The DNA Y-STRs Profile of Louis XVI ( 1754-1793 )

We have obtained, for the first time, the Y-STRs profile of the King of France Louis XVI (1754-1793). His genomic DNA was extracted from his authentic hairs, that were studied by optic and electronic microscopy. Louis XVI’s Y-STRs profile is very similar to those of three living Bourbons previously published , differing from them by three Y-STRs allele values only. Dating estimates of the divergence time of the common ancestor (Louis XIII) between Louis XVI and the living Bourbons correspond to the observed genealogical time. K.W. Naundorff, the famous pretender, is certainly not a Louis XVI’s natural son.


INTRODUCTION
In 1793, during the French Revolution, the King of France Louis XVI (1754-1793) and Queen Marie-Antoinette (1755-1793) were beheaded. Before their deaths, both the King and the Queen remained imprisoned in the Temple (in Paris). The only surviving son of Louis XVI and Marie-Antoinette, Louis-Charles (born in 1785), remained also imprisoned in the Temple where he survived to the death of his parents ; he (officially) died in the Temple in 1795.
So, the establishment of the Y-STRs profile (profile of the Y-Chromosome based on the DNA genetic markers named Short Tandem Repeats) of Louis XVI remains a difficult work, depending crucially on the authenticity of the starting material. The first attempt to obtain Louis XVI's Y-STRs profile (1) by the Lalueza-Fox group was quickly contradicted by a further study (2)

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But the real Y-STRs profile of Louis XVI remains unknown until now. In the present study we find the Y-STRs profile of Louis XVI, based on his authentic hairs ; the methodology used here is similar to the one we adopted recently (4)for the study of K.W.
Naundorff (1785?-1845), who was the most famous pretender to Louis XVI's throne. Table 1 summarizes datas about all Bourbons subjects and allied (and about the pretender) under study.

The hairs
The lock of hairs (designated as number 2) is attached to a cardboard located in a glass-frame, comprising from top to bottom ( Figure 2) : a drawing (designated as number 1) showing the left profiles of Louis XVI, Marie-Antoinette and the Dauphin ; a small envelope (designated as number 3) ; a folded portion of the tie (designated as number 4) ; a large envelope (designated as number 5) ; a red seal of sealing wax (designated as number 6) representing Louis XVI's right profile, and located (designated as number 7) on another portion of the tie.
The small envelope contained the lock of hair, because it is written on its surface : "cheveus du Roi et de la reine" (see Figure 20). The large envelope contained both the other portion of the tie and the small envelope, because it is written on its surface, on five lines : "doné à toulancravate du Roi -Louis XVIet cheveus du Roiet de la reine." The texts are drawn up in old french, because there is one n in the word donné (given) and a s, instead of a x, at the end of the word cheveus (hairs). In both texts the word Roi (King) is written with the letter r in capital, while the word reine (Queen)that is not namedis written with the letter r in small letter ; that corresponds to some conventions used at that time.
Interestingly, we know that Toulan was a "Conventional", who had the responsibility of the surveillance of the members of the royal family during the imprisonment in the Temple ; he was very grown fond to the King and the Queen (and to the royal family in general) during this period.

Microscopy and elementary analysis
The hairs and other objects were examined and analysed in confocal stereoscopic micrography, and by SEM (Philips XL30 model, environmental version) -EDX (probe Bruker AXS energy dispersive X-ray ; PGT system analysis ; Spirit model, Princeton Gamma technology).

DNA extraction
Three cut portions of the brown hair number 1 containing dandruffs and the whole hair number 2 were submitted to DNA extraction to obtain genomic DNA. The genomic DNA of these hairs was extracted using a standard method (0.5M EDTA, sarcosyl 20% and proteinase K 10mg/ml), and purified using a commercial kit (NucleoSpin + Kit ; Macherey-Nagel, Duren, Germany) in accordance with the manufacturer's instructions (with some modifications)
The experimenter involved in molecular biology experimentations used protective clothing, sterile gloves and facemask, to prevent exogenous contamination. DNA extraction and purification were performed according to our previously protocol (5), in a dedicated laboratory. The laboratory performed DNA typing under strict precautions, following previously published criteria for a-DNA authentication (6). Only one of the authors of this paper (G.L.) proceeded to molecular biology experimentations, and his Y-STRs profile is known.

Dating
Time estimation for each chromosomal lineage was made using the ASD (Average Squared Distance) method of Goldstein et al. , 1995 (7) on the 15 Y-STRs (excluding DYS385. a and .b) ; this method is based on a strict single stepwise mutation model. The set of Y-STR mutation rates we applied in the estimations is that of Burgarella and Navascuès, 2011 (8). We used the generally accepted generation time of 25 years, that corresponds to that we observe in the tree (Figure 1), to produce a time estimate in years. Figure 3 shows optical views of a cut up portion of the tie ; it is an "armure-simple" cloth, threads being disposed in the mode 1-2-1 relatively to weft threads. Both sorts of threads are composed of linen fibres, but there is evidence of some cotton fibres ( Figure 4) disposed externally to linen threads. Morphologically, both sorts of fibres are easily distinguishable ( Figure 5) : the linen fibres are cylindric, with nodes regularly spaced along the fibre length ; cotton fibres, flatter, are short with pointed extremities (and there are characteristic twists along the fibres). The same results were obtained for a cut up portion of the tissue (part 7, in Figure 2). Figure 6 shows two photographs (in optical and in SEM) of the upper-left corner of the small envelope paper. As shown in Figure 7, this paper is a "papierchiffon", where we can distinguish linen and hemp fibres. This sort of luxurious paper was used in the past for the writing of official and fiduciary documents. The same results were obtained for a cut up portion of the large envelope ( Figure 2). Figure 8 shows optical views of two ink blots (number 1 and 2), located on the top of the small envelope paper. Examined in retrodiffused SEM ( Figure 9) the ink blot number 1 is constituted of a set of bright particles (this indicating that they are constituted of heavy elements). EDX analysis of one of them ( Figure 10) shows that the ink is a "ferrogallic" ink, iron sulphate particles added darkening again the blackness of the organic matter constituting the ink. This sort of ferro-gallic ink was commonly used in Europe, from the Middle-Age to the end of the 18 th Century (and even after).

The hairs
One of the difficulties of such a study is to recognize hairs belonging to the King from the Queen's hairs, because the lock of hair ( Figure 2) contains both these hairs (as indicated : "cheveus du Roi et de la reine").
So we have analysed separately blond hairs and brown hairs. Three blond hair portions (numbers 1, 2 and 3) are represented on the optical photography of Figure 11 ; in the enlarged photography of hair number 3, we can observe (by transparency) the medullar canal of the hair. All the blond hairs observed are cut at both extremities. Figure 12 shows a SEM photography of the extremity of the of the blond hair number 3 ; its elementary composition is characteristic of one hair (C : carbon, and O : oxygen represent the organic matter of the hair; S: sulphur peak corresponds to the keratin; Cl: chloride and Na: sodium correspond to sweat salt). The mean feature of blond hairs is that they are very clean: we can observe only very few deposit particles at the scale surface; so they correspond to well prepared washed and cleaned hairs. Figure 13 shows residual particles of the "black soap" potassium chloride that was used in the cleaning process.
Three brown hairs (numbers 1,2 and 3) are represented on the optical photography of Figure 14. All the brown hairs observed are also cut at both extremities.
Both extremities of the cut brown hair number 1 are shown on the SEM photographs of figure 15. A surface portion of brown hair number 1 is shown on the MEB photography of Figure 16. We can distinguish at this surface a great number of mineral deposits particles (from D1 to D17); the mean feature of brown hairs-contrary to blond hairs-is that they are "dirty".
Numerous deposits of particles can be observed at the surface of hair number 1 (figure 17): they can correspond to modern deposits of synthetic fibres, spores and even acari egg-layings, or mineral deposits of calcium carbonate (Figure 17'

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Among the mineral (and metallic) deposits shown on Figure 17, EDX spectrum analysis (figure 18) establishes that one particle (number10) is a clay, another one (number 9) is a cement, and a last one (number 8) is a titanium dioxide metallic microplaque.
The SEM photography of a surface portion of hair number 1 (Figure19) shows a dandruff. We can observe a total of five dandruff particles along the surface of hair number 1. Table 2 gives the main features (observations based on 10 hairs of each category) of blond and brown hairs. And we can attribute brown hairs to Louis XVI ; they were cut during his imprisonment in the Temple ( "cheveux de coiffeur"). We know that, at this time, his hairs were brown (and bleach). It is secondarily that the two sorts of hairs were mixed, in the lock of hair under study ( Figure 2).
It is possible to extract genomic DNA from hairs with bulbs (5, 10). On cut hairs, it is relatively easy to obtain mitochondrial-DNA (mt-DNA) only (11) ; but it is possible to obtain some large amounts of genomic DNA from dandruffs joined to hairs (12).

The Y-STRs profile of brown hairs
It is the same DNA profile ( Table 3) that was obtained, repeatedly, for genomic DNA extracted from the three cut portions of the hair number one and from the hair number 2. Results about amplification of the amelogenin gene show that the man with these hairs is XY. For a total of 15 sites compared (DYS19, DYS385.a and .b, DYS389. I and .b, DYS390, DYS391, DYS392, DYS393, DYS438, Y-GATA-A4, DYS448, DYS456, DYS458 and Y-GATA-H4), the Y-STRs profile of the man bearing brown hairs is greatly similar to those of the TLB : identical allele values are found for 12 of these (a percentage homology of 12/15 = 80%). This profile differs from the Y-STRs profiles of the TLB for three Y-STR allele values only : allele values at DYS391 of 11 versus 10, at Y-GATA-A4 of 13 versus 12, and at DYS456 of 16 versus 17 : moreover these three are one-step mutation only, in the plus sense for DYS391 and for Y-GATA-A4, and in the minus sense for DYS456.
So we conclude that the Louis XVI Y-STRs profile is perfectly compatible to that of the common Bourbon Y-STRs profile previously established (2) ; allele Y-STR values that differ between the two are easily explained by accumulation during time of single-step mutations since the common ancestor Louis XIII (see Figure 1) between the two lineages. The Louis XVI Y-haplotype, submitted to the Wit Athey's Haplogroup Predictor test (9), had more than 95% of chance to be of the sub-haplogroup R1b.

Dating
Because there is a time gap between Louis XVI and the three living relatives, we calculated the ages based on the genetic datas of the three living Bourbons and they plus Louis XVI, respectively (Table 4) ; the TLV and Louis XVI trace their common ancestor to Louis XIII (1601-1643). The former can trace common ancestor to 220 ya by using genealogical mutation rates, and the latter accordingly gives 405 ya. So, the two approaches give similar TMRCA times, the second being relatively more conform to the real time estimate (Figure 1).

DISCUSSION
We have established in the present study, based on his hairs, the Y-STRs profile of Louis XVI. The first problem to take in consideration is that of the authenticity (and the traceability) of the hairs examined.
The small envelope (object 3, Figure 2) contained hairs. We showed here that the paper constituting the envelope is a "papier-chiffon" (a special sort of paper used in the past for the official documents) and that the ink used for the writing is a "ferro-gallic" ink (commonly used at that time). The large envelope (object 5, Figure 2) contained both the tie fragment (object 7, Figure 2) and the small envelope.
On the apparent side of the large envelope (of the same paper as the small envelope) is indicated (same writing as the small envelope, and same ink) both origins of the tie and of the hairs. Toulan is explicitly designated as the second donator ("doné à toulan…") of both the tie and the hairs. We know (13) that the bookseller François-Adrien Toulan (1761-1794), Conventional but supporter of the royal family members (Madame Elisabeth, the King's sister, called him "Fidèle"), broke the seals appended to the last Louis XVI objects (a ring, his hairs and the seal that he intended to give to his son), retrieved these rests and handed them to Marie-Antoinette. The Queen will be further successful in getting these precious relics out of the Temple, and gave them to the Count of Provence and to the Count d'Artois (both the King 's brothers).
We depict on Figure 20 the writing located at the other side of the large envelope paper. It is in fact a letter-paper, where the name and the address of the last addresser ("Madame Teulière, à Montauban") were written. After the lock of hair was left out to the small envelope and was sewn to the cardboard located in the glass-frame, to constitute the object 2 ( Figure  2). The second difficulty surmounted concerns the parting between Louis XVI and Marie-Antoinette 's hair. Probably the mixture between the two sorts of hairs was realized by Toulan himself : he had Louis XVI's hair, and he probably obtained Marie-Antoinette's hair by another way. Hair mixture in a lock between hair coming from two affectively linked persons is not an uncommon procedure used to make these sorts of relics ; for instance we had recently access (Lucotte, unpublished) to a peculiar relic where hair from the Dauphin and the one from his sister Madame Royale were mixed together.
We have separated Louis XVI's hair from the one of Marie-Antoinette on the basis (Table 2) of colour, thickness and general aspect. Contrary to Louis XVI's hair, Marie-Antoinette's blond hair was carefully washed, cleaned and prepared; it was kept separately from Louis XVI 's hair , and it is in a second time that both sorts of hair was mixed. Both sorts of hairs are cut-hairs, that excludes to obtain genomic DNA from them. Fortunately, because Louis XVI hair are "unprepared hair" ; their surfaces are literally covered by numerous deposit particles. It is the dandruffs that adhere to these hairs that allows us to obtain Louis XVI 's Y-STR profile.
Louis XVI 's Y-STR profile obtained here (Table 3) is greatly similar to those of the TLV previously studied (2) : it differs from them by three allele values only, at the Y-STRs DYS391, Y-GATA-A4 and DYS456 (alleles 11 instead of 10, 13 instead of 12, and 16 instead of 17, respectively) ; moreover, these three variant values of Louis XVI's Y-STRs profile differ each from those of the TLV by one-step mutation only. This allows us to estimate securely the divergence time (Table 4) between the two main branches (the one that goes to Louis XVI , and the one that goes to TLV), time (in ya) that corresponds to the observed genealogical time (Figure 1).
We ignore for the moment what is the Y-STRs profile of the common ancestor of these two branches, Louis XIII. But, in a similar manner to the one we have used for Napoléon's Y-STRs reconstruction profile (14)