It is believed that shortly after the night of July 16th, 1918, Tsar Nicholas II, his wife, Tsarina Alexandra, their four daughters, Olga, Tatyana, Maria and Anastasia and their only son Alexei, were herded into the cellar together with three of their servants and the family doctor, Eugene Botkin. They were all shot by the Bolshevik firing squad, although a number of the victims were allegedly stabbed to death when gunfire failed to kill them.

The bodies were placed onto a truck with the intention of disposing them down a mine shaft. However, the truck broke down during the trip to the mine. The Bolsheviks reportedly dug a shallow pit and buried the bodies.

After referring to archival materials and photographs, which gave an indication of a burial site, two Russian amateur historian investigators, Gely Ryabov and Alexander Avdonin, announced that they had discovered a communal grave approximately 20 miles from Ekaterinburg. Consequently, the Russian government authorized an official investigation coordinated by the Chief Forensic Medical Examiner of the Russian Federation.

The grave consisted of a shallow pit (less than a yard deep) and contained human skeletal remains. Many of the bones were badly damaged. All of the skeletons showed evidence of violence before death. Some of the skulls had bullet wounds. Facial areas of the skulls were destroyed, rendering classical facial identification techniques difficult.

The Chief Forensic Medical Examiner has requested your assistance in verifying the authenticity of the remains. It is believed that eleven (11) individuals were buried by the Bolsheviks (the Romanov family, 3 servants and their doctor) How many skeletons were found in the pit? Who was buried? Is this the grave of the Romanov family? Some think that Anastasia and Alexei survived the family's assassination. What evidence can you gather about whether Anastasia's body was in the pit?

Analyze the evidence provided to provide proof of your answers

Normal genomes contain many extremely variable regions. These regions of the genome can have a specific sequence of nitrogenous bases repeated any number of times. We can often trace an individual's genetic sequence to their parents by comparing the number of copies of a genetic sequence in a region. For every chromosome we have, we get one copy of that chromosome from our mother and one copy from our father. Therefore, we expect that, given any two specific variable regions of the genome, one of our chromosomes will have the same number of repeats as our father and one will have the same number of repeats as our mother. Using this information, can you establish (from the table below) who are the two parents of the three children found in the pit?

Skeleton	Tandem Repeats	Length of Femur (cm)	Length of Humerus (cm)	Length of Radius (cm)
1	9, 10	44.79	31.5
2	6, 10	37.64	26.15
3 (Child)	8, 10	38.9	27.1
4	7, 10	42.5	30.0
5 (Child)	7, 8	38.9	27.21	20.34
6 (Child)	8, 10	37.6	26.2	23.0
7	8, 8	39.4	27.40
8	6, 9	38.32	26.68
9	6, 6	37.32	25.92

Use Microsoft® Excel to find the approximate height of all of the individuals found in the grave. You will need these equations:

• 2.38(femur length) + 61.41 cm = height in centimeters +/- 3.27
• 3.08 (humerus length) + 70.45 = height in centimeters +/- 4.05
• 3.78 (radius length) + 79.01 = height in centimeters +/- 4.32

Mitochondrial DNA is often used in forensic investigation to determine relationships between individuals. Many investigators believe that mitochondrial DNA is only passed down through the mother (but see Zouros, et al., 1992, and Ankel -Simons & Cummins, 1996). If we assume that mitochondrial DNA is inherited through the maternal line, we can gather more information about the relationships of the individuals in the burial site. Use the handout (see link below) to figure out relationships based on mitochondrial DNA sequences. On your handout, when the base present is the same as the reference sequence, this is denoted with a dash (-). If a different base is present, it is given.

Questions:

1. Which skeleton(s) can be immediately ruled out as a possible parent of the other children?
2. Which pair of adults could be the parents of all three children?
3. Given that Anastasia was 5'4 3/4", do you think that her skeleton is among those found in the pit?
4. Assuming that the Tsar is taller than the Tsarina, how tall is the Tsar?
5. Using the mitochondrial DNA evidence from the handout (Adobe PDF Reader required), can you tell who is the tsar? Who is the tsarina? Which skeletons are the three maids and the doctor?

When you are finished, search the Internet and see if you can find out the truth. Who was actually found in the pit?