Although discovered before, the Y-SNP (Single Nucleotide Polymorphism of the Y-chromosome) Z-381 is a useful Y-DNA polymorphism that was taken into consideration in the 1000 Genomes Project (Rocca et al., 2012) . It was intensively studied for the frequencies by the Larmuseau group in West-European populations (Larmuseau et al., 2012, 2013a) .
Furthermore, it was shown (Larmuseau et al., 2013b) , studying three living male members of the Bourbon family (Axel of Bourbon-Parma, Sixte-Henri of Bourbon-Parma, and Joao Henrique of Orléans-Braganza), that the Z-381 DNA marker is the Y-SNP characteristic of the French Bourbon patrilineal lineage.
The goals of the present study are: 1) To construct an accurate Z-381 allele map frequency (Lucotte, 2015a) of West-Europe; 2) To show that Louis XVI, the last French King, is Z-381+; 3) To verify that Sixte-Henri Bourbon-Parma (S-HB-P) is well Z381+ and to give a corrected version of his complete Y-STRs (Short Tandem Repeats of the Y-chromosome) profile; taking S-HB-P as the living representative individual of the French Bourbon patrilineal lineage, we have explored other Y-SNP DNA markers located under Z-381.
2. Subjects and Methods Used
The population sample (16 populations) consisted of 1118 unrelated adult males from West-Europe (Table 1); they originate from 13 countries. All samples of blood were collected from volunteer donors, with informed consent; their classifications were based on their grandfather’s birthplaces. The geographic locations of the populations analysed are shown in Figure 1.
Genomic DNA of volunteer donors was extracted from whole blood by a classic method (Gautreau et al., 1983) . At least 5 µg of genomic DNA were restricted with Taq I enzyme, separated by electrophoresis on a 1.5% agarose gel, transferred to Hybond N+ membranes by the Southern blot (SB) method, and hybridized with two probes (p49f and p49a) according to Lucotte et al. (1994) ; the Taq I fragments (most of which being male-specific) permit to detect those of the volunteers who are of the haplotype XV (=haplogroup M269).
Table 1. Geographic characterization of the sixteen populations studied (N: numbers of subjects in each population).
Figure 1. Sampled populations (population numbers refer to table 1) and their locations in West-Europe in this map, country (or main region) outlines are drawn approximately.
By PCR (Polymerase Chain Reaction) it is possible, using the Family Tree DNA (FT-DNA) procedure (https://www.geni.com/projects/R-U106-Y-DNA/12031) to detect those of them that are of the R-S21 = U106 haplogroup. Genotypes at Z-381 (Z381− or Z381+) were also determined (Figure 2) using the method of FTDNA, according to the instructions given by the Company.
Y haplotype and SNPs maps were realized with the Spatial Analyst program (Arcview software) using the classical Kringing procedure (Dieterlen & Lucotte, 2005) . We used the inverse distance weighting (IDW), which performs well with scarce data. The IDW method was computed for the five nearest neighbours (the grid has 250 rows and 355 columns) and we used a power of 2 (so that the influence is greater at large distance than with a high power).
The Z-381 genotype was tested on genomic DNA extracted from Louis XVI hairs (Lucotte et al., 2016) . Z-381 genotype and the complete Y-STRs profile of S-HB-P were obtained, after his informed consent, according to the FT-DNA procedure.
In the determination of Y-SNP markers of S-HB-P located under Z-381, the terminal Y-SNP detected that we obtained is DF-98 (Figure 2 shows also the PCR method we used for genotyping DF98).
3. Haplotype XV, Haplogroup R-S21 and Z-381 Isofrequency Maps in West-Europe
Table 2 gives numbers of donors being of haplotype XV, of haplogroup S21 (=U106) and who are Z381+ (who are positive in the PCR reaction for the Z381 target indicated on the above Figure 2) in each population.
Table 2. Numbers (and %) of donors being of haplotype XV, of haplogroup S21 and Z381+ in each population.
Figure 2. Above: genotyping (primers and DNA sequence) for R-381. Below: genotyping (primers and DNA sequences) for DF98 (according to FT-DNA).
Figure 3 shows the isofrequency map of haplotype XV in West-Europe; there is a slight cline of decreasing haplotype XV frequencies from West to East; that corresponds to the general cline of decreasing haplotype XV European frequencies already described (Lucotte & Hazout, 1996; Lucotte & Loirat, 1999) . This figure shows also the isofrequency map of haplogroup S21 that is, corresponding to that reported previously (Lucotte, 2015a) , more packty in distribution: S21 frequencies are more elevated in the northern continental part of Europe (corresponding to Belgium, the Netherlands, Denmark and the North of Germany) than that in the peripheral southern countries.
The Z381+ isofrequency map (Figure 4) accentuates this last distribution, Z381+ frequencies being slightly lower in the French Belgian side than in that of the Netherland side of the country. So we confirm, on a more rational basis, results previously obtained (Larmuseau et al., 2013a) concerning the general pattern of Z381+ frequencies already described for six Belgium selected regions.
4. Louis XVI Is Z381+
We have obtained previously the genomic DNA of the King Louis XVI (Lucotte et al., 2016) . Testing for Z-381on his DNA shows that Louis XVI is Z381+. Figure 5 represents that on the simplified masculine genealogy of the French Bourbons, together with the Z381+ status of three living Bourbons already tested (Larmuseau et al., 2013b) .
5. The Y-STRs Profile of Sixte-Henri of Bourbon-Parma
We confirm that Sixte-Henri of Bourbon-Parma (S-HB-P) is Z381+. Table 3 shows the Y-STRs profile of S-HB-P), compared to that already obtained (Larmuseau et al., 2013b) . Our own study concerns 58 Y-STR markers, for a total of 67 allele values; it represents a complete Y-STRs profile. We consider that this profile is the characteristic of one of that we name “the living French Bourbon of reference”.
Figure 3. Above: isofrequency map of haplotype XV. Isofrequency lines indicate the artificial limits of the areas with various nuances of blue (white rectangle: no data available). Below: isofrequency map of haplogroup S21. Isofrequency lines indicate the artificial limits of the areas with various nuances of brown to pink.
Figure 4. Isofrequency map of Z381+. Isofrequency lines indicate the artificial limits of the areas with various nuances of black to grey.
Figure 5. Masculine genealogy of the French Bourbons and relatives, descending from the common male ancestor Louis XIII. The propositus Louis XVI is arrowed (the three living Bourbons already tested are represented as open squares).
Table 3. The complete Y-STRs profile (allele values are determined by the use of the FT-DNA kit Y-DNA-37) of Sixte-Henri of Bourbon-Parma (ND: allele value not determined). The three allele values in italics do not correspond to those reported previously in Larmuseau et al. (2013b) .
Comparison of 37 allele values (for 30 Y-STR markers) so obtained to those previously published shows that there are three mistakes in the initial study: allele value = 12 (instead of 11) at Y-GATA-H4, allele value = 19 (instead of 15) at DYS607, and allele value = 18 (instead of 13) at DYS442. The two last of them (differing from 4 and 5 units, respectively) cannot be considered as simple technical errors.
6. Y-SNPs of Sixte-Henri of Bourbon-Parma Located under Z-381
With a first set of detection for S-HB-P genomic DNA ‘Y-SNP markers located under Z-381, we found that it is U198−, L47− and L48−; this excludes that it be part of this corresponding Z-301 sub-lineage of Z-381. On the other hand, it is Z156+; this establishes that it is part of this corresponding Z-381 sub-lineage.
With a second set of detection, we found that it is L1/S26−; but it is Z306+, Z305+ and Z304+. With a third set, it is DF96−and DF98+. This last result is of some importance: Sixte-Henri of Bourbon-Parma, being R-DF98, is so identified through DNA as being of the “King Cluster” (Little, 2012) .
Table 4 summarizes what we know concerning the main successive Y-SNP markers of the DNA Sixte-Henri of Bourbon-Parma; their corresponding haplogroups, and their approximate ages (according to Iain Mc Donald) are also given in the table.
Prince Sixte-Henri of Bourbon-Parma is a highly representative living descendant of the royal French family of Bourbons. We establish here his complete (based on fifty-eight different Y-STR markers and sixty-seven corresponding allele values) Y-STRs profile.
We also show in the present study that the main successive SNP markers of his Y-chromosome are, in order: M269 (haplotype XVI) > U106 > Z381 > Z156 > (Z306, Z305 and Z304) and > DF98. This last SNP marker confers to S-HB-P the status of being presently of the R1b1a1a2a1a1c1a1haplogroup.
Being R-DF98, S-HB-P belongs to the King Cluster, becoming to the House of Wettin. The house of Wettin belongs to a clade defined by the following Y-SNP mutations: U106 > Z381 > Z156 > Z306 > Z304 > DF98, and other mutations downstream.
This Y-DNA lineage comes from Prince Albert of Saxe-Coburg and Gotha (1819-1861), who was the husband of Queen Victoria (1819-1901). The paternal Winsor DNA line continues back to Franz Josias (Germany, 1697-1764), John (Elector of Saxonomy, 1468-1532) and further to Dietrich I of Wettin (Germany, 916-976). Numerous royal lineages (https://www.the-kings-son.com/royal_wettin_haplogroup) are from the House of Wettin. The Y-DNA signature from the House of Wettin is characterized as haplogroup R1b-U106, with terminal Y-SNPs Z305 and DF98 (https://www.surnamed.com/?articles=y-dna-of-the-british-monarch).
Table 4. Successive main Y-SNPs of S-HB-P’DNA, their corresponding haplogroups (from ISOGG-R chart, 2018) and ages (in years BC: Before Christ).
Detection of further Y-SNPs downstream (beyond DF98) of S-HB-P’DNA necessitates to obtain the full-length sequence of his Y-chromosome. So the Big Y test procedure, previously used by us in French dynasty to a living descendant of the Emperor Napoléon the First (Lucotte et al., 2015b) , is now in progress to establish the Y-chromosome sequence of Sixte-Henri de Bourbon-Parma.
In conclusion, we have obtained a complete Y-STRs profile of Sixte-Henri of Bourbon-Parma. Like the French King Louis XVI, Prince Sixte-Henri of Bourbon-Parma is Z381+, an Y-SNP that is found at appreciable frequencies in the North of Germany, in Belgium, the Netherlands and Denmark. Exploration of the Sixte-Henri of Bourbon-Parma ‘Y-SNPs located under Z-381 shows that DF98, the main DNA marker of the “King cluster”, is the terminal Y-SNP of his differentiation.
We thank Sixte-Henri of Bourbon-Parma for the donation of his DNA sample. This study is a part of the genetic investigations developed in the Institute of Molecular Anthropology concerning royal families in France. The authors thank very much Camila Wasserbach and Dr. Daniel Edgeworth in making our genetic investigations possible and for their competent advice.