DNA Lesson Series: The mtDNA and its role in Ancestry
mtDNA Part I - mtDNA 101
mtDNA Part II - Facts about mtDNA
mtDNA Part III - mtDNA Structure
mtDNA Part IV - Ancestral Markers
mtDNA Part V - Detecting Mutations in the mtDNA
mtDNA Part VI - mtDNA Ancestral Markers
mtDNA Part VII - The Cambridge Reference Sequence
mtDNA Part VIII - mtDNA Test Types
mtDNA Part IX - mtDNA Haplogroup Determination
mtDNA Part X - mtDNA Subclades
mtDNA Part XI - mtDNA Haplogroup H
mtDNA Part XII - Subclades of mtDNA Haplogroup H
mtDNA Part XIII - Distribution of Subclades of H
mtDNA Part XIV - Descendents of Maria-Theresa
mtDNA Part XV - Luke the Evangelist
mtDNA Part XVI - Empress Feodorovna  <<– you are here
mtDNA Part XVII - James “Earthquake McGoon” McGovern

In the last few blogs, we have been discussing the topic of how anthropologists have used mtDNA to solve historical questions.  In this blog, we will continue our analysis of historical figures who may have belonged to haplogroup H.

Case #3:  Empress Alexandra Feodorovna of Russia

Who was she?

Empress Alexandra Feodorovna of Russia, the granddaughter of Queen Victoria was born on June 6, 1872 as Princess Alix of Hess and by Rhine in Darmstadt (Germany).  She was the sixth child of Grand Duke Louis IV of Hesse and by Rhine and Princess Alice of the United Kingdom.  She was the princess consort of Nicholas II, and the last Tsaritsa of Russia. 

What were the anthropologists trying to find out? ie why was her DNA tested?

At the end of the February Revolution of 1917, Tsar Nicholas II was forced to abdicate.  The Romanov family were imprisoned in Ipatiev House at Ekaterinburg in the Urals of Central Russia in 1918 with three servants and the family doctor.  On the night of July 16, 1918, the entire Romanov family was shot and killed by the Bolshevik firing squad. 

According to legend, two bodies were burnt and the others were buried in a roadside pit.  To hinder identification, sulphuric acid was said to have been thrown into the open grave and a truck was driven back and forth over the grave. 

The case remained a mystery until July 1991, when nine human skeletons were discovered in a shallow pit around 20 miles from Ekaterinburg, Russia.  Historians speculated that the remains belonged to the Romanov family.  To help solve the mystery, DNA testing was performed to identify the remains.

Who’s who in researching the history of the Romanov family:

• Gill et al. from the Central Research and Support Establishment, Forensic Science Service, Aldermaston, Reading, Berkshire, UK
• Ivanov et al. from Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
• Hagelberg et al. from Department of Biological Anthropology, University of Cambridge, Cambridge, UK
• Wadhams et al. from Armed Forces DNA Identification Laboratory, Office of the Armed Forces medical Examiner, Armed Forces Institute of Pathology, Rockville, Maryland, USA
• Hofreiter et al. from Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
• Loreille et al. from Armed Forces DNA Identification Laboratory, Office of the Armed Forces medical Examiner, Armed Forces Institute of Pathology, Rockville, Maryland, USA
• Knight et al. from Department of Anthropological Sciences, Stanford University, Stanford, USA
• Zhivotovsky et al. from Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
• Kass et al. from Department of Biology, Eastern Michigan University, Ypsilanti, USA
• White et al. from Bioscience Division, Los Alsmos national Laboratory, Los Alamos, USA
• Mountain et al. from Department of Genetics, Standford University, Stanford, USA

Top peer reviewed research publications for Tsarina Alexandra and the Romanov family:

This table lists the most significant papers for the Romanov family and the DNA tests that were performed to identify their remains in peer reviewed journals.  Links are provided to access the original articles.

These papers provide the extent of what is known today about the DNA type of Tsarina Alexandra and provides answers to the question of whether the bodies discovered in the grave belong to the Romanov Family.

Name of Scientific Article	Scientific Journal
Identification of the remains of the Romanov family by DNA analysis.  Gill et al, Forensic Science Service, Berkshire, UK.  Click here to read abstract	Nat Genet. 1994 Feb; 6(2):130-5.
DNA forensics. Buried, recovered, lost again? The Romanovs may never rest.Stone et alThis publication is published in Science magazine, a subscriptions only magazine.  Click here to go to Science magazine.	Science. 2004 Feb 6; 303(5659):753.
Molecular, forensic and haplotypic inconsistencies regarding the identity of the Ekaterinburg remainsKnight et al, Stanford University, Stanford, USA.  Click here to read abstract	Ann Hum Biol.  2004 Mar-Apr; 31(2):129-38.
Ongoing controversy over Romanov remains.Hofreiter et al, Gill et al, Knight et alThis publication is published in Science magazine, a subscriptions only magazine.  Click here to go to Science magazine.	Science. 2004 Oct 15;306(5695):407-8, 408-9, 409-10

As more articles become available, they will be listed here.

Solving the case:  Collecting DNA samples from the Ekaterinburg remains

The first step in solving this case is to test the DNA of the Ekateringburg remains.  After the DNA type of the remains is known, it can be compared to the DNA type of known relatives in the royal family.  Since Tsarina Alexandra is the maternal granddaughter of Queen Victoria (Queen Victoria is her mother’s mother), Tsarina Alexandra must have inherited her mtDNA from Queen Victoria.  A living descendent of Queen Victoria who carries Queen Victoria’s mtDNA type is Prince Philip.  The following diagram shows in purple how the mtDNA is passed down from Queen Victoria to her descendents and which one of her descendents carry her mtDNA:

Individuals who inherited the mtDNA type of Queen Victoria are highlighted in purple.

The descendent tree for Queen Victoria indicates that Tsarina Alexandra should have the same mtDNA profile as Prince Philip as well as other living and deceased descendents from the same line.  By collecting the DNA sample from known family members, scientists can find out the expected mtDNA type for Tsarina Alexandra and her children and use it to identify the remains.

Who was tested to solve this case?

1. A blood sample was collected from Prince Philip, a living descendant of Queen Victoria (maternal grandmother of Tsarina Alexandra).
2. Bone fragments from each of the nine skeletal remains found in Ekaterinburg.

Three Types of DNA tests were performed to confirm the identity of the Ekateringburg remains:

1. Gender determination:  To identify the gender of each of the nine skeletal remains.
2. Autosomal STR testing:  To determine whether the nine skeletons are related to each other, ie in the same family.
3. Mitochondrial HVR1 and HVR2 sequencing:  To examine whether the skeletal remains are members of the Romanov family by comparing the mtDNA profile of the remains to the mtDNA of known royal family members.

Step 1:  Autosomal STR Testing:  To determine whether the 9 skeletons are related to each other

Autosomal STR testing (the same technology used for paternity testing) was used to determine whether the nine skeletons are from the same family. 

Skeleton #	Autosomal DNA Markers					Relationship
	VWA	TH01	F13A01	FESFPS	ACTBP2
3	15, 16	8, 10	5, 7	12, 13	11, 32	Child
4	15, 16	7, 10	7, 7	12, 12	11, 32	Parent
5	15, 16	7, 8	5, 7	12, 13	11, 36	Child
6	15, 16	8, 10	3, 7	12, 13	32, 36	Child
7	15, 16	8, 8	3, 5	12, 13	32, 36	Parent

The results of the autosomal test confirmed the following:

1. Skeletons 3, 4, 5, 6, and 7 belonged to the same family.
2. Skeletons 3, 4, 5, 6 and 7 are likely a family group consisting of three children and both of their parents. 
3. Skeletons 1, 2, 8, 9 are not related to skeletons 3, 4, 5, 6, and 7.

This information is consistent with the skeletons belonging to members of the Romanov family. 

Step 2:  Determining the gender of the skeletons

The next set of tests sets out to determine the gender of the skeletons.

Skeleton #	Sample Type	Sample Quality	Gender	Relationship
3	Bone	Good	Female	Daughter
4	Bone	Good	Male	Father
5	Bone	Good	Female	Daughter
6	Bone	Good	Female	Daughter
7	Bone	Good	Female	Mother

The results of the gender determination tests showed that four of the skeletons are male and one was female.  The combined results of the autosomal test and gender test leads to the speculation that skeleton #4 is the father, skeleton #7 is the mother, and skeletons 3, 5, and 6 are their daughters.

Step 3:  What if the skeletons belonged to the Romanov family?

If the remains truly belong to the Romanov family, then:

• Skeleton 4 “father” should belong to Tsar Nicholas II
• Skeleton 7 “mother” should belong to Tsarina Alexandra
• Skeletons 3, 5, and 6 are three of their 4 daughters

Skeleton #	Relationship	Speculated Identity (if the Romanovs)
3	Daughter	Daughter #1
4	Father	Tsar Nicholas II
5	Daughter	Daughter #2
6	Daughter	Daughter #3
7	Mother	Tsarina Alexandra

The next step is to confirm the speculated identities by comparing each skeleton to living royal family members. 

Step 4:  Solving the mystery.  Confirming the identity of Skeleton #7, Tsarina Alexandra and her daughters, Skeletons 3, 5, and 6

Now that the preliminary studies indicate the possible identities of each of the skeletons, the next step is to confirm the identity by comparing to the DNA of living royal family members.  Tsarina Alexandra is the grandaughter of Queen Victoria, and her mtDNA should be identical to the mtDNA of Prince Philip, a living descendent of Queen Victoria. 

The next step is to test the mtDNA of Prince Philip and compare it to the mtDNA of the skeletons.

Prince Philip’s mtDNA:

Name	HVR1 Region	HVR2 Region	Coding Region	Publication
Prince Philip	16111,16357	263,315.1	Not tested	Gill et al 1994

Prince Philip is a direct descendent of Queen Victoria and he carries the mtDNA type of Queen Victoria. 

The mtDNA of the Skeletons:

Name	HVR1 Region	HVR2 Region	Coding Region	Publication
Skeleton #3:Putative daughter	16111,16357	263,315.1	Not tested	Gill et al 1994
Skeleton #4:Putative Tsar Nicholas II	16126,16169Y,16294,16296	73,263,315.1	Not tested	Gill et al 1994
Skeleton #5:Putative daughter	16111,16357	263,315.1	Not tested	Gill et al 1994
Skeleton #6:Putative daughter	16111,16357	263,315.1	Not tested	Gill et al 1994
Skeleton #7:Putative Tsarina Alexandra	16111,16357	263,315.1	Not tested	Gill et al 1994

 Conclusion:  The results of the mtDNA test confirm the following:

1. The putative Tsarina Alexandra’s mtDNA (skeleton 7) is a perfect match to the mtDNA profile of Prince Philip, indicating that it is the actual skeleton of Tsarina Alexandra.
2. Tsarina Alexandra’s mtDNA is a perfect match to the mtDNA profile of Skeletons 3, 5, and 6, her putative daughters.
3. Skeletons 3, 5, and 6 are also identical to Prince Philip’s mtDNA, indicating that they are the actual skeletons of Tsarina Alexandra’s daughters.
4. The putative Tsar Nicholas II’s mtDNA (skeleton 4) does not match the mtDNA of Prince Philip.  This is expected since Tsar Nicholas II is not a direct descendent of Queen Victoria.  We will discuss the set of analysis that was conducted to confirm the identity of Tsar Nicholas II in a separate blog.

In summary, the DNA studies confirm that five out of the nine skeletons discovered truly belonged to the Romanov family, namely Tsar Nicholas II, Tsarina Alexandra, and 3 of their 4 daughters.  The skeleton of one of their daughters and their only son, Alexei, remain missing.

The Aftermath:  Doubts and Controversies

Since the findings, the skeletons were reburied with honors in the imperial-era capital of St. Peterburg.  The two remaining children remain missing.

The scientific findings and results have since been challenged by Knight et al, suggesting irregularities and sample handling deficiencies.

Recent Findings from the summer of 2007

In the summer of 2007, using metal detectors and metal rods as probes, two burned partial skeletons were discovered at a bonfire site near Ekaterinburg, 900 miles east of Moscow.  15 intact bone fragments and more than 40 pieces of charred bone were discovered.

The site appeared to match the site described in Yurovsky’s (the Bolshevfik officer in charge of the Romanovs’ captivity) memoirs.  According to his memoirs, the bodies of nine victims were doused with sulphuric acid and buried along a road.  Alexei’s body and one of his sister’s bodies was burned and left in a pit nearby

Preliminary analysis of the bones showed that the remains were from a boy roughly between the ages of 10 to 13 years old at the time of death, the putative Alexei and a young woman roughly between the ages of 18 to 23 years old at the time of death, attributed to one of the Romanov daughters, likely Maria or  Anastasia.

Genetic investigations were performed in the Sverdlovsk Regional Forensic Medicine Bureau, a lab in Moscow, and a US laboratory, the University of Massachusettes Medical School. 

On April 30, 2008, the groups claimed that DNA testing (mainly mitochondrial DNA analysis) proved that the remains belonged to Tsarevich Alexei and one of his sisters (likely Maria) were released in several major newspapers and news sources (including New York Times, BBC, and MSNBC).

Article	Year
Bones turn up in hunt for last czar’s son	2007
Probe reopened into death of last Russian czar	2007
DNA tests may solve mystery of last czar’s heirs	2008
Report: DNA proves link to last czar’s kids	2008
Russia dig finds ‘tsar’s family’	2007
Lost Romanov bones ‘identified’	2007
Tsar’s lost children identified	2008
Experts May Have Found Remains of Czar’s Children	2008
Amateurs Unravel Russia’s Last Royal Mystery	2007
DNA Tests Confirm the Deaths of the Last Missing Romanovs	2007

What is the haplogroup of Queen Victoria’s descendents?

Now that the mtDNA type of Queen Victoria’s line is known, amateur genealogists from around the world have used the data to compare to their own families to see if they may have links to royalty.

Although the original researchers never attempted to use the data from the study to determine the haplogroup of this royal family line, amateur genealogists have tried t use the data from the study to determine the mtDNA haplogroup of Queen Victoria’s line, concluding that Queen Victoria and her descendent Tsarina Alexandra belonged to Haplogroup H.  Let’s take a look at the raw data and see how accurate they are.

Step 1:  Click here to download and print the mtDNA Haplogroup map so that you can follow along with the discussion.

Step 2:  Identify the presence and absence of HVR1 markers on the map.

Tsarina Alexandra only had two HVR1 markers, namely 16111 and 16357.  On the map, all HVR1 markers are in blue.  Starting from the CRS, move outwards and cross off all of the HVR1 markers that Tsarina Alexandra does not have, circle the ones that she does have and put a question mark next to the ones that are unknown:

The results of the HVR1 test helps to eliminate the haplogroups that Tsarina Alexandra definitely does not belong to, and shows that Tsarina Alexandra must belong to either Haplogroup R, Pre-HV, HV, H, or the CRS branch of H.

Step 3:  Identify the presence or absence of HVR2 markers on the map:

Tsarina Alexandra has 2 markers in her HVR2 region, namely 263 and 315.1.  On the map, all HVR2 markers are red.  Starting from the CRS, move outwards and cross off all of the HVR2 markers that Tsarina Alexandra does not have and circle all of the HVR2 markers that she does have:

The results of the HVR2 test helps to eliminate Haplogroup R and shows that Tsarina Alexandra must belong to either Haplogroup Pre-HV, HV, or H.  Tsarina Alexandra has marker 263 which brings her away from CRS, indicating that she is unlikely to belong to CRS, but the presence of 263 cannot eliminate her from CRS as such a conclusion woudl require more data from the coding region.

In conclusion, the results of the scientific studies show that Tsarina Alexandra can belong to any one of the following Haplogroups:

• H
• HV
• Pre-HV

These results are similar to those of Marie Antoinette, even though the exact mtDNA mutations that they carry are slightly different.  To confirm whether Tsarina Alexandra is really a member of Haplogroup H, one would need to test the coding region of her mtDNA to confirm that she does not have markers 7028 and 14766.  However, no coding region data was ever available for Tsarina Alexandra or for any of her living or deceased relatives, so there is no way to confirm that she is a member of Haplogroup H.  Since the original researchers were aiming to identify her remains rather than to determine her haplogroup, the coding region was never examined and in this particular case, coding region SNP test is required to confirm her haplogroup.

In conclusion, it is pre-mature to conclude that Tsarina Alexandra is a member of Haplogroup H, but the data from the studies to date do serve to narrow down her possible haplogroups to H, HV, and Pre-HV.  Further studies woud need to focus on markers 7028 and 14766 in the coding region which would provide a determination of which Haplogroup Tsarina Alexandra actually belongs to, and if she does in fact belong to Haplogroup H, further H subclade testing using the H subclade test panel should confirm which branch of H she falls into.