were buried together, except for one adult woman, buried separately from her mother and young sister. Y-chromosome analysis revealed a burial pattern linked to paternal lineages, with men bearing closely related Y-haplotypes buried on the same sites. Inversely, various mtDNA lineages can be found on each site. Y-chromosomal and mtDNA haplogroups were almost equally distributed between Western and Eastern Eurasian haplogroups. These results suggest that Siberian Scythians were organized in patrilocal and patrilineal societies with burial practices linked to both kinship and paternal lineages. It also appears that the group analyzed shared a greater genetic link with Asian populations than Western Scythians did.

Determined about sex: sex-testing in 45 primate species using a 2Y/1X sex-typing assay.

Sex-testing using molecular genetic technique is routinely used in the fields of forensics, population genetics and conservation biology. However, none of the assay used so far allows a non-ambiguous and successful sex determination for human and non-human primate species. The most widely used method, AMELY/X, and its alternatives suffer from a set of drawbacks in humans and can rarely be used in New World primate species. Here, we designed a new sex-typing assay using a multiplexed PCR amplification of UTX and UTY-homologous loci and combined male-specific SRY locus. This method was successfully tested on 1048 samples, including 82 non-human primates from 45 Anthropoidea and Lemuriformes species and 966 human samples from 24 populations (Africans, Europeans, and South Americans). This sex-typing method is applicable across all primate species tested from Hominoidea to Indriidae, and also on various populations with different background origins; it represents a robust and cheap sex-typing assay to be used both by the anthropologist and primatologist communities.

The ancient Yakuts: a population genetic enigma.

This study is part of an ongoing project aiming at determining the ethnogenesis of an eastern Siberian ethnic group, the Yakuts, on the basis of archaeological excavations carried out over a period of 10 years in three regions of Yakutia: Central Yakutia, the Vilyuy River basin and the Verkhoyansk area. In this study, genetic analyses were carried out on skeletal remains from 130 individuals of unknown ancestry dated mainly from the fifteenth to the nineteenth century AD. Kinship studies were conducted using sets of commercially available autosomal and Y-chromosomal short tandem repeats (STRs) along with hypervariable region I sequences of the mitochondrial DNA. An unexpected and intriguing finding of this work was that the uniparental marker systems did not always corroborate results from autosomal DNA analyses; in some cases, false-positive relationships were observed. These discrepancies revealed that 15 autosomal STR loci are not sufficient to discriminate between first degree relatives and more distantly related individuals in our ancient Yakut sample. The Y-STR analyses led to similar conclusions, because the current Y-STR panels provided the limited resolution of the paternal lineages.

Comparing chromosomal and mitochondrial phylogenies of the Indriidae (Primates, Lemuriformes).

The Malagasy primate family Indriidae comprises three genera with up to 19 species. Cytogenetic and molecular phylogenies of the Indriidae have been performed with special attention to the genus Propithecus. Comparative R-banding and FISH with human paints were applied to karyotypes of representatives of all three genera and confirmed most of the earlier R-banding results. However, additional chromosomal rearrangements were detected. A reticulated and a cladistic phylogeny, the latter including hemiplasies, have been performed. Cladistic analysis of cytogenetic data resulted in a phylogenetic tree revealing (1) monophyly of the family Indriidae, (2) monophyly of the genus Avahi, (3) sister-group relationships between Propithecus diadema and Propithecus edwardsi, and (4) the grouping of the latter with Indri indri, Propithecus verreauxi, and Propithecus tattersalli, and thus suggesting paraphyly of the genus Propithecus. A molecular phylogeny based on complete mitochondrial cytochrome b sequences of 16 species indicated some identical relationships, such as the monophyly of Avahi and the sister-group relationships of the eastern (P. diadema and P. edwardsi) to the western Propithecus species (P. verreauxi, Propithecus coquereli, and P. tattersalli). However, the main difference between the molecular and cytogenetic phylogenies consists in an early divergence of Indri in the molecular phylogeny while in the chromosomal phylogeny it is nested within Propithecus. The similarities and differences between molecular and cytogenetic phylogenies in relation to data on the species' geographic distributions and mating systems allow us to propose a scenario of the evolution of Indriidae. Chromosomal and molecular processes alone or in combination created a reproductive barrier that was then followed by further speciation processes.

Comparing chromosomal and mitochondrial phylogenies of sportive lemurs (Genus Lepilemur, Primates).

In recent years several new sportive lemur species (genus Lepilemur) have been described. In contrast to other lemur taxa, the genus shows comparatively high chromosomal variability, which, in addition to molecular data, can be used to infer phylogenetic relationships within the genus. By comparing R-banding patterns and fluorescence in-situ hybridization data, we detected chromosomal rearrangements that occurred during speciation within the genus. The analysis of these data with cladistic methods resulted in a dichotomic phylogenetic tree comparable to that obtained from mitochondrial sequence data. However, a phase of reticulation can not be excluded from the evolution of Lepilemur. Although some incongruences were detected, both phylogenies show similar patterns concerning relationships of the basal and terminal splits. We therefore hypothesize that both, chromosome rearrangements and molecular mutations, alone or in combination, contributed to the speciation process in sportive lemurs.

Molecular phylogeny and taxonomic revision of the sportive lemurs (Lepilemur, Primates).

BACKGROUND: The number of species within the Malagasy genus Lepilemur and their phylogenetic relationships is disputed and controversial. In order to establish their evolutionary relationships, a comparative cytogenetic and molecular study was performed. We sequenced the complete mitochondrial cytochrome b gene (1140 bp) from 68 individuals representing all eight sportive lemur species and most major populations, and compared the results with those obtained from cytogenetic studies derived from 99 specimens. RESULTS: Interspecific genetic variation, diagnostic characters and significantly supported phylogenetic relationships were obtained from the mitochondrial sequence data and are in agreement with cytogenetic information. The results confirm the distinctiveness of Lepilemur ankaranensis, L. dorsalis, L. edwardsi, L. leucopus, L. microdon, L. mustelinus, L. ruficaudatus and L. septentrionalis on species level. Additionally, within L. ruficaudatus large genetic differences were observed among different geographic populations. L. dorsalis from Sahamalaza Peninsula and from the Ambanja/Nosy Be region are paraphyletic, with the latter forming a sister group to L. ankaranensis. CONCLUSION: Our results support the classification of the eight major sportive lemur taxa as independent species. Moreover, our data indicate further cryptic speciation events within L. ruficaudatus and L. dorsalis. Based on molecular data we propose to recognize the sportive lemur populations from north of the Tsiribihina River, south of the Betsiboka River, and from the Sahamalaza Peninsula, as distinct species.

Placenta-specific INSL4 expression is mediated by a human endogenous retrovirus element.

The human insulin-family genes regulate cell growth, metabolism, and tissue-specific functions. Among these different members, only INSL4 gene shows a predominant placenta-specific expression. Here, we show that the human INSL4 gene is tightly clustered with three other members of the human insulin superfamily (RLN1, RLN2, and INSL6) within a 176-kilobase genomic segment on chromosome region 9p23.3-p24.1. We also report evidence that INSL4 is probably the only insulin-like growth factor gene to be primate-specific. We identified an unexpected human endogenous retrovirus (HERV) element inserted into the human INSL4 promoter with a sequence similar to that of env gene, flanked by two long terminal repeats(LTRs). The emergence of INSL4 gene and genomic insertion of HERV appear to have occurred after the divergence of New World and Old World monkeys ( approximately 45 million years ago). Transient transfection experiments showed that the placenta-specific expression of INSL4 is mediated by the 3' LTR of the HERV element, and that the latter may have a major role in INSL4 up-regulation during human cytotrophoblast differentiation into syncytiotrophoblast. Finally, we identified an INSL4 alternatively spliced mRNA species that encodes putative novel INSL4-like peptides. These data support the view that ancient retroviral infection may have been a major event in primate evolution, especially in the functional evolution of the human placenta.

Asymmetrical morphogenesis and medio-lateral positioning of molars during mouse development.

The functionality of the dentition depends on occlusal relationships between opposing crown surfaces. To investigate the relative changes in positioning of upper and lower molar germs during mouse development, we used serial histological sections of late day 13 (embryonic day (ED)13.5) to early day 18 (ED18) foetus heads and performed computer-aided 3D reconstructions. From ED13.5 to ED15.5. the first lower molar (M1) got a less medial position relative to its upper counterpart (M1); superimposition progressed postero-anteriorly. From ED14.5, the apparent medial displacement of M(1) vs. M1 was partly due to the asymmetrical growth of the M(1) to give rise to the lingual row of cusps, conspicuous at ED17. The superimposition of M(2)/M2 along the medio-lateral axis was observed from their bud stage (ED14.5), and the one of M(1)/M1 was almost complete at ED15.5. However, this was not the final position. as at ED 18, M1 and M2 had a more lateral location than their upper counterparts. Immunostaining showed that differential expression of antigens associated to desmosomes but not to adherens junctions might be involved in the asymmetrical development of M(1) thus contributing to the relative medio-lateral positioning of the first molars at early stages.

Phylogenetic relationships between Hapalemur species and subspecies based on mitochondrial DNA sequences.

BACKGROUND: Phylogenetic relationships of the genus Hapalemur remains controversial, particularly within the Hapalemur griseus species group. In order to obtain more information on the taxonomic status within this genus, and particularly in the cytogenetic distinct subspecies group of Hapalemur griseus, 357 bp sequence of cytochrome b and 438 bp of 12S mitochondrial DNAs were analyzed on a sample of animals captured in areas extending from the north to the south-east of Madagascar. This sample covers all cytogenetically defined types recognized of the genus Hapalemur. RESULTS: Phylogenetic trees and distances analyses demonstrate a first emergence of Hapalemur simus followed by H. aureus which is the sister clade of the H. griseus subspecies. Hapalemur griseus is composed of 4 subspecies separated into two clades. The first contains H. g. griseus, H. g. alaotrensis and H. g. occidentalis. The second consists of H. g. meridionalis. A new chromosomal polymorphic variant from the region of Ranomafana, H. griseus ssp, has been analysed and was found in both clades. CONCLUSIONS: Our results support the raising of H. g. meridionalis to the specific rank H. meridionalis, while neither cytogenetic nor molecular evidences support the raising of H. g. alaotrensis to a species rank despite its morphological characteristics. The new cytotype H. g. ssp which has been previously characterized by cytogenetic studies contains animals clustering either with the group of Hapalemur griseus griseus or with that of Hapalemur meridionalis. This suggests the existence of an ancestral polymorphism or an introgression of mitochondrial DNA between subspecies.