DNA tests confirm that human remains found in Leicester, England, belong to King Richard III. With 527 years passing this is the oldest human identification case of a known individual to date. Walther Parson from the Institute of Legal Medicine, Medical University of Innsbruck, has been invited to join the research group as expert in mitochondrial DNA analysis. Mitochondrial DNA is inherited directly from mother to offspring and can therefore be used to test relatedness between individuals that lived many generations apart.
King Richard III died at the battle of Bosworth on August 22nd 1485 and was buried in the medieval church of the Grey Friars in Leicester. In September 2012 a human skeleton was excavated at the presumed site. The archaeological, osteological and radiocarbon dating evidence were all consistent with the remains being those of Richard III.
Teeth and bone samples were genetically tested at the DNA laboratories of the Universities of Leicester, York, Toulouse and Copenhagen. The DNA results were compared to living descendants of Richard III to clarify the identity of the skeleton. Genetic as well as earlier established non-genetic evidence were statistically evaluated.
Genetic analysis of the skeletal remains yielded a male result.
Mitochondrial DNA analyses
Mitochondrial DNA (mtDNA) is inherited from mother to offspring. Relatedness along the maternal lineage can therefore be directly tested. Refernce DNA samples were available from two living individuals: Michael Ibsen and Wendy Duldig, who are 19 and 21 generations removed from King Richard III, respectively.
MtDNA testing resulted in a complete match of the entire mitochondrial genome (approx. 16,570 nucleotides) between the skeleton (total of 16 samples) and Michael Ibsen’s mtDNA. Wendy Duldigs mtDNA differed only at a single position to that of Michael Ibsen (and the skeleton). This position (8994) has been observed to mutate frequently during human evolution.
For statistical evaluation of the mtDNA results the internationally renowned mtDNA database EMPOP was used. EMPOP has been developed and maintained by Walther Parson at the Institute of Legal Medicine in Innsbruck. The database is also used as quality control tool in forensic science.
The so-called control region (approx. 1,100 nucleotides) is the relevant portion of the analyzed mtDNA for statistical evaluation, as population data are available for this region in EMPOP. The control region haplotype of the skeleton matches both that from Ibsen and Duldig and has not been observed in EMPOP holding 27,126 data from Europe. It has also not been found in 1,832 partial control region data from the British isles, which indicates that this mtDNA type is rare. This is supporting the evidence that the skeleton is that of Richard III.
Y-chromosomal markers can be used to investigate relatedness along the paternal line, as this genome is inherited from fathers to their sons. For DNA testing reference samples from five living men were available that are 24 to 26 generations removed from King Richard III.
One man was found to have deviant Y-results from the other four. This indicates that a false-paternity event had occurred within the time span considered (four generations).
The skeletal remains yielded Y-results that did not match any of the five living references. Another false-paternity event that may have happened in the 19 generations between Richard III and the ancestor of the five men cannot be excluded. The likelihood for that was estimated as 16% in the present pedigree based on an average false-paternity rate of 1-2% in the general population (conservative assumption).
Statistical evaluation of the results For statistical evaluation likelihoods of the observed data were computed under hypothesis H1 (that the skeleton is Richard III) and under hypothesis H2 (that the skeleton is not Richard III). A likelihood ratio (LR) below 1 favors H2, while an LR exceeding 1 favors H1. Using genetic and non-genetic evidence the overall LR is 6.7 millions and thus strongly supporting, beyond reasonable doubt, that the skeleton is Richard III.