Editor’s Note: This is part two of a two-part series about FamilyTreeDNA’s involvement in the “Unearthing Who and Y at Harewood Cemetery and inference of George Washington’s Y-chromosomal haplotype.” Read part one here:

Take a closer look at the revolutionary genetic testing methods used to confirm the Washington family genealogy and the test results for Y-DNA, autosomal DNA, and mtDNA.

Samuel Walter Wahsington's family tree, limited to ancestors relevant to the relationships discussed in the new study about the genetic analysis of George Washington's family
Pictured: Samuel Walter Washington’s family tree, limited to ancestors relevant to the relationships discussed in the new study. Based on Figure 3 from Cavagnino et al. 2024.

Y-DNA Analysis Confirmed The Washington Family’s Direct Paternal Lineage

The Y-DNA test results from the study confirm the unbroken patrilineal relationship between Big Y tester Samuel Walter Washington (S.W.W.) and the Washington family, including two different brothers of President George Washington.

Understanding The Washington Paternal Lineage

One important and somewhat confusing thing to note is that even though S.W.W. shares his Y-DNA signature with his great-great-grandfather, that lineage is not strictly paternal, and instead, they both inherited their Y chromosomes from S.W.W.’s 5th great-grandfather and Dr. Samuel Walter Washington’s great-grandfather, Augustine Washington (1694–1743).

Samuel Walter Washington's family tree with the Y-DNA inheritance highlighted to show that the lineage is not strictly paternal

Augustine Washington was also the father of George Washington, meaning that as long as George Washington was a biological son of Augustine, we can reliably infer the detailed Y-DNA haplogroup of George Washington based on the confirmed haplogroup of his two brothers.

Big Y-700 Analysis Led to the Discovery of Subclade R-FTE201

The paper describes the Washington Y-DNA haplogroup as R-BY32422, dated to circa 1850 BCE, or about 3,850 years ago. The initial Big Y haplogroup was very old because S.W.W. was the first Big Y tester from this lineage.

After the manuscript was written and while it was undergoing the scientific peer-review process, one new Big Y-700 customer result unexpectedly arrived, which distantly matched S.W.W.’s lineage, causing the subclade R-FTE201 to be created and refining the Washington lineage by around 1,800 years, meaning that it can now be tracked down to the Common Era. Now that the paper is published, we can finally reach out to the customer and inform him that he is this far the closest patrilineal Big Y match of the presidential Washington lineage (although still 2,000 years removed).

Ancestral Path report for R-FTE201, George Washington's haplogroup
Pictured: Ancestral Path report for R-FTE201. The new Family Finder Y-DNA haplogroup feature can confirm intermediate haplogroups down to R-BY2151, just 3 steps above the most refined Washington haplogroup.

Once the Presidential Washington lineage’s Y-STR profile is confirmed, we can also invite Y-STR matches to perform a Big Y-700 upgrade and refine the Washington haplogroup further. There is also one mystery connection with a different surname that may uncover a previously unknown Washington descendant lineage. How exciting!

Autosomal Analysis and Adjusted Relationship Predictions

Autosomal DNA confirmed the expected relationship between S.W.W. and the remains in the burials after being adjusted for pedigree collapse due to the multiple distant cousin marriages within the family (a common practice at the time.)

It was also used to infer which of the brothers were interred in each of the two male burials. Present-day Samuel Walter Washington was predicted to be one degree closer to his great-great-grandfather, Dr. Samuel Walter Washington (1797-1831), than his brother, George Steptoe Washington Jr. (1806-1831,) an expected result for those two relationships.

The three Washington ancestors can now be properly buried in marked graves - Courtney Cavagnino

Pedigree Collapse Within the Washington Family Tree

Samuel Walter Washington's family tree showing the autosomal inheritance and pedigree collapse in the Washington family line

The autosomal analysis was challenging because three-way pedigree collapse caused the relationship prediction to be closer than expected when considering only the closest relationship between S.W.W. and his great-great-grandfather. Three of S.W.W.’s great-grandparents were Washingtons. The three known relationships are:

      • S.W.W. was the great-great-grandson of Dr. Samuel Walter Washington through his father’s father’s mother.
      • S.W.W. was also the second cousin, four times removed, of his great-great-grandfather and his brother George Steptoe Washington Jr., through his father’s father’s father.
      • S.W.W. was also the second cousin five times removed to the two brothers through his father’s mother.

Additional unknown, distant relationships cannot be completely ruled out.

Stated another way, three of S.W.W.’s great-grandparents were Washington family descendants through two different sons of Augustine Washington and Mary Ball.

This pedigree collapse meant that S.W.W is likely to have more Washington DNA autosomal segments than if he were descended from only one Washington ancestor.

In the article Pedigree Collapse and DNA – Plus an Easy-Peasy Shortcut, Roberta Estes has written about:

        • the effects of pedigree collapse
        • where the same ancestors appear in one’s family tree multiple times, on shared DNA
        • how it can be accounted for.

What’s Different About These Autosomal Results

One important technical difference to note is the test method that was used in the study. Family Finder matching is based on autosomal test chips that cover around 700,000 genome-wide SNPs. Armed Forces DNA Identification Laboratory, a division of the Armed Forces Medical Examiner System (AFMES-AFDIL) used their NGS-based test with around 95,000 SNPs for the autosomal matching of the degraded historical remains, and the present-day tester S.W.W.

The AFMES-AFDIL kinship prediction is based on shared or overlapping SNPs instead of the shared centimorgans (cM) that most genetic genealogists are used to. Their method, which utilizes a genotype likelihood approach initially developed for ancient DNA applications, was described in the 2022 paper Extended kinship analysis of historical remains using SNP capture by Gorden et al.

FamilyTreeDNA was pleased to be able to help with the genealogical analysis and pedigree collapse adjustments to assist with confirming the identities of the burials in this very interesting study.

A limited version of Samuel walter Washington's pedigree and the results from the kinship predictions
the results from the pairwise kinship predictions between S.W.W. and the three burials.

Mitochondrial Analysis of the Lucy Payne Family

The mitochondrial DNA signatures from the three burials helped exclude certain possible close relationships (for example, fathers and sons typically have different mtDNA signatures) and confirm the matrilineal relationship between the two sons and their mother, Lucy Payne.

The Million Mito Project’s preliminary TMRCA (Time to Most Recent Common Ancestor) estimate for mtDNA haplogroup J1c1b1a1 is circa 3500 BCE. A preliminary subclade of J1c1b1a1, which includes the Lucy Payne family and several FamilyTreeDNA mtDNA testers, has also been identified based on a shared mutation (G13707A). This new subclade has a preliminary TMRCA estimate of circa 150 BCE.

Comparing their mitochondrial signatures to the FamilyTreeDNA mtFull Sequence database, a few close connections within the haplogroup could be identified, and the commonality between them is maternal ancestry from Ireland, suggesting Irish matrilineal ancestry for Lucy Payne.

Of note, Lucy was the sister of Dolley (Payne) Madison who was the first lady of President James Madison.

Discover Your Connection to Lucy Payne in the upcoming Mito Discover™ Reports

Although this was not the focus of the study, Lucy’s mtDNA haplogroup will be included in the upcoming Mito Discover reports, allowing users to see if they are matrilineally related to Lucy Payne.

Paul Maier, the population geneticist at FamilyTreeDNA, is hard at work with the Million Mito Project and has produced the preliminary Mitotree structure and TMRCA estimates shown here.

The Irish mtDNA haplogroup cluster (F654503) closley matches Lucy Payne and her two Washington sons on the preliminary Mito Tree
Pictured: The Irish mtDNA haplotype cluster (F6549503) closely matches Lucy Payne and her two sons on a preliminary version of the Mito Time Tree. (Formatting and design of the report are subject to change.)

Continuing to Test the Washington Line

Importantly, knowing the detailed Y-DNA profile of the Presidential Washington lineage will allow future research to confirm or reject different hypotheses about relationships to the Washington family, including the identification of unknown remains or the authentication of other artifacts.
Any genetic male who thinks they may be a direct patrilineal descendant of a Washington ancestor can take a Big Y-700 test and see if they belong to the presidential Washington lineage.

Take the Big Y-700 Test