# Genomic analyses of hair from Ludwig van Beethoven

Tristan James Alexander Begg, Axel Schmidt, Arthur Kocher, Maarten H D Larmuseau, Göran Runfeldt, Paul Andrew Maier, John D Wilson, Rodrigo Barquera, Carlo Maj, András Szolek, et al.

► To cite this version:

Tristan James Alexander Begg, Axel Schmidt, Arthur Kocher, Maarten H D Larmuseau, Göran Runfeldt, et al. Genomic analyses of hair from Ludwig van Beethoven. Current Biology, 2023, 33, pp.1431-1447.e22. 10.1016/j.cub.2023.02.041. hal-04917722

HAL Id: hal-04917722

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<figure><img src="image_1.png" /></figure>
# Current Biology

## Genomic analyses of hair from Ludwig van Beethoven

### Graphical abstract

<figure><img src="image_2.png" /></figure>

### Authors

Tristan James Alexander Begg,
Axel Schmidt, Arthur Kocher, ...,
Robert David Attenborough,
Toomas Kivisild, Johannes Krause

### Correspondence

tristanj.a.begg@gmail.com (T.J.A.B.),
toomas.kivisild@kuleuven.be (T.K.),
krause@eva.mpg.de (J.K.)

### In brief

Begg et al. perform genomic analyses of eight locks of hair attributed to composer Ludwig van Beethoven. They sequence a high-coverage genome, finding strong genetic risk for liver disease that may have been compounded by alcohol and an infection with hepatitis B. An extra-pair paternity event is discovered in Beethoven's direct patrilineage.

### Highlights

* Eight locks of hair attributed to Ludwig van Beethoven underwent genomic analyses

* We deemed five of these authentic and sequenced Beethoven's genome to high coverage

* Beethoven had a predisposition for liver disease and became infected with hepatitis B

* We also discovered an extra-pair-paternity event in Beethoven's paternal line

<figure><img src="image_3.png" /></figure>
Article

# Genomic analyses of hair from Ludwig van Beethoven

Tristan James Alexander Begg,<sup>1,6,24,*</sup> Axel Schmidt,<sup>2</sup> Arthur Kocher,<sup>9,22,24</sup> Maarten H.D. Larmuseau,<sup>3,14,15,16</sup>

Göran Runfeldt,<sup>4</sup> Paul Andrew Maier,<sup>4</sup> John D. Wilson,<sup>5,28</sup> Rodrigo Barquera,<sup>9</sup> Carlo Maj,<sup>2,27</sup> András Szolek,<sup>18,19</sup>

Michael Sager,<sup>4</sup> Stephen Clayton,<sup>6,24</sup> Alexander Peltzer,<sup>26</sup> Ruoyun Hui,<sup>8,20</sup> Julia Ronge,<sup>12</sup> Ella Reiter,<sup>6</sup> Căcilia Freund,<sup>24</sup>

Marta Burri,<sup>24</sup> Franziska Aron,<sup>24</sup> Anthi Tiliakou,<sup>9,24</sup> Joanna Osborn,<sup>1</sup> Doron M. Behar,<sup>21</sup>

(Author list continued on next page)

<sup>1</sup>Department of Archaeology, University of Cambridge, CB2 3ER Cambridge, UK

<sup>2</sup>Institute of Human Genetics, University Hospital of Bonn, Bonn 53127, Germany

<sup>3</sup>Department of Human Genetics, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

<sup>4</sup>FamilyTreeDNA, Gene by Gene, Houston, TX 77008, USA

<sup>5</sup>Austrian Academy of Sciences, 1030 Vienna, Austria

<sup>6</sup>Institute for Archaeological Sciences, University of Tübingen, 72070 Tübingen, Germany

<sup>7</sup>American Beethoven Society, San Jose State University, San Jose, CA 95192, USA

<sup>8</sup>MacDonald Institute for Archaeological Research, University of Cambridge, Cambridge CB2 3ER, UK

<sup>9</sup>Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany

<sup>10</sup>Ira F. Brilliant Center for Beethoven Studies, San Jose State University, San Jose, CA 95192, USA

<sup>11</sup>School of Music and Dance, San Jose State University, San Jose, CA 95192, USA

<sup>12</sup>Beethoven-Haus Bonn, 53111 Bonn, Germany

<sup>13</sup>School of Archaeology & Anthropology, Australian National University, Canberra, ACT 0200, Australia

<sup>14</sup>Laboratory of Human Genetic Genealogy, Department of Human Genetics, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

<sup>15</sup>ARCHES - Antwerp Cultural Heritage Sciences, Faculty of Design Sciences, University of Antwerp, 2000 Antwerp, Belgium

<sup>16</sup>Histories vzw, 9000 Gent, Belgium

<sup>17</sup>Department of Internal Medicine I, University Hospital Bonn, 53127 Bonn, Germany

<sup>18</sup>Applied Bioinformatics, Department for Computer Science, University of Tübingen, Sand 14, 72076 Tübingen, Germany

<sup>19</sup>Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany

<sup>20</sup>Alan Turing Institute, 2QR, John Dodson House, London NW1 2DB, UK

<sup>21</sup>Estonian Biocentre, Institute of Genomics, University of Tartu, Tartu, Estonia

(Affiliations continued on next page)

## SUMMARY

Ludwig van Beethoven (1770–1827) remains among the most influential and popular classical music composers. Health problems significantly impacted his career as a composer and pianist, including progressive hearing loss, recurring gastrointestinal complaints, and liver disease. In 1802, Beethoven requested that following his death, his disease be described and made public. Medical biographers have since proposed numerous hypotheses, including many substantially heritable conditions. Here we attempt a genomic analysis of Beethoven in order to elucidate potential underlying genetic and infectious causes of his illnesses. We incorporated improvements in ancient DNA methods into existing protocols for ancient hair samples, enabling the sequencing of high-coverage genomes from small quantities of historical hair. We analyzed eight independently sourced locks of hair attributed to Beethoven, five of which originated from a single European male. We deemed these matching samples to be almost certainly authentic and sequenced Beethoven's genome to 24-fold genomic coverage. Although we could not identify a genetic explanation for Beethoven's hearing disorder or gastrointestinal problems, we found that Beethoven had a genetic predisposition for liver disease. Metagenomic analyses revealed furthermore that Beethoven had a hepatitis B infection during at least the months prior to his death. Together with the genetic predisposition and his broadly accepted alcohol consumption, these present plausible explanations for Beethoven's severe liver disease, which culminated in his death. Unexpectedly, an analysis of Y chromosomes sequenced from five living members of the Van Beethoven patrilineage revealed the occurrence of an extra-pair paternity event in Ludwig van Beethoven's patrilineal ancestry.

## INTRODUCTION

On March 27, 1827, the day after Beethoven's death, two of his associates discovered several documents stored within a hidden

compartment in his writing desk, including an unusual document written in 1802 and addressed to his brothers, which is now known as the Heiligenstadt Testament. In it, Beethoven confessed that he had been “hopelessly afflicted” with a progressive

<figure><img src="image_4.png" /></figure>
Malte Boecker,<sup>12</sup> Guido Brandt,<sup>24</sup> Isabelle Cleynen,<sup>3</sup> Christian Strassburg,<sup>17</sup> Kay Prüfer,<sup>9</sup> Denise Kühnert,<sup>22,23,24</sup> William Rhea Meredith,<sup>7,10,11</sup> Markus M. Nöthen,<sup>2</sup> Robert David Attenborough,<sup>8,13</sup> Toomas Kivisild,<sup>1,3,25,29,*</sup> and Johannes Krause<sup>6,9,24,29,30,*</sup>

<sup>22</sup>Transmission, Infection, Diversification and Evolution Group, Max Planck Institute for the Science of Human History, 07745 Jena, Germany

<sup>23</sup>European Virus Bioinformatics Center (EVBC), Jena, Germany

<sup>24</sup>Max Planck Institute for the Science of Human History, Kahlaische Str. 10, 07745 Jena, Germany

<sup>25</sup>Estonian Biocentre, Institute of Genomics, University of Tartu, Tartu 51010, Estonia

<sup>26</sup>Quantitative Biology Center (QBiC) University of Tübingen, Tübingen, Germany

<sup>27</sup>Center for Human Genetics, University Hospital of Marburg, Marburg, Germany

<sup>28</sup>University of Vienna, 1010 Vienna, Austria

<sup>29</sup>These authors contributed equally

<sup>30</sup>Lead contact

*Correspondence: tristanj.a.begg@gmail.com (T.J.A.B.), toomas.kivisild@kuleuven.be (T.K.), krause@eva.mpg.de (J.K.)

https://doi.org/10.1016/j.cub.2023.02.041

hearing loss. Stating that only virtue and his art held him back from committing suicide, he explained that he could not leave the world “before I had produced all the works that I felt the urge to compose.” Beethoven then requested that following his death, his disease be described by his favorite physician, Dr. Johann Adam Schmidt (1759–1809), and made public.¹

Although Beethoven outlived Dr. Schmidt by 18 years, medical biographers have since attempted to determine the most likely causes of Beethoven's various health complaints. Such research has relied principally on documentary sources, including Beethoven's letters, diaries, and conversation books, and accounts from Beethoven's contemporaries including physicians' notes, an autopsy report, and descriptions of skeletal material following exhumations in 1863 and 1888. In addition, analyses of tissue sources claimed to originate from Beethoven have been performed, including toxicological analyses of hairs of unknown authenticity²⁻⁴ and paleopathological and toxicological examinations of skull fragments,⁵ at least two of which are inauthentic.⁶

These sources attest to a number of health complaints varying in severity and impact on Beethoven's life and career. Foremost among these were a bilateral, late-onset, progressive, and predominantly sensorineural form of hearing loss, as well as chronic gastrointestinal problems and, toward the end of Beethoven's life, liver disease. Beethoven's hearing loss began in his mid-to late 20s, characterized initially by tinnitus, loudness-recruitment, and the loss of high-tone frequencies, and would end his career as a performing artist by his mid-40s.⁷,⁸ From at least the age of 22, Beethoven suffered from debilitating abdominal complaints that continued throughout his adult life, characterized primarily by abdominal pains (“Kolik”) and attacks and remissions of often prolonged bouts of diarrhea. In the summer of 1821, Beethoven began to exhibit symptoms of liver disease when the first of at least two attacks of jaundice occurred, culminating in his death, considered most likely due to cirrhosis,⁷,⁸ on March 26, 1827. Several lines of evidence indicative of the regular consumption of moderate to large quantities of alcohol⁹ have led some medical biographers to conclude that Beethoven was alcohol dependent,⁸,¹⁰ which is a known risk factor for liver cirrhosis.¹¹ While several of Beethoven's contemporaries insisted that Beethoven usually consumed alcohol in moderation,¹²⁻¹⁴ one close friend is alleged to have stated that in ca. 1825–1826, Beethoven had been consuming at least a liter (“Mass”) of wine with lunch every day.⁹ Although little is known with certainty about the medical history of Beethoven's

immediate family, a family history of alcohol dependence and liver disease has been noted.⁷,⁸

In addition to the three areas of illness mentioned above, Beethoven also showed other symptoms during his life, somatic and possibly also psychological.⁷⁻⁹ In clarifying possible genetic causes of Beethoven's illnesses, we limit our investigation to the three somatic disease areas that dominate the medical biographical literature because they represent Beethoven's main health restrictions and are widely documented by Beethoven's own reports, as well as reports from Beethoven's contemporaries and physicians.

We sought to sequence Beethoven's whole genome to high coverage from authenticated strands of hair in order to improve our understanding of his health. On the basis of genetic data and supporting provenance information, we assessed the authenticity of eight locks of hair claimed to originate from Beethoven. We required authentic samples to derive from a single male individual and to exhibit DNA damage patterns consistent with the reported antiquity of the samples. We sequenced a 24-fold coverage genome from the best-preserved sample among five matching samples using a highly sensitive protocol for historical hair. We then performed ancestry analyses on the expectation that this individual's ancestry would be consistent with Beethoven's documented genealogy. As part of our ancestry analyses, we introduce a novel geo-genetic triangulation (GGT) technique using long identity-by-descent (IBD) segments shared with individuals in FamilyTreeDNA's genealogically explicit consumer database to determine the likely locations of Beethoven's ancestors. In addition, we compared this genome to two groups of genealogically documented living relatives. We extensively analyzed Beethoven's genome for genetic causes of and risk for somatic disease, in addition to metagenomic screening for evidence of infections, followed by targeted DNA capture.

## RESULTS

### Authentication of hair samples

Strands of hair from eight locks attributed to Ludwig van Beethoven were acquired from public and private collections for analysis, which were determined to have had independent provenances (Figure 1; STAR Methods). We refer to these as the Müller, Bermann, Halm-Thayer, Moscheles, Stumpff, Cramolini-Brown, Hiller, and Kessler Locks (Methods S1A–S1H). These locks can conservatively be estimated to fall within
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1. Provenance summaries and authenticities of hair locks**</figcaption></figure>

Timelines, sex determination, mitochondrial and Y chromosome haplogroup determinations, and authenticity assessments for eight tested locks of hair claimed to have originated from Beethoven. The Kessler lock has a date of acquisition 36 to 61 years after Beethoven's death, during one of his exhumations in 1863 or 1888. See also Tables S1 and S2 and Figure 5.

a transect of Beethoven's life between ca. November of 1821 and his death in March of 1827, with the provenance histories for two of these locks, the Stumpff and Halm-Thayer Locks, bearing intact chains of custody (Figure 1). We expected authentic and independently sourced locks of Beethoven's hair to derive from a single male with predominantly Central European ancestry. We further expected the presence of terminal C-T deamination caused by DNA degradation over time, consistent with their provenances in the early 19th Century.

We performed shallow shotgun sequencing to permit assessments of DNA preservation and authenticity (Figure 1; Data S1A). Five samples, the Müller, Bermann, Halm-Thayer, Moschelles, and Stumpff Locks, shared identical mitochondrial genomes of haplogroup H1b1+16,362C with a private mutation at C16,176T, and had male XY karyotypes (Figure 1; Table S2; STAR Methods). Relatedness testing of autosomal and X chromosome DNA demonstrated that these five matching samples originated either from a single individual or monozygotic twins (Figure 2; Table S5; STAR Methods). All matching samples had DNA damage patterns consistent with their provenances in the early 19th Century (Data S1A).

In light of their provenance histories, we considered these genetic findings to be compelling evidence for the identity of these five independent locks of hair and proceeded under the working hypothesis that they are authentic. We determined that the Stumpff Lock (Figure 3) was marginally the best preserved of the five matching samples (Methods S1I) and sequenced a nuclear genome to an average of 24-fold coverage, incorporating laboratory and bioinformatics protocols optimized for the ultra-short DNA fragments characteristic of historical hair samples (mean fragment length 29.62 bp)¹⁵⁻²¹ (STAR Methods). We restricted further analyses to the 1.64 Gb of the genome to which short reads could be confidently mapped ("accessible genome"; STAR Methods).

Principal component analyses (PCAs) performed on the high-coverage Beethoven genome placed it among Europeans, clustering with modern Germans (Figures S1–S3). Testing for admixture among five global populations using ADMIXTURE²² revealed that Beethoven's ancestry was >99% European (Figures S4 and S5). We assessed geographic clustering of ancestors of 665 FamilyTreeDNA customers who share long (≥6 cM) autosomal IBD segments with Beethoven and used a novel GGT method (STAR Methods) to analyze the place names

documented in their genealogical records. We found strong geographic clustering of matches along the Rhine River and within present-day North Rhine-Westphalia in Germany (Figure 4; Methods S1L–S1O), largely consistent with the geographic distribution of the birthplaces of Beethoven's German ancestors.²³ We were unable to identify any shared ancestors with Beethoven among genealogical records for 30 customers with the longest shared IBD segments. Beethoven's I1a-Z139 Y chromosome haplogroup is common and widespread in Europe (Data S1D).

Of the non-matching hair samples tested, our sequence data show that the highly publicized Hiller Lock originated from a woman with close autosomal affinity in PCA space to present day North African, Middle Eastern, and Jewish populations²⁴ (Methods S1K). Its mitochondrial haplogroup, K1a1b1a (Table S2), is highly prevalent among Ashkenazi Jews.²⁵ Toxicological analyses of hairs extracted from this lock have been used to argue that Beethoven's health problems were caused or compounded by plumbism, and to refute suggestions that he was administered opiates during the course of his final illness and mercury for a hypothesized infection with syphilis.²⁻⁴,²⁶,²⁷ We now conclude that these findings do not apply to Beethoven. We additionally demonstrate that patterns of longitudinally distributed lead isotope concentrations believed to have been shared between hair strands from the Hiller, Halm-Epstein, and Erdödy Locks³ do not constitute proof of their authenticity, as the Hiller Lock is inauthentic.

We found that the Cramolini-Brown Lock originated from a male of European autosomal ancestry (Figure 1; Methods S1J), belonging to the mitochondrial haplogroup H79 and the Y chromosome haplogroup R1a-Z283 (Figures 1 and S4). As this sample differs from our five matching samples and we could not confirm its provenance prior to 2012, we conclude that it is almost certainly inauthentic.

Both the Hiller and Cramolini-Brown Locks exhibited levels of DNA damage similar to the five matching Beethoven samples (Data S1A).²⁸,²⁹

The Kessler Lock lacked sufficient DNA preservation for sex chromosomal karyotyping or ancestry determination, mitochondrial contamination estimation, or mitochondrial haplogroup assignment (Data S1A). We were therefore unable to assess its authenticity.

On the basis of these genetic data, and in light of their known provenance histories, we conclude that the Müller, Bermann,
<figure><img src="image_7.png" /><figcaption>Figure 2. Relatedness testing of eight locks of hair attributed to Beethoven</figcaption></figure>

Relatedness testing of eight locks of hair attributed to Beethoven, relative to an external reference panel of 41 medieval Bavarians. The proportions of non-matching alleles (P0) per pair and estimated degrees of relatedness are calculated from pseudo-haploid genotype calls using READ. The numbers of SNVs in each comparison are denoted by n. *Known medi- eval Bavarian sibling pair (STR355c/STR491). See also Table S5.

Halm-Thayer, Moscheles, and Stumpff Locks almost certainly authentically derive from Beethoven, the Cramolini-Brown Lock is almost certainly inauthentic, the Hiller Lock is definitely inauthentic, and the authenticity of the Kessler Lock could not be determined.

In order to support the authenticity of the matching samples further, we compared the Y chromosome from Beethoven's high-coverage genome against high-coverage Y chromosomes sequenced from five living men belonging to the Van Beethoven patrilineage. These individuals were identified through analyses of genealogical records, which document Aert van Beethoven (1535-1609) as a patrilineal ancestor shared by Ludwig van Beethoven and our research participants (Methods S1P).²³ Consistent with genealogical records, these five individuals share nearly identical Y chromosomes falling within the R-FT446200 haplogroup within R1b, with an average of 4.8 [3–7] private mutations having arisen along each lineage (Figure 5; STAR Methods). The pedigree reconstructed on the basis of these private mutations reproduced the documented pedigree among the participants (Figure 5). These Y chromosomes did not, however, match the Y chromosomes from either the five matching Beethoven hair samples within I1a-Z139 or the Cramolini-Brown Lock within R1a-Z283 (Figure 5).

Considering the strong historical and genetic evidence for the authenticity of the five matching hair samples, and our Y chromosome evidence for the lack of discontinuity in the paternal lineage between Aert van Beethoven and the five living descendants, we conclude that the most plausible explanation for our observations entails that at least one extra-pair paternity (EPP) event occurred on Beethoven's direct paternal line, between the conception of Aert van Beethoven's son Hendrik in Kampenhout, Belgium, in c.1572, and the conception of Ludwig van Beethoven seven generations later in 1770, in Bonn, Germany.

In order to further investigate the details of an EPP scenario and possibly ascertain Beethoven's genetic patrilineage, we queried the FamilyTreeDNA Y chromosome database, including >52,500 user records at high sequence resolution.³⁰ We identified five closely related profiles descending from the I-FT396000 lineage within I1a-Z139, with a mean time to most recent common ancestor (TMRCA) of 1,018 (95% CI 714–1,419) years before present (Figure 5; Methods S1Q; Table S6). However, all five of these participants carried dissimilar

surnames, consistent with the fixation of surnames in many parts of Europe occurring several centuries after the most probable TMRCA for I-FT396000. We were

therefore unable to establish Beethoven's direct genetic patrilineage and the surname of the individual involved in an EPP event.

In addition to testing the Y chromosomes of living Van Beethovens, we tested for IBD segment sharing among three living descendants of Beethoven's nephew, Karl van Beethoven, who are documented as 7th-degree genetic relatives to Beethoven.²³ Using IBIS,³¹ which can accurately detect IBD segments ≥7 cM, we detected no IBD segments ≥7 cM shared between Beethoven and the three participants. The IBD-sharing and mitochondrial relatedness detected among the participants internally, however, was consistent with their documented genealogy.

In order to better interpret this result, we performed 100,000 simulations on a reconstructed pedigree using pedSIM,³² including the three living descendants of Karl van Beethoven, and including Ludwig and Karl's father, Kaspar Anton Karl van Beethoven (1774–1815), as full siblings. These simulations estimated an average of 47.23 detectable cM among 2.46 IBD segments ≥7 cM shared with Beethoven per descendant. The simulated probability for detecting zero IBD segments ≥7 cM shared between Beethoven and each individual participant averaged 9.08% (95% CI 8.9%–9.26%), while the probability of detecting zero ≥7 cM IBD-segment sharing with Beethoven among all three participants combined was 0.851% (95% CI 0.79%–0.91%).

However, given the virtual certainty that an EPP event occurred in Beethoven's direct paternal ancestry, we could not assume with confidence that Ludwig and Kaspar Anton Karl were full siblings. In the event that Ludwig and Kaspar Anton Karl were half-siblings, the probability of detecting zero ≥7 cM IBD-segment sharing with Beethoven among all three participants combined was 8.34% (95% CI 7.81%–8.9%). As a result, we are unable to conclusively prove or disprove relatedness between Beethoven and the descendants of Karl van Beethoven, and are unable to provide further verification of sample authenticity on this basis.

## Genetic variants associated with somatic disease

Diseases differ in terms of the degree of genetic causation and the number of genes involved. In monogenic and some complex diseases, an accurate diagnosis can be made by identifying the responsible mutation(s) in the patient. In multifactorial diseases, many genes are involved in interaction with non-genetic factors,
<figure><img src="image_8.png" /><figcaption>Figure 3. The Stumpff Lock</figcaption></figure>

The Stumpff Lock from which Beethoven's high-coverage genome was sequenced. The lock is affixed to a letter from Johann Andreas Stumpff to Patrick Stirling, dated May 7, 1827. Stumpff's poem reads, "The **head**<sup>8</sup>, these hair's have grac'd lies low; But what it wrought — will ever grow. <sup>9</sup>of Lud. v. Beethoven." Photographed in July of 2018 by American Beethoven Society member Kevin Brown.

and without exception, not all of the genes involved are yet known. For multifactorial diseases, the genetic contribution known so far can be summarized in a polygenic risk score (PRS). When attempting to determine an individual's polygenic predisposition for a multifactorial disease, the individual's PRS is usually compared against a distribution of PRSs generated from a suitable reference population. As a rule, an individual PRS does not provide sufficient predictive accuracy for a diagnosis. This must be kept in mind when interpreting the molecular genetic findings reported in the following.

Several diseases have been proposed to account for Beethoven's hearing loss, including otosclerosis,⁸ Paget's disease of bone (PDB),³³ complications from Crohn disease (CD) or ulcerative colitis (UC),³⁴ sarcoidosis,³⁵ and systemic lupus erythematosus (SLE).³⁶ Genome-wide association study (GWAS) summary statistics with sufficient power for meaningful disease

risk stratification via PRS could not be obtained for otosclerosis, PDB, or sarcoidosis, limiting our assessments of Beethoven's polygenic risk to CD, UC,³⁷ and SLE³⁸ (Data S1H; Methods S1R–S1X; STAR Methods). Only Beethoven's PRS for SLE was found to confer notably elevated polygenic risk, placing him within the 93rd polygenic risk percentile, and conferring an odds ratio (OR) relative to the mean polygenic score among controls of approximately 2.96 (1.54–5.67) (Methods S1U; Data S1H).

In addition to evaluating polygenic risk for multifactorial diseases underlying Beethoven's hearing loss, we evaluated the hypothesis of a monogenic etiology. We prioritized 55 genes³⁹,⁴⁰ in which variants could cause monogenic post-lingual hearing loss and further analyzed an extended set of an additional 209 genes predominantly related to pre-lingual hearing loss (Data S1F). In addition, we analyzed 137 genes causing rare monogenic
<figure><img src="image_9.png" /><figcaption>**Figure 4.** Geo-genetic triangulation of Beethoven's probable ancestor locations</figcaption></figure>

Geo-genetic triangulation, showing probable locations of Beethoven's autosomal ancestors. Regional intensity per hexagon represents number of shared ancestors between Beethoven and modern FTDNA customers, with the following criteria: (1) IBD segments (n = 665) must be shared (triangled) between at least three individuals to be genetically validated and (2) ancestor locations (n = 89) of matching individuals must occur within the same hexagon to be geographically validated. Areas of Western and Central Europe that were considered but lack any probable ancestors are shown in gray. See also Methods S1L-S1O.

subforms of SLE, inflammatory bowel disease-like syndromes, PDB, sarcoidosis, and otosclerosis (Data S1F; STAR Methods). To estimate the sensitivity of our analysis, we analyzed the coverage of these prioritized genes. On average, 83% (SD ±19%) of the protein or tRNA/rRNA coding sequences of these genes (n = 390) were within the accessible genome, and 64% (SD ±24%) were within the accessible genome and covered by at least 20 reads (Figure S6). While acknowledging the limited sensitivity of our analyses, we were unable to identify any unambiguous disease-causing variants. Several variants of uncertain significance and with weak evidence for pathogenicity were, however, identified (Data S1G). In summary, we could not reliably evaluate most hypothesized multifactorial causes of Beethoven's hearing loss, nor did we identify a monogenic origin.

In the overwhelming majority of cases worldwide, cirrhosis of the liver can be attributed to the effects of alcohol⁴¹ or infections by the hepatitis B virus (HBV) or the hepatitis C virus (HCV),⁴² acting on a background of individual genetic predisposition. Both singly and acting in combination, alcoholic liver disease (ALD) and viral hepatitis are the most frequently proposed

hypotheses for Beethoven's liver disease.⁷ However, liver cirrhosis may also occur in the context of specific underlying diseases, which are typically multifactorial in origin. Primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), SLE, sarcoidosis, and complications from CD or UC have previously been hypothesized for Beethoven.⁷ Several monogenic etiologies have also been considered as underlying conditions, including hereditary hemochromatosis (HH), alpha-1 antitrypsin deficiency (AATD), Wilson's disease, and cystic fibrosis (CF).⁷

We analyzed Beethoven's polygenic risk for liver cirrhosis,⁴³ which placed Beethoven within the 96th risk percentile (Methods S1X; Data S1H; STAR Methods). Consistent with his PRS polygenic risk for liver cirrhosis, Beethoven was found to be homozygous for the variant consistently implicated as the most strongly associated locus for liver cirrhosis in GWASs, at rs738409 in *PNPLA3*.⁴⁴ A significant modulating effect on rs738409 has been observed at rs2294918, also in *PNPLA3*, which attenuates risk among rs738409 carriers.⁴⁵ Beethoven lacked the risk attenuating allele and was homozygous for the highest known risk diploid type in *PNPLA3*, I148M-K434E. Beethoven's polygenic scores for PBC⁴⁶ and PSC⁴⁷ did not confer disease risk, placing him
<figure><img src="image_10.png" /><figcaption>**Figure 5.** *Y chromosome phylogeny of all male samples tested*</figcaption></figure>

Y chromosome phylogeny containing all male samples tested, including Ludwig van Beethoven, the six closest known living Y chromosome relatives of Ludwig van Beethoven within the FamilyTreeDNA database (FT1–FT6), five living descendants of Aert van Beethoven (VB1–VB5), and the Cramolini-Brown Lock.

(A) Deep phylogenetic overview within the last 50,000 years of three major clades harboring Ludwig van Beethoven and his closest living Y chromosome relatives (I-Z139), the living Aert van Beethoven descendants (R-Z2565), and the Cramolini-Brown Lock (R-Z283).

*(legend continued on next page)*
within the 22nd and 59th polygenic risk percentiles, respectively (Methods S1V and S1W; Data S1H).

Additionally, in order to investigate a possible monogenic condition underlying Beethoven's liver cirrhosis, we analyzed the genes responsible for hypothesized monogenic diseases including AATD, HH, Wilson's disease, and CF, as well as 47 genes that may cause rare monogenic forms of hypothesized multifactorial diseases already analyzed via PRS (Figure S6; Data S1F; STAR Methods). While no mutations causal to AATD, Wilson's disease, or CF were identified, Beethoven was found to be a compound heterozygote for two variants causal to HH, at rs1799945 (p.His63Asp) and rs1800562 (p.Cys282Tyr) in the *HFE* gene.

In order to assess the combined effect of Beethoven's heritable risk factors for liver disease with heavy alcohol consumption, retrospective cohort analyses were performed on cohorts of UK BioBank males matching Beethoven's genotypes at rs738409 and rs2294918 in *PNPLA3*, and rs1799945 and rs1800562 in *HFE*, including males meeting ICD-10 criteria for "Harmful use" and "Alcohol dependence" (ICD-10 codes F10.1 and F10.2), whom we term heavy drinkers. The disease prevalence of all-cause liver disease (ICD-10 code K7*) and liver cirrhosis<sup>43</sup> (Data S1H) among heavy drinking males homozygous for Beethoven's *PNPLA3* diplototype was found to be 30.95% (95% CI 24.77%-37.68%) and 21.43% (95% CI 16.08%-27.6%) compared to a prevalence among all male heavy drinkers of 20.64% (95% CI 19.43%-21.89%) and 10.34% (95% CI 9.44%-11.3%), respectively (Figure 6; Data S1K and S1L). Prevalence for all-cause liver disease (24% [95% CI 16.02%-33.57%]) and cirrhosis (14% [95% CI 7.87%-22.37%]) was also significantly elevated among male heavy drinkers with compound heterozygosity in *HFE*, as compared to baseline rates among heavy drinkers (Figure 6; Data S1M). The combined effect of Beethoven's *PNPLA3* and *HFE* genotypes among heavy drinking males could not, however, be accurately determined owing to a low number of matching individuals (n = 4).

Although many medical biographers favor irritable bowel syndrome (IBS) as the cause of Beethoven's gastrointestinal symptoms, several have proposed one of the two inflammatory bowel diseases, i.e., CD or UC.<sup>7,8</sup> In the vast majority of cases, all three of these diseases are multifactorial in origin. We additionally queried for genetic origins for other causes of gastrointestinal distress, including celiac disease, and monogenic diseases and conditions such as lactose intolerance, CF, and monogenic forms of inflammatory bowel disease or inflammatory bowel disease-like syndromes.<sup>48</sup>

Beethoven's polygenic scores for CD and UC<sup>37</sup> placed him in the 36th and 61st percentiles, respectively (Methods S1R and S1S; Data S1H). Beethoven's polygenic score for IBS<sup>49</sup> placed him within the 9th polygenic risk percentile, conferring a protective status against IBS with an OR of 0.39 (0.15-1) (Methods S1T; Data S1H). However, due to our use of UK BioBank IBS cases during parameter optimization, which were also included

during the generation of the GWAS summary statistics we used,<sup>49</sup> this result may be confounded by overfitting.

Beethoven additionally lacked the HLA-DQ2 and DQ8 alleles, which are a prerequisite for celiac disease (STAR Methods). Beethoven was most likely lactose tolerant, carrying heterozygous genotypes for both lactase persistence alleles at rs4988235 and rs4988235 near the *LCT* gene.<sup>50,51</sup> Furthermore, we were unable to identify any disease-causing variants for CF (Data S1F).

## Hepatitis B virus DNA recovered from Beethoven's hair

An infection with viral hepatitis has been considered by several of Beethoven's medical biographers<sup>7,52-55</sup> as a plausible cause for his liver disease. Using the metagenomic screening pipeline MALT,<sup>56,57</sup> we screened all sequence data from the Müller, Bermann, Halm-Thayer, and Stumpff Locks for HBV DNA. Three DNA libraries prepared from the Stumpff Lock yielded putative traces of HBV DNA (HEB001.B0102, HEB001.F0101, and HEB001.M0103; Data S1I). Although only four HBV-mapping reads were identified, these appeared to represent specific matches and were well distributed along the genome. We therefore performed hybridization capture to enrich libraries for HBV DNA. After sequencing, deduplication, and filtering of low-copy-number reads of HBV-DNA-enriched libraries, 92 unique reads from 20 libraries remained (Data S1I), resulting in a mean HBV genome coverage of 1.26-fold (Figure S7A), with all positive libraries deriving from the Stumpff Lock (STAR Methods). No clear damage pattern was observed, which likely resulted from the relatively low number of recovered reads (Figure S7B). Our phylogenetic analysis placed the reconstructed sequence within subgenotype D2 with 100% support (Figure 7), irrespective of the HBV reference genome used for read mapping (STAR Methods). Following HBV-DNA enrichment of the 25 extraction and library preparation blanks, three unique reads were found from three libraries (LIB002.A0116, LIB002.A0139, and LIB002.A0141; Data S1J), all mapping to an ~200-bp section of the S gene in the HBV genome (pos. 296-487).

## DISCUSSION

### Authenticity of Beethoven's genome

Of the eight locks of hair analyzed here, seven yielded sufficient DNA for interpretation, and we found five of those to derive from a single male individual with ancestry and DNA damage patterns consistent with originating from Ludwig van Beethoven. Four main considerations support our conclusion that the individual in question is indeed Ludwig van Beethoven.

First, the documentary evidence supporting the authenticity of these five locks (Figure 1; STAR Methods) is very strong. In particular, the Halm-Thayer Lock is recorded to have been presented in 1826 by Beethoven himself to fellow musician Anton Halm and his wife; Halm presented it in 1859 to the Beethoven scholar, Alexander Thayer; and it remained with the Thayer family in the United States until American Beethoven Society member Kevin Brown

<footnote>(B) Detailed phylogeny within the last thousand years outlining Ludwig van Beethoven's Y chromosome lineage (blue outline; haplogroup I-FT396000), the living Van Beethoven Y chromosome lineage (orange outline; haplogroup R-FT446200), and the approximate hypothesized location of an extra-pair paternity (EPP) event within the Van Beethoven Y chromosome tree (dotted line). In blue parentheses are the numbers of confidently ascertained private variants (for Ludwig van Beethoven, see those with a 3-star rating in Data S1E). See also Methods S1P and S1Q.</footnote>
<figure><img src="image_11.png" /><figcaption>**Figure 6.** Liver disease prevalence in cohorts sharing Beethoven's risk factors</figcaption></figure>

Prevalence and 95% confidence intervals of (A) all-cause liver disease (ICD-10 code K7*) and (B) liver cirrhosis following Emdin et al.⁴³ among cohorts of UK BioBank (UKB) men (n = 227,602) with compound heterozygosity at rs1799945 and rs1800562 in *HFE* (HFE; n = 5,004), homozygosity at rs738409 and rs2294918 in *PNPLA3* (PNPLA3; n = 10,485), and heavy drinkers (HD; ICD-10 codes F10.1 and F10.2; n = 4,235) by individual risk factors and in combinations of risk factors (HFE & PNPLA3, n = 233; HD HFE, n = 100; HD PNPLA3, n = 210; HD HFE & PNPLA3, n = 4). See also Data S1K–S1M.

purchased it in 2017 for the Society, which made a sample from it available for the present study. Similarly, following Beethoven's death in 1827, the Stumpff Lock was sent by family friend Johann Streicher, acting for Johann Schickh, who was organizing Beethoven's funeral, to harp-maker Johann Stumpff in London, who, within a month, sent it to Patrick Stirling, member of a prominent Scots family of musical patrons; when put up for auction in 2016, still attached to a document bearing Stumpff's signature, Sotheby's described the lock as having "come down to the present owners by direct descent," and at that auction it was purchased by Kevin Brown for the American Beethoven Society, which made a sample available. By contrast, there are gaps in the known ownership and whereabouts of the Müller, Bermann, and Moscheles Locks: before 1851 when Thayer acquired the Bermann Lock; between 1917 and 1940 in the case of the Moscheles Lock; and between 1820 and 1956 when the Beethoven-Haus Bonn acquired the Beethoveniana collection of Hans Conrad Bodmer, including the Müller Lock, on his death. Nonetheless, these three locks all have documented 19th Century origins. In the cases of the Müller and Moscheles Locks, like the Halm-Thayer and Stumpff ones, the associated documentation is original. In all recorded cases, the historical custodians of the locks have been known Beethoven acquaintances, musicians, scholars, collectors, enthusiasts, and institutions.

Second, the histories of these locks are, with one small exception, independent of each other. The exception is that the Bermann and Halm-Thayer Locks were held together in a picture frame while in Thayer's ownership. That apart, there are no historical opportunities for the locks to have been confused, amalgamated, contaminated, or replaced by one another. The Bermann and Moscheles Locks could not share a source later than Beethoven himself, as the chronological gaps in their documentation do not overlap. Yet all these five almost entirely

independent locks, two of them with impeccable provenance, two with good provenance, and a fifth with moderate provenance, are genetically identical.

Third, the two locks that do not genetically match the five matching locks not only fail to match each other, too, but also have weaker supporting documentation than the matching locks. Although a genuine Cramolini Lock may exist, the lock known as Cramolini-Brown has no secure documentation before 2012. Similarly, while there is earlier documentation of a Hiller Lock, there is no surety that the lock that came to light in the difficult circumstances of 1943 is that same lock.

Fourth, plausible hypotheses whereby five separate locks attributed to Beethoven could share the same genetic source individual other than him are extremely hard to construct. Any fraudulent manipulation of the locks and documentation would have had to pre-date by many decades any concept, verified in 1985, that genetic data could be derived from hair.⁵⁵ It would require a coordinated effort to disperse at least five locks of hair derived from a single individual among a diverse group of Beethoven's close affiliates and/or subsequent collectors, most likely within Beethoven's lifetime or immediately after his death, in tandem with forgery of supporting provenance documents. Any later effort, undertaken during gaps in their known custodianship, would have to have been made before 1850 for the Bermann Lock but after 1915 for the Moscheles Lock. The feasibility of such complex operations seems impossible to credit, as does any apparent motivation for it, whether financial or otherwise.

## Comparison with living individuals' genomes

We were not able to confirm Beethoven's genetic relationship with either a set of five Belgian male research participants sharing his surname or a set of three Austrian research participants genealogically documented as his collateral descendants. These two analyses are very different.

The five living men belonging to the Van Beethoven patrilineage are only distantly interrelated genealogically but have matching Y chromosome haplogroups, consistent with descent from the common patrilineal 16th century ancestor Aert van Beethoven, also identifiable as such from genealogical data. Genealogical data also identify Aert as ancestral to Ludwig van Beethoven,
<caption>Figure 7. Phylogenetic tree of HBV</caption>

Phylogenetic tree of HBV with branches in sub-
stitutions per site estimated using RAxML.⁵⁸ Clades
corresponding to the main genotypes were colla-
psed and annotated with their typical geographic
location, except for subgenotype D2, in which the
HBV genome recovered from Beethoven was
placed. Bootstrap supports are reported on the
nodes. See also Figure S6 and Data S1I and S1J.

<figure><img src="image_12.png" /></figure>

but the Y chromosome data do not match in this case. What sce-
narios could explain this? We have concluded above against the
possibility that an individual other than Ludwig van Beethoven
could be the source of the five genetically matching hair samples.
Could there have been an error in Beethoven's legal genealogy?
We conclude against this possibility also, given the well-estab-
lished paternal genealogy for Ludwig van Beethoven.²³,⁶⁰ With
the exception of his father, Johann van Beethoven (ca. 1739-
1792), each step in the patrilineage is documented in at least
two different archival records, which were reviewed thoroughly

on observing the mismatch. Moreover, the
strong concordance between surname
and Y chromosome haplogroup observed
in historical Belgium,⁶¹ supporting the ac-
curacy of local record-keeping, renders
the mismatch very difficult to account for
by genealogical error. The possibility that
remains, therefore, is that an EPP event
took place in one of the generations be-
tween Aert and Ludwig van Beethoven. In
Western Europe over the but last 400 years,
such events were rare but did take place,
at an average frequency of 1%-2% per
generation.⁶² One Beethoven biographer⁶³
has previously suggested, on circumstan-
tial grounds, that Ludwig senior may not
have been Johann van Beethoven's biolog-
ical father. Our genetic findings, however,
do not allow us to favor any particular gen-
eration for the occurrence of an EPP event.

A second analysis focused on three
closely related living descendants of Lud-
wig van Beethoven's nephew Karl, docu-
mented as 7th-degree genetic relatives of
Ludwig. Because Karl's patriline is now
extinct, Y chromosome analysis was
impossible, so a more probabilistic method
involving analysis of autosomal IBD seg-
ments was adopted. We were unable to
detect any IBD-segment sharing ≥ 7 cM
between Beethoven and these living
individuals and could not determine
conclusively whether this finding reflects
an unusually low level of IBD sharing for
this level of relatedness or an EPP event.

Origins of Beethoven's diseases

The genomic sequence data are a novel
and unbiased primary source that offer the potential to improve
our understanding of Beethoven's health problems. It must be
emphasized, however, that our approach has important limita-
tions. First, the ultra-short read data characteristic of historical
hair samples significantly impact the data quality and coverage
of analyzed genes. For example, deletions and duplications,
which have been shown to cause a relevant proportion of cases
of monogenic non-syndromic hearing loss,⁶⁴⁻⁶⁶ were not consid-
ered in our analyses due to these limitations. Second, despite
great advances in medical genetics, the genetic causes of
many diseases are not yet fully understood, especially in the case of multifactorial diseases, which are further complicated by the fact that non-genetic causes may also substantially contribute to the development of disease.

Taking these limitations into account, we did not find a molec-
ular genetic cause for Beethoven's hearing loss. However,
important differential diagnoses, such as otosclerosis, which is
frequently suggested in the literature, could not be evaluated
due to a lack of reference data. Substantiation of the previously
hypothesized role of plumbism as a causative or contributory
factor to Beethoven's hearing loss<sup>3,4,26,27</sup> must await analyses
of samples authenticated via genetic testing.

Similar to the results for hearing loss, we did not find a molec-
ular genetic explanation for Beethoven's gastrointestinal
complaints. However, we were able to render some important di-
agnoses less likely. For example, celiac disease and lactose
intolerance can almost be ruled out as causes. IBS, often sus-
pected as a cause, is less likely on the basis of the PRS findings
but, given the limited diagnostic power of the IBS PRS, is still
possible.

Our most significant results concern Beethoven's liver dis-
ease. The elevated PRS for liver cirrhosis, which includes homo-
zygosity for a risk variant in *PNPLA3*, the strongest known
genetic risk factor, suggests that Beethoven inherited a consid-
erable genetic predisposition. Compound heterozygosity in the
*HFE* gene may have made an additional contribution. Due to
the low penetrance of the *HFE* diplotype,<sup>67-69</sup> it cannot be
assumed that Beethoven suffered from clinically relevant hemo-
chromatosis. Nonetheless, the role of iron overload caused by
mutations in the *HFE* gene, for which excessive alcohol con-
sumption is a co-morbid risk factor<sup>69</sup> (*Data S1M*), may have
had an additional, unfavorable effect on liver health<sup>70</sup> (*Figure 6*;
*Data S1K* and S1L). Our retrospective cohort analyses demon-
strated that Beethoven's risk for liver disease would have been
heavily contingent on the extent of his alcohol consumption (*Fig-
ure 6*; *Data S1K-S1M*). If Beethoven was regularly consuming
sufficiently large quantities of alcohol, the combined risk
conferred by alcohol consumption and his substantial genetic
predisposition may constitute a plausible causative explanation
for his liver disease. In addition, we demonstrated that Beet-
hoven had an HBV infection at least during the months leading
to his death. Our analyses presently lack the sensitivity to deter-
mine the nature and timing of this infection, which would have
strongly influenced the extent of its causal involvement with
Beethoven's liver disease. A chronic perinatal or childhood
HBV infection would have been a strong driver of liver disease,
no doubt exacerbated by his genetic risk and alcohol consump-
tion, whereas an HBV infection closer to the end of Beethoven's
life would have been of lesser relevance (*Hepatitis B virus DNA in
Beethoven's hair; STAR Methods*). Nonetheless, we conclude
that Beethoven's substantial genetic predisposition, HBV infec-
tion, and alcohol consumption all present plausible causal fac-
tors in his liver disease, although the exact causal pattern cannot
presently be determined.

Hepatitis B virus DNA in Beethoven's hair

HBV is an important current global public health problem as a ma-
jor cause of liver cirrhosis and cancer.⁷¹ This virus can be trans-
mitted from mother to child during birth, via sexual contacts, or

through surgery with contaminated instruments. It may cause
chronic infections (especially when contracted during childhood),
which result in liver complications after decades in a large propor-
tion of cases. Acute HBV infections are usually asymptomatic or
mild but can lead to lethal fulminant hepatitis in rare cases.

Hair has recently been revealed as a potential reservoir of HBV DNA in individuals suffering from both chronic and acute HBV infections,⁷²,⁷³ supporting the plausibility of HBV DNA fragments surviving in ancient and historical hair samples from HBV-positive individuals. Screening of shotgun sequencing data and the hybridization capture experiment indicated the presence of HBV DNA in several libraries prepared from the Stumpff Lock (*Data S1L*). Ninety-two sequencing reads mapping on the HBV genomes were recovered, after filtering of low-copy-number reads and deduplication. These were well distributed along the HBV genome sequence, as expected from fragments genuinely originating from a target organism. This allowed the reconstruction of a significant proportion of it (63%; *Figure S7A*). In contrast, only three reads mapping on the HBV genome were recovered from the 25 negative controls after HBV-DNA enrichment, and these were all found within a small (~200-bp-long) section of the HBV genome, more indicative of cross-mapping due to the presence of another DNA molecule sharing a local similarity with the HBV genome (*Data S1J*). Of note, extraction and library blanks corresponding to the hair samples yielding the strongest signal (i.e., HEB001.A and HEB001.B) did not contain any HBV-mapping reads. Therefore, it appears unlikely that the signal recovered from the Stumpff Lock arose from laboratory contamination, which is, in general, not expected for HBV. Furthermore, our phylogenetic analysis indicated that the HBV genome recovered from the Stumpff Lock belonged to a single subgenotype D2 (*Figure 7*). Subgenotype D2 is one of the most prevalent HBV variants in Europe today⁷⁴,⁷⁵ and has been shown to be present in the region since at least the Middle Ages.⁷⁶ These results are consistent with an authentic HBV infection.

Only hairs from the Stumpff Lock were proven positive for
HBV, plausibly spanning a period of growth no later than the
summer to winter of 1826, and likely earlier (STAR Methods).
However, owing to differential sequencing efforts between the
four samples tested and random fluctuations in HBV viremia,⁷⁷
the absence of detection in older samples does not necessarily
imply that the infection was acquired toward the end of Beet-
hoven's life. Owing to these limitations, we are unable to deter-
mine how or when Beethoven's infection with HBV occurred.

Future directions

Genomic sequence data from authenticated locks of Beet-
hoven's hair provide Beethoven studies with a novel primary
source, already revealing several significant findings relating to
Beethoven's health and genealogy, including substantial herita-
ble risk for liver disease, infection with HBV, and EPP. This dataset additionally permits numerous future lines of scientific inquiry.
This initial series of five hair samples, spanning approximately the
last 7 years of Beethoven's life, is hoped to be expanded through
the authentication testing of additional independent locks of hair,
and enables future testing for infections, informative biomarkers,
and exposures to environmental causes of or contributors to dis-
ease. The further development of bioinformatics methods for risk
stratification and continued progress in medical genetic research
will allow more precise assessments both for Beethoven's disease risk and for the genetic inference of additional phenotypes of interest. Increases in the size of consumer genetics databases, as well as the testing of additional hypothesized relatives both living and deceased, will lend further clarity to our understanding of Beethoven's genetic genealogy. This study illustrates the contribution and further potential of genomic data as a novel primary source in historical biography.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY

* Lead contact

* Materials availability

* Data and code availability

* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* Beethoven hair samples

* Müller Lock

* Bermann Lock

* Halm-Thayer Lock

* Moscheles Lock

* Cramolini-Brown Lock

* Stumpff Lock

* Hiller Lock

* Kessler Lock

* Identification of living patrilineal descendants of Aert van Beethoven

* Identification of living descendants of Karl van Beethoven

* Permits

* METHOD DETAILS

* Decontamination, extraction and purification

* Library preparation, indexing and sequencing

* QUANTIFICATION AND STATISTICAL ANALYSIS

* Bioinformatics processing of initial low-coverage data

* Mitochondrial contamination estimation, haplogroup assignment, and regional haplogroup frequency analysis

* High-coverage Beethoven autosomal genome sequencing and genotype calling

* HLA genotyping

* Sex chromosomal karyotyping

* Relatedness testing of autosomal and X chromosome DNA among locks of hair

* Damage rate assessment

* Principal components analyses

* ADMIXTURE analyses

* Geo-genetic triangulation with IBD-segments

* Y chromosome analyses

* Time to most recent common ancestor estimates

* Y-STR imputation

* Analyses of living descendants of Karl van Beethoven

* Polygenic risk scoring

* Variant Effect Predictor

* Analysis of coverage

* Retrospective cohort studies

* Screening, capture, sequencing and analysis of hepatitis B virus DNA

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cub.2023.02.041.

## ACKNOWLEDGMENTS

We are grateful to American Beethoven Society member Kevin Brown for providing generous funding and access to the Bermann, Halm-Thayer, and Stumpff Locks, as well as to Victoria Korkoras, Arthur Lund, and the Hugh Stuart Center Charitable Trust for additional funding. We also thank Alfredo Guvara for providing access to the Hiller Lock, William Kindermann and the Illinois State University Music Department for providing access to the Kessler Lock, and the Beethoven-Haus Bonn for providing access to the Müller Lock. G.R. and P.A.M. thank Katy Rowe for contacting FamilyTreeDNA customers to acquire consent for Big Y upgrades and Ulf Engstrand for helping to optimize and improve the segment triangulation software used for the GGT analysis. M.H.D.L. thanks the five living Van Beethoven males and their families for providing DNA samples, Walter Sluydts for his valued help in contacting some of these families, and several FamilyTreeDNA customers for providing their genealogical data. J.D.W. thanks the three living descendants of Karl van Beethoven and their families for providing DNA samples. A.S. has received a scholarship from the BONFOR program of the Medical Faculty of the University of Bonn (Gerok grant, account number O-149.0134). T.J.A.B. thanks Clare Hall, Cambridge, for providing funding for the sequencing of the living Van Beethovens; Kevin Brown and the American Beethoven Society for providing research scholarships; Katerina Harvati for permitting access to ancient and historical hair samples during testing of the laboratory protocol; Alison Galloway for comments on examinations of Beethoven's skeletal remains; Stephan Schiffels for comments on preseq; Harald Ringbauer and Amy Williams for comments on pedSIM; Henrike O. Heyne for comments on polygenic risk scoring; Verena Schünemann for comments on early laboratory protocol testing; Kirsten Bos for comments on metagenomic screening; Theodore Albrecht for comments on Beethoven's conversation books, Grant Cook for comments on sample provenance histories; and Susanna Sabin for wet lab assistance. Computational analyses were carried out using the facilities of the High-Performance Computing Center of the University of Tartu and the Max Planck Institute for the Science of Human History in Jena.

## AUTHOR CONTRIBUTIONS

T.J.A.B., W.R.M., and J.K. conceptualized the research. T.J.A.B., J.K., W.R.M., T.K., M.H.D.L., M.M.N., A. Schmidt, and D.K. designed the research. T.J.A.B., P.A.M., A.K., and A. Schmidt visualized the research. W.R.M., T.J.A.B., M.H.D.L., M. Boecker, and J.D.W. acquired samples. T.J.A.B., W.R.M., and T.K. acquired funding. T.J.A.B., E.R., G.B., A.K., C.F., A.T., M. Burri, J.O., R.B., and F.A. performed the laboratory work. T.J.A.B., A. Schmidt, A.K., G.R., P.A.M., M.S., K.P., T.K., A. Szolek, A.P., R.B., C.M., and S.C. performed bioinformatics analyses. W.R.M., J.D.W., M.H.D.L., J.R., and T.J.A.B. performed historical research. T.K., R.D.A., M.M.N., D.K., J.K., and W.R.M. supervised the research. T.J.A.B., T.K., and R.D.A. wrote the original draft. T.J.A.B., R.D.A., T.K., A. Schmidt, C.M., A.K., G.R., P.A.M., M.H.D.L., J.D.W., R.B., A. Szolek, M.S., C.S., K.P., D.K., W.R.M., M.M.N., and J.K. reviewed and edited the final draft with contributions from all authors.

## DECLARATION OF INTERESTS

T.J.A.B. has received scholarships from the American Beethoven Society in support of his graduate studies. G.R., M.S., and P.A.M. are employees of GeneByGene. G.R. and M.S. hold stock options in MyDNA, Inc. M.M.N. has received fees for membership in an advisory board from HMG Systems Engineering GmbH (Fürth, Germany), for membership in the Medical-Scientific Editorial Office of the Deutsches Ärzteblatt, and for serving as
a consultant for EVERIS Belgique SPRL in a project of the European Commission (REFORM/SC2020/029). M.M.N. receives salary payments from Life & Brain GmbH and holds shares in Life & Brain GmbH.

Received: May 29, 2022

Revised: October 11, 2022

Accepted: February 13, 2023

Published: March 22, 2023

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#EP0052</td></tr><tr><td>10% Criterion TBE-Urea Polyacrylamide Gel, 18 well, 30 µl</td><td>BioRad</td><td>Cat#3450089</td></tr><tr><td>10x GeneAmp PCR Gold Buffer and MgCl2</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#4379874</td></tr><tr><td>2-Propanol</td><td>Merck</td><td>Cat#1070222511</td></tr><tr><td>20% SDS Solution</td><td>Serva</td><td>Cat#39575.01</td></tr><tr><td>20x SCC Buffer</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#AM9770</td></tr><tr><td>2x TBE-Urea Sample Buffer</td><td>BioRad</td><td>Cat#1610768</td></tr><tr><td>3M Sodium Acetate buffer pH 5.2</td><td>Sigma-Aldrich</td><td>Cat#S7899-500ML</td></tr><tr><td>5 M Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>Cat#S5150-1L</td></tr><tr><td>5 M Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>Cat#S5150-1L</td></tr><tr><td>ATP</td><td>New England Biolabs</td><td>Cat#P0756S</td></tr><tr><td>BSA 20 mg/ml</td><td>New England Biolabs</td><td>Cat#B9000S</td></tr><tr><td>Bst 2.0 DNA Polymerase</td><td>New England Biolabs</td><td>Cat#M0537S</td></tr><tr><td>Buffer Tango 10x</td><td>Thermo Scientific</td><td>Cat#BY5</td></tr><tr><td>D1000 Reagents</td><td>Agilent Technologies</td><td>Cat#5067-5583</td></tr><tr><td>D1000 ScreenTapes</td><td>Agilent Technologies</td><td>Cat#5067-5582</td></tr><tr><td>Denhardt's solution</td><td>Sigma-Aldrich</td><td>Cat#D9905-5MI</td></tr><tr><td>dNTP Mix 25 mM each</td><td>Thermo Scientific</td><td>Cat#R1121</td></tr><tr><td>DTT 5g</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#R0861</td></tr><tr><td>Dynabeads MyOne Streptavidin C1</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#65002</td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#65601</td></tr><tr><td>Ethanol</td><td>Merck</td><td>Cat#1009832511</td></tr><tr><td>FastAP Thermosensitive Alkaline Phosphatase</td><td>Thermo Scientific</td><td>Cat#EF0652</td></tr><tr><td>GeneRuler Ultra Low Range DNA Ladder</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#SM1211</td></tr><tr><td>Guanidine hydrochloride</td><td>Sigma-Aldrich</td><td>Cat#G3272-500g</td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent Technologies</td><td>Cat#600679</td></tr><tr><td>Human Cot-1 DNA</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#15279011</td></tr><tr><td>Klenow fragment</td><td>Thermo Scientific</td><td>Cat#EP0052</td></tr><tr><td>Oligo Length Standards 20/100 Ladder</td><td>IDT</td><td>Cat#51-05-15-02</td></tr><tr><td>PEG 8000 Powder, Molecular Biology Grade</td><td>Promega</td><td>Cat#V3011</td></tr><tr><td>Pfu Turbo Cx Hotstart DNA Polymerase</td><td>Agilent Technologies</td><td>Cat#600412</td></tr><tr><td>Polyethyleneglycol 8000 50%</td><td>Jena Bioscience</td><td>Cat#CSS-256</td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>Cat#P2308-100MG</td></tr><tr><td>Sera-Mag Speed CM</td><td>GE Healthcare Lifescience</td><td>Cat#GE65152105050250</td></tr><tr><td>Silica Magnetic Beads</td><td>G-Bioscience</td><td>Cat#GENO786-915</td></tr><tr><td>Sodiumhydroxide Pellets</td><td>Fisher Scientific</td><td>Cat#10306200</td></tr></tbody></table>

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**Continued**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>SYBR Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO)</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#S33102</td></tr><tr><td>T4 DNA Polymerase</td><td>New England Biolabs</td><td>Cat#M0203L</td></tr><tr><td>T4 DNA-Ligase</td><td>Thermo Scientific</td><td>Cat#EL0013</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England Biolabs</td><td>Cat#M0201L</td></tr><tr><td>T4 RNA ligation Buffer</td><td>New England Biolabs</td><td>Cat#B0216L</td></tr><tr><td>TE buffer pH 8.0 low EDTA</td><td>Panreac AppliChem</td><td>Cat#A8569,0500</td></tr><tr><td>Tris-HCl, pH 8.0</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#15568025</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>Cat#P9416-50ML</td></tr><tr><td>UltraPure Salmon Sperm DNA Solution</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#15632011</td></tr><tr><td>UltraPure TBE Buffer, 10X</td><td>Thermo Fisher Scientific (Life Technologies)</td><td>Cat#15581044</td></tr><tr><td>USER Enzyme</td><td>New England Biolabs</td><td>Cat#M5505L</td></tr><tr><td>Water HPLC Plus</td><td>Sigma-Aldrich</td><td>Cat#34877-2.5L-M</td></tr></tbody></table>

**Critical commercial assays**

<table><tbody><tr><td>Oragene OG-500 Saliva collection kit</td><td>Oragene DNA</td><td>N/A</td></tr><tr><td>Infinium GSA-24 v2.0 Kit</td><td>Illumina</td><td>Cat#20024446</td></tr><tr><td>High Pure Viral Nucleic Acid Large Volume Kit</td><td>Roche</td><td>Cat#5114403001</td></tr><tr><td>DyNAmo Flash SYBR Green qPCR Kit</td><td>Thermo Fisher Scientific</td><td>Cat#F415L</td></tr><tr><td>MinElute PCR Purification Kit</td><td>QIAGEN</td><td>Cat#28006</td></tr><tr><td>Quick Ligation Kit</td><td>New England Biolabs</td><td>Cat#M2200L</td></tr><tr><td>Oligo aCGH/Chip-on-Chip Hybridization Kit</td><td>Agilent Technologies</td><td>Cat#5188-5220</td></tr><tr><td>HiSeq 4000 SBS Kit (50/75 cycles)</td><td>Illumina</td><td>Cat#FC-410-1001/2</td></tr><tr><td>HiSeq 3000/4000 PE Cluster Kit</td><td>Illumina</td><td>Cat#PE-410-1001</td></tr><tr><td>QIAquick Nucleotide Removal Kit</td><td>Quiagen</td><td>Cat#28304</td></tr><tr><td>Oligo aCGH/Chip-on-Chip Hybridization Kit</td><td>Agilent Technologies</td><td>Cat#5188-5220</td></tr><tr><td>NextSeq 500 High Output Flow Cell Cartridge v2</td><td>Illumina</td><td>Cat#15065973</td></tr><tr><td>DNBseq (100 cycles) PE Kit</td><td>BGI</td><td>N/A</td></tr></tbody></table>

**Deposited data**

<table><tbody><tr><td>Human reference genome NCBI build 37, GRCh37</td><td>Genome Reference Consortium</td><td>http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/</td></tr><tr><td>Downloadable genotypes of present-day and ancient DNA data</td><td>Allen Ancient DNA Resource</td><td>https://reich.hms.harvard.edu/allen-ancient-dna-resource-aadrdownloadable-genotypes-present-day-and-ancient-dna-data</td></tr><tr><td>Thousand genomes project phase 3 release</td><td>The International Genome Sample Resource</td><td>http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase3/</td></tr><tr><td>FamilyTreeDNA consumer database</td><td>goranr@genebygene.com</td><td>N/A</td></tr><tr><td>GWAS summary statistics: Cirrhosis</td><td>Emdin et al. 2021<sup>43</sup></td><td>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035329/bin/NIHMS1680867-supplement-1.pdf</td></tr><tr><td>GWAS summary statistics: Systemic lupus erythematosus</td><td>Julià et al. 2018<sup>38</sup></td><td>http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST005001-GCST006000/GCST005831/</td></tr><tr><td>GWAS summary statistics: Primary biliary cirrhosis</td><td>Cordell et al. 2015<sup>46</sup></td><td>http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST003001-GCST004000/GCST003129/</td></tr><tr><td>GWAS summary statistics: Primary sclerosing cholangitis</td><td>Ji et al. 2016<sup>47</sup></td><td>http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST004001-GCST005000/GCST004030/</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>GWAS summary statistics: Crohn disease</td><td>de Lange et al. 2017<sup>37</sup></td><td>http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST004001-GCST005000/GCST004131/</td></tr><tr><td>GWAS summary statistics: Ulcerative colitis</td><td>de Lange et al. 2017<sup>37</sup></td><td>http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST004001-GCST005000/GCST004131/</td></tr><tr><td>GWAS summary statistics: Irritable bowel syndrome</td><td>Eijsbouts et al. 2021<sup>49</sup></td><td>http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST90016001-GCST90017000/GCST90016564/</td></tr><tr><td>HLA reference panel</td><td>Szolek et al. 2014<sup>78</sup></td><td>https://github.com/FRED-2/OptiType</td></tr><tr><td>HBV reference genome; genotype D</td><td>GenBank</td><td>GenBank: NC003977</td></tr><tr><td>HBV reference genome; genotype F</td><td>GenBank</td><td>GenBank: FJ657525</td></tr><tr><td>Phylotree Build 17</td><td>van Oven 2015<sup>79</sup></td><td>https://www.phylotree.org/builds/mtDNA_tree_Build_17.zip</td></tr><tr><td>Mediavel Bavarian reference panel</td><td>Veeramah et al. 2018<sup>80</sup></td><td>https://www.ebi.ac.uk/ena browser/view/PRJEB23079?show=reads</td></tr><tr><td>Hg19</td><td>UCSC Genome Browser</td><td>http://hgdownload.cse.ucsc.edu/goldenpath/hg19/bigZips/</td></tr><tr><td>hg38</td><td>UCSC Genome Browser</td><td>ftp://hgdownload.soe.ucsc.edu/goldenPath/hg38/</td></tr><tr><td>UK BioBank</td><td>UK BioBank</td><td>Approved Research ID: 54169 'The Beethoven Genome Project'</td></tr><tr><td>ClinVar build 2021-03-15</td><td>Clinical Genome Resource</td><td>https://ftp.ncbi.nlm.nih.gov/pub/clinvar/</td></tr><tr><td>OMIM</td><td>Online Mendelian Inheritance in Man</td><td>https://omim.org/downloads</td></tr><tr><td>Deafness Variation Database version 9, build 2021-01-04</td><td>Deafness Variation Database</td><td>https://deafnessvariationdatabase.org/public/releases/v9/</td></tr></tbody></table>

Oligonucleotides

<table><tr><td>IS5 (AATGATACGGCGACCACCGA)</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>IS6 (CAAGCAGAAGACGGCATACGA)</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>IS7 (ACACTCTTCCCTACACGACGC)</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>IS8 (GTGACTGGAGTTCAGACGTGTGC)</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>BO4.P7.part1.R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT[Phos])</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>BO6.P7.part2.R (CAAGCAGAAGACGGCATACGAGAT[Phos])</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>BO8.P5.part1.R (GTGTAGATCTCGGTGGTCGCCGTATCATT[Phos])</td><td>Sigma-Aldrich</td><td>N/A</td></tr><tr><td>BO10.P5.part2.R (AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT[Phos])</td><td>Sigma-Aldrich</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>Bcftools version 1.10.2</td><td>Li, 2011<sup>81</sup></td><td>https://samtools.github.io/bcftools/</td></tr><tr><td>Burrows-Wheeler Aligner (BWA) version 0.7.12</td><td>Li and Durbin, 2009<sup>82</sup></td><td>http://bio-bwa.sourceforge.net/</td></tr><tr><td>Samtools version 1.9</td><td>Li et al. 2009<sup>83</sup></td><td>http://samtools.sourceforge.net/</td></tr><tr><td>GATK version 3.5</td><td>McKenna et al. 2010<sup>84</sup></td><td>https://software.broadinstitute.org/gatk/</td></tr><tr><td>mapDamage2.0</td><td>Jónsson et al. 2013<sup>28</sup></td><td>https://ginolhac.github.io/mapDamage/</td></tr><tr><td>Haplogrep2.0</td><td>Weissensteiner et al. 2016<sup>85</sup></td><td>http://haplogrep.uibk.ac.at/</td></tr><tr><td>PLINK version 1.9</td><td>Purcell et al. 2007<sup>86</sup></td><td>http://pngu.mgh.harvard.edu/purcell/plink/</td></tr><tr><td>Schmutzi</td><td>Renaud et al. 2015<sup>87</sup></td><td>https://github.com/grenaud/schmutzi</td></tr><tr><td>IGV version 2.14</td><td>Thorvaldsdóttir et al. 2013<sup>88</sup></td><td>https://software.broadinstitute.org/software/igv/</td></tr><tr><td>PRSice2.0 version 2.3.3</td><td>Choi et al. 2019<sup>89</sup></td><td>https://www.prsice.info</td></tr><tr><td>Variant Effect Predictor</td><td>McLaren et al. 2016<sup>90</sup></td><td>http://grch37.ensembl.org/Homo_sapiens/Tools/VEP/Edit</td></tr></table>

(Continued on next page)
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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>pedSIM</td><td>Caballero et al. 2019<sup>32</sup></td><td>https://github.com/williamslab/ped-sim</td></tr><tr><td>snpAD version 0.3.4</td><td>Prüfer, 2019<sup>20</sup></td><td>https://bioinf.eva.mpg.de/snpAD/</td></tr><tr><td>FlashPCA2.0</td><td>Abraham et al., 2017<sup>91</sup></td><td>https://academic.oup.com/bioinformatics/article/33/17/2776/3798630</td></tr><tr><td>R</td><td>R Core Team, 2021<sup>92</sup></td><td>https://www.r-project.org</td></tr><tr><td>AdaptorRemoval</td><td>https://github.com/MikkelSchubert</td><td>https://github.com/MikkelSchubert/adapterremoval</td></tr><tr><td>ANGSD</td><td>Korneliussen et al. 2014<sup>93</sup></td><td>http://www.popgen.dk/angsd/index.php/ANGSD</td></tr><tr><td>MALT version 0.3.8</td><td>Herbig et al. 2016<sup>56</sup></td><td>https://software-ab.informatik.uni-tuebingen.de/download/malt/welcome.html</td></tr><tr><td>READ</td><td>Kuhn et al., 2018<sup>94</sup></td><td>https://bitbucket.org/tguenther/read</td></tr><tr><td>BEAGLE version 5.1</td><td>Browning et al. 2011<sup>95</sup></td><td>https://faculty.washington.edu/browning/beagle/b5_1.html</td></tr><tr><td>ADMIXTURE</td><td>Alexander et al. 2009<sup>22</sup></td><td>https://www.genetics.ucla.edu/software/admixture/</td></tr><tr><td>Preseq</td><td>Daley and Smith, 2014<sup>96</sup></td><td>https://github.com/smithlabcode/preseq</td></tr><tr><td>EAGER version 1.92.38</td><td>Peltzer et al. 2016<sup>97</sup></td><td>https://eager.readthedocs.io/en/latest/</td></tr><tr><td>EIGENSOFT</td><td>Patterson et al. 2006<sup>98</sup></td><td>https://github.com/DReichLab/EIG</td></tr><tr><td>IBIS</td><td>Seidman et al. 2020<sup>31</sup></td><td>https://github.com/williamslab/ibis</td></tr><tr><td>Rx_identifier</td><td>Mittnik et al. 2016<sup>99</sup></td><td>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5051676/bin/pone.0163019.s003.r</td></tr><tr><td>leehom version 1.1.5</td><td>Renaud et al. 2014<sup>100</sup></td><td>https://github.com/grenaud/leeHom</td></tr><tr><td>PATHd8</td><td>Britton et al. 2007<sup>101</sup></td><td>https://rdrr.io/github/fkeck/diatobc/man/pathd8.html</td></tr><tr><td>BEAST version 2.5.2</td><td>Bouckaert et al. 2014<sup>102</sup></td><td>https://www.beast2.org</td></tr><tr><td>MEGAN version 6.13.1</td><td>Huson et al. 2016<sup>103</sup></td><td>https://software-ab.informatik.uni-tuebingen.de/download/megan6/welcome.html</td></tr><tr><td>Picard tools</td><td>http://broadinstitute.github.io/picard/</td><td>http://broadinstitute.github.io/picard/</td></tr><tr><td>DamageProfiler</td><td>Neukamm et al. 2021<sup>104</sup></td><td>https://github.com/Integrative-Transcriptomics/DamageProfiler</td></tr><tr><td>MAFFT version 7.475</td><td>Katoh and Standley, 2013<sup>105</sup></td><td>https://mafft.cerc.jp/alignment/software/</td></tr><tr><td>Gblocks</td><td>Castresana, 2000<sup>106</sup></td><td>https://home.cc.umanitoba.ca/~psgendb/doc/Castresana/Gblocks_documentation.html</td></tr><tr><td>RAxML version 8.2.12</td><td>Stamatakis, 2014<sup>58</sup></td><td>https://github.com/stamatak/standard-RAxML</td></tr><tr><td>OptiType</td><td>Szolek et al. 2014<sup>78</sup></td><td>https://github.com/FRED-2/OptiType</td></tr></tbody></table>

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Johannes Krause (krause@eva.mpg.de).

Materials availability

This study did not generate new unique reagents.

Data and code availability

* Sequence data generated from the five matching locks have been deposited at the European Nucleotide Archive under the accession number ENA: PRJEB56343 and are publicly available as of the date of publication. The raw data from non-matching locks of hair reported in this study cannot be deposited in a public repository because the identity of the donors could not be confirmed and consent could not be provided.
* Original code for the Geo-genetic triangulation method has been deposited at Dryad: https://datadryad.org/stash and is publicly available as of the date of publication. https://doi.org/10.5061/dryad.k0p2ngfc4.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## EXPERIMENTAL MODEL AND SUBJECT DETAILS

### Beethoven hair samples

We identified 34 locks of hair attributable to Ludwig van Beethoven that have been described in primary sources and/or reside in public and private collections around the world, of which 25 are believed to have independent provenances (Table S1; Methods S1A–S1H). We tested eight locks of hair of independent provenance, namely the Müller (ca. 1815-1820; Methods S1A), Bermann (ca. 1821; Methods S1B), Halm-Thayer (April 25th, 1826; Methods S1C), Moscheles (March 24th, 1827; Methods S1D), Cramolini-Brown (March 27th, 1827; Methods S1E), Stumpff (March 26th-28th, 1827; Figure 3; Methods S1F), Hiller (March 27th, 1827; Methods S1G) and Kessler Locks (October 13th-23rd, 1863 or June 22nd, 1888; Methods S1H). Of the five genetically matching locks of hair, the Halm-Thayer and Stumpff Locks bear perfect chains of custody, the Müller and Moscheles Locks have incomplete chains of custody, but intact documentary links to their original provenances, while the Bermann Lock has no documentation attesting to its initial acquisition (Figure 1). The Müller, Moscheles and Stumpff Locks remain affixed to or associated with documents bearing the signatures of their original name-sake recipients (Methods S1A, S1D, and S1F). Of the non-matching locks of hair, the Hiller Lock lacks a contemporary account of its acquisition, while Ludwig Cramolini's (1805-1884) account of the Cramolini Lock's acquisition was first published after his death in 1907. The provenance of the Hiller Lock is unknown prior to the 1880s, and that of the Cramolini-Brown Lock unknown prior to 2012. The Kessler Lock was reputedly recovered during one of Beethoven's two exhumations in 1863 or 1888, with the earliest known reference to its acquisition dating to 1948.

Hair locks were named according to their first intended recipient or long-term owner, rather than the initial acquirer, who in a number of cases acted only as a brief intermediary (eg. Anton Schindler for the Moscheles Lock; Johann Valentin Schick and Johann Baptist Streicher for the Stumpff Lock). In cases where either a lock of hair is documented to have been split into two or more locks, or additional locks of hair bearing the same name are known to exist elsewhere, the last name of a subsequent confirmed owner is appended to the name of the first owner to specify the lock of hair in question (eg. Halm-Thayer Lock; Cramolini-Brown Lock). Below we describe the provenance histories of each tested lock.

### Müller Lock

The Müller Lock has a moderately documented historical provenance, lacking a clear first-hand account of its original acquisition from Beethoven, in addition to having an incomplete chain of custody. An accompanying provenance (Methods S1A) note nonethe-less associates the lock with two individuals, Nannette Streicher and Elise Müller, whose interactions with Beethoven are well documented within the time-frame of the lock's documented acquisition, on November 4th, 1820, by Elise Müller. The Müller Lock would eventually be acquired by the renowned collector of Beethoven memorabilia, Hans Conrad Bodmer, after which it would be bequeathed to the Beethoven-Haus Bonn, where it permanently resides. The Müller Lock is one of three locks of hair tested which remains associated with a provenance note bearing the signature of its namesake owner. Genetic testing has demonstrated that the Müller Lock is almost certainly authentic.

The Müller Lock is currently part of the collections of the Beethoven-Haus, Bonn, where it is cataloged and briefly described under the identification HCB V 12 (Hans Conrad Bodmer collection):

Lock of Beethoven's hair; brown strands with some white hairs; enclosed in [folded] paper and wrapped in two paper covers. On the inner paper cover [is found] the handwritten remark: "Beethoven's hair / obtained by [Mrs.] Streicher / on Nov. 4. 1820'; on the outside paper cover [exists] the handwritten remark "Beethoven's hair. / Elise Müller'; the second signature of this piece: R 1 b.

1815-1820

Concerning the dating: Nannette Streicher was particularly close to Beethoven after 1815. It remains an open question, whether she obtained the strands of hair from Beethoven at the request of Elise Müller, or whether she had already possessed it earlier.

Provenance: Nannette Streicher in Vienna; Elise Müller in Bremen; unknown owner(s); Hans Conrad Bodmer in Zürich.

Date of accession: 1956, Hans Conrad Bodmer Bequest.

[translation William Meredith]

The first name in the set of paper covers, Nannette Streicher (1769-1833; née Stein), was an esteemed fortepiano manufacturer and important figure in Beethoven's life. She was born in Augsburg in 1769 to the famous organ and fortepiano manufacturer, Johann
Andreas Stein (1728-1792). After her father's death, she took over the firm. In 1794 she married Johann Andreas Streicher (1761-1833) and they moved, with her brother Matthäus, to Vienna. In 1802 Nannette and her brother separated as business partners and the firm was renamed "Nannette Streicher, née Stein." Nannette probably first met Beethoven in Augsburg in 1787 when he visited the city; her husband, who frequently assisted Beethoven in his business dealings, stated in letters that he had known the composer since 1788. Unusually for Beethoven, his relationship with the couple has been described as "uniformly serene", but Nannette was especially helpful to him in the years 1817-18, as documented in numerous letters and notes. At this time he was living near them in the Landstraße, and Nannette greatly assisted Beethoven in advising him on how to hire a housekeeper and a kitchen maid. Professionally, he greatly admired the fortepianos her firm produced, writing in 1817 that they had been his preferred instruments since 1809, an extraordinary statement.¹ That same year, he asked them to rent him one of their instruments that had been specially altered to accommodate his hearing loss.

The second name in the set of paper covers, Elise Müller (1782-1849), was a pianist, piano teacher, and song composer. She was born to Dr. Wilhelm Christian Müller (1752-1831), an important teacher and writer on music, and his wife Maria Amalia, in Bremen. Elise gave her first public concert at the age of ten; in 1807 the famous Leipzig music periodical, the *Allgemeine musikalische Zeitung*, said that her playing was distinguished by "fluency, assuredness, and expression."¹⁰⁷ She revered the works of Beethoven, which were one of her specialties, and her father characterized her as "Pianoforte-Spielerin Beethoven'scher Werke" (piano player of Beethoven's works). After Dr. Müller retired in 1817, he traveled extensively. In 1820 he and his daughter traveled to Vienna on their way to Italy, arriving in the city in October.

During their time in Vienna, the Müllers visited Beethoven twice. The first visit was on October 26th, documented in a book about Dr. Müller's travels.¹⁰⁸,¹⁰⁹ The second is an account of Dr. Müller's observations of Beethoven eating in a restaurant, published in an obituary for Beethoven in the *Allgemeine musikalische Zeitung* No. 21 on May 23rd, 1827, entitled "Etwas über Ludw. van Beethovcn" ("Some Things About Ludwig van Beethovcn").¹⁴,¹¹⁰ In his book, Müller stated that he and his daughter had been in correspondence with Beethoven for several years before meeting him. It is likely that a statement in the diary of Fanny Giannatasio on January 31st, 1817, that Beethoven had received letters and a gift from a lady in Bremen, refers to Elise.¹,¹¹¹ Little of this correspondence survives, except a collection of poems commemorating Beethoven's 49th birthday authored by Elise Müller and Dr. Carl Iken, editor of the *Bremer Zeitung*, in December of 1819,¹¹² and a brief note Beethoven wrote during the Müllers' stay in Vienna.¹ In 1822 Beethoven asked the Berlin publisher of his Sonata in E Major, Op. 109, to send Elise a copy of the first edition.¹¹² The date of Elise Müller's acquisition of the lock of hair from Nannette Streicher on November 4th, 1821, was the Müllers' last day in Vienna during their trip.¹⁰⁸

The Beethoven-Haus' provenance description is careful in its dating of when Nannette cut the lock of hair, only giving a probable five-year timespan when she and the composer were closely connected. The handwritten note thus only gives a *terminus ante quem* for the date of the cutting. The date does, however, indicate that Elise received the locket from Nannette that day, and it would have been natural for a dedicated pianist and piano teacher to visit the famous manufacturer at her shop, which also served as a famous music salon.

Nothing is known about the provenance of the lock between its ownership by Elise in 1820 (though one might presume it was in her collection until her death in 1849) and the unknown date upon which it was acquired by the great Swiss doctor and Beethoven collector Hans Conrad Bodmer (1891-1956). Bodmer also owned a lock of hair given to Robert Schumann in 1845 (HCB Br 115), one that belonged originally to Beethoven's friend Karl Holz (HCB V 11), one that belonged to Peter Simrock (HCB BBi 11/28), a lock from 1825 given to Schlesinger (HCB V 6), a lock given by "Cramolini in Darmstadt" to someone (HCB V 10), and a lock with a miniature portrait of the composer (HCB Mh 49).

Two DNA extractions, each consisting of 25cm of hair, were performed on the Müller Lock, for a total of 50cm of hairs sampled.

## Bermann Lock

The original provenance of the Bermann Lock is almost entirely unknown. It was most likely acquired by Jeremias Bermann, a publisher of one of Beethoven's compositions in 1821, whose direct associations with Beethoven are likewise largely unknown. The American-born Beethoven biographer, Alexander Wheelock Thayer (1817-1897), records receiving the lock from a "Mr. Bermann" in 1851 during Thayer's biographical research on Beethoven. The Bermann Lock would remain within Thayer's family until its acquisition by the American Beethoven Society in 2017. Genetic testing has demonstrated that the Bermann Lock is almost certainly authentic.

The Bermann Lock's first documented owner, a "Mr. Bermann," is believed to have either been Jeremias Bermann (1770-1855), or Jeremias' son, Joseph Bermann (1810-1886). Jeremiah had married Anna Eder (1790-1859), the daughter of Joseph Eder (1760-1835), a publisher of several of Beethoven's early compositions in the late 1790s. The two most significant of the father's editions are the first edition of the three Fortepiano Sonatas, Op. 10 and the first Titelauflage of the *Pathétique* Sonata, Op. 13. The firm's specialty was the printing of visiting cards and New Year's cards. The Edersche Kunsthandlung was in a building called Zum schwarzen Elephanten (Black Elephant) on the famous Graben street.

In 1811, Bermann joined Eder's publishing firm, taking over operations in 1815 and publishing further Beethoven compositions. In June of 1821 Jeremias Bermann published the first edition of Beethoven's Bagatelles, Op. 119, Nos. 7-11 (published as Nos. 28-32), as part of the third volume of the Wiener Piano-Forte-Schule von Friedrich Starke, Kapellmeister. The manuscript of No. 7 is dated "am 1 = ten Jenner 1821." This date helps us locate Bermann's direct connection to Beethoven to between late 1820 and the early summer of 1821. Unfortunately, there is a gap in the conversation books and correspondence of Beethoven from 1820-22, and
Bermann is not mentioned in any of the surviving conversation books, nor is he mentioned in any Beethoven letter. Thus, while we know Bermann had a direct connection to the composer in 1820-21, there is no surviving document connecting them or detailing how or when Bermann acquired the lock. A New York Tribune Illustrated Supplement article from February 13th, 1898 states describing the recent auctioning of Thayer's library, and details that on May 15th, 1851, Alexander Wheelock Thayer visited the Edersche Kunsthandlung, purchasing a number of Beethoven autographs and acquiring the Bermann Lock. By 1851, the ownership of the firm, then named Jeremias Bermann & Sohn, had been passed on to Jeremias' son, Joseph.

Although we can deduce that Bermann most likely directly interacted with Beethoven ca. 1821 concerning the publishing of Op. 119, Nos. 7-11, the lock itself may have been acquired at any time between 1815 and 1821, and possibly later than 1821. Thayer records in an accompanying provenance note, "This lock of Beethoven's hair received from a Mr. Bermann. May 15, 1851," (Methods S1B).

The picture frame, containing both the Bermann Lock and the Halm-Thayer Lock described below, as well as several other items belonging to Alexander Wheelock Thayer including a military uniform and hat, dress sword, a shirt believed to have belonged to Beethoven, and numerous documents, were acquired as a single collection from living descendants of Thayer in the summer of 2017 by American Beethoven Society members Kevin Brown, William Meredith, and other members of the American Beethoven Society. Two DNA extractions, each consisting of 25cm of hair, were performed on the Bermann Lock, for a total of 50cm of hairs sampled. The Bermann Lock is currently in the collection of American Beethoven Society member Kevin Brown.

## Halm-Thayer Lock

Among the eight locks of hair tested, the Halm-Thayer Lock is peerless in the completeness of his provenance history, having a perfectly documented chain of custody, as well as a detailed first-hand account of its acquisition (Figure 1). Evidence of the interactions and events leading up to its original acquisition are furthermore corroborated in numerous letters and conversation book entries. The Halm-Thayer Lock is the only lock of hair tested which is documented to have been received personally from Beethoven. Genetic testing has demonstrated that the Halm-Thayer Lock is almost certainly authentic.

The lock originated between March and April of 1826 as a practical joke perpetrated by Beethoven's friend and then-secretary, Karl Holz (1798-1858), on the wife of one of Beethoven's acquaintances, Anton Halm (1789-1872). Karl Holz was a close friend of Beethoven's and a skilled violinist, from 1823 playing second violin in the famous Schuppanzigh Quartet, which premiered many of Beethoven's string quartets. During the years 1825 to 1826, Holz acted as an unpaid secretary to Beethoven, replacing Beethoven's former secretary, Anton Schindler (1795-1864). Anton Halm was formerly a Lieutenant in the Austrian military who, in 1811, resigned his commission in order to pursue a career as a pianist and composer.¹¹³ At the time of the lock's acquisition, Halm was completing a four-hand piano transcription of Beethoven's *Große Fuge*, Op. 133, having been commissioned to do so by Mathias Artaria (1793-1835), cousin to the founder of the eminent publishing house, Artaria & Co.¹¹² Halm's wife wanted a lock of Beethoven's hair, and Halm asked Holz to convey the wish to Beethoven in a conversation book. Several days later, Frau Halm received a lock of hair, which Holz had removed from a goat. On April 24th, Halm wrote to Beethoven that he would deliver the manuscript of the piano transcription to him, and when he visited the next day, Beethoven told Halm they had been deceived and gave Halm a white sheet of paper with "a significant quantity of his hair," saying "Das sind meine Haare!" ("That is my hair!").¹¹⁴ Halm regarded this as a triumph but his wife was indignant with Holz about the dirty trick.

Several accounts of Halm's acquisition of the lock exist. The most authoritative account was told to Alexander Wheelock Thayer by Anton Halm, presumably during the same interview in 1859 when Thayer records receiving the lock from Halm. This account was published posthumously in 1908 from Thayer's notes in the fifth volume of his biography of Beethoven:

> Around the time of the preparations for and performance of the B-flat major quartet, there took place a mischievous joke, whereby a liberty was taken with a lock of Beethoven's hair.

> We have the first-hand account of the immediate recipient, as told by the piano player and composer Anton Halm to Thayer, who recorded it in his notes. Schindler's narrative is somewhat corrected and supplemented by this account. In the rehearsal for Schuppanzigh's concert, Frau Halm, "née Sebastiani from Trier, whom Beethoven always referred to as his fellow countrywoman", was also present. She had wished to own a lock of Beethoven's hair, a favor of which few could boast; Beethoven usually replied: "Leave me alone!"

> "My wife asked me to ask Beethoven for a lock of hair during this favorable occasion. But since Beethoven could not hear, and several people were present, I declined out of politeness to negotiate personally with Beethoven through his notebook. I therefore asked Karl Holz to present my wife's request to Beethoven. After a few days, my wife received a lock of hair from a third party, which was supposed to be Beethoven's hair."

In the interim, Beethoven asked Halm to arrange the quartet fugue for the pianoforte four-hands.

> "Meanwhile, Karl Groß, an accomplished amateur cellist, had asked me, shrugging his shoulders: 'Who knows whether the hair is genuine?,' And yet I had no suspicions. After the piano arrangement was finished, I brought it to [Beethoven]." [...]
"When I was about to leave, he met me with a fearfully serious expression on his face, saying: 'You have been cheated with the lock of hair! You see, I am surrounded by such terrible creatures that they put aside all the respect they owe respectable people. You have the hair of a nanny-goat.' And speaking like this, he gave me a significant quantity of his hair in a sheet of white paper, which he had cut entirely from the back of his head, with the words: 'That is my hair!' - He had probably cut off the hairs from behind because they were still black there, while in front everything was already snow-white. - So I went home with this seldom-received gift, in triumph. - Not so my wife. She was indignant about Karl Holz's wickedness and immediately wrote a letter appropriate to the circumstances."

"One or two years later, my wife was standing by Beethoven's open grave, on the 29th of March, 1827, and she saw Holz, standing on the other side weeping, too ashamed to look at her directly. Touched by that, she held out her hand to him across the grave as reconciliation."¹¹⁴ [translation Tristan Begg & Robert Attenborough]

Halm's account begins during the first rehearsals of Beethoven's String Quartet No. 13 in B major, which would first be premiered by the Schuppanzigh Quartet on March 21st, 1826. ¹¹⁵ This account is corroborated by numerous lines of evidence, including letters and conversation book entries. The involvement of Karl Holz is recorded in an entry in Beethoven's conversation books around March 31st, 1826, confirming that Holz had indeed given a lock of hair to Halm's wife:

> I have already given the hair to Halm. ¹¹⁶ [translation Tristan Begg]

In a conversation book entry on April 16th, Halm confirms his receipt of the initial, inauthentic lock of hair, and, on behalf of his wife, thanks Beethoven for the lock:

> My wife respectfully thanks you as a fellow countrywoman for the exceedingly dear memento (the hairs), and if we are not too irritating, we will call on you for a visit. ¹¹⁶ [translation Tristan Begg]

Interestingly, the words “die Haare” (the hairs) were added to this entry after it was initially written; Halm may have had to clarify what the memento was.

On the 24th of April, 1826, after the completion of his arrangement for the *Große Fuge*, Op. 133 for piano four-hands, Anton Halm wrote to Beethoven, confirming their intention to meet the following day:

> I have finished your Fugue, which I have the honor of sending along, with the greatest possible diligence and care! At every bar, I was amazed at your power of harmony and its flow, as well as the musical motives that you used and their development to the point of exhaustion!

Concerning my arrangement, it was unfortunately not possible always to keep the subjects in their original shape; rather more frequently they had to be broken. Otherwise it is so brilliant, so advantageously playable, and, as I hope, still intelligible enough, that your most elevated masterwork will be acknowledged as that which it is. I shall take the liberty of delivering your manuscript at a quarter past three tomorrow afternoon, at the latest, to get your kind opinion of my arrangement. ¹¹² [translation Theodore Albrecht]

In addition to Halm's first hand account as recorded by Thayer, as well as supporting lines of evidence from the letters and conversation books, two accounts of Halm's acquisition of the lock were related by Anton Schindler in different editions of his biography of Beethoven. These accounts contain several inconsistencies, both internally, and when compared to Halm's account. The accuracy of Schindler's discrepancies cannot be confirmed. The first account, from 1840 reads:

> Though Beethoven was throughout his whole life a prey to misfortune and disappointment, yet there were moments in which he did not scruple to inflict pain and disappointment on others. Nevertheless, it must be observed that in most cases of this kind he acted under some other influence than that of his own feelings. The following circumstance occurred in the latter years of his life.

The wife of M. Halm, an esteemed piano-forte player and composer, residing in Vienna, was a great admirer of Beethoven, and she earnestly wished to possess a lock of his hair. Her husband, anxious to gratify her, applied to a gentleman who was very intimate with Beethoven, and who had rendered him some service. At the instigation of this person, Beethoven was induced to send the lady a lock of hair cut from a goat's beard; and Beethoven's own hair being very gray and harsh, there was no reason to fear that the hoax would be very readily detected. The lady was overjoyed at possessing this supposed memorial of her saint, proudly showing it to all her acquaintance; but when her happiness was at its height, some one, who happened to know the secret, made her acquainted with the deception that had been practised on her. In a letter addressed to Beethoven, her husband warmly expressed his feelings on the subject of the discovery that had been made. Convinced of the mortification which the trick must have inflicted on the lady, Beethoven determined to make atonement for it. He immediately cut off a lock of his hair, and enclosed it in a note, in which he requested the lady's forgiveness of what had occurred. The respect which
Beethoven previously entertained for the instigator of this unfeeling trick was now converted into hatred, and he would never afterwards receive a visit from him.

This is not the only instance that could be mentioned, in which our great master was influenced by vulgar-minded persons to do things unworthy of himself.¹¹⁷ [translation Ignaz Moscheles]

Anton Schindler's first account contains several inconsistencies with Halm's account. Schindler is initially uncertain of the date of the episode. Although it is unclear whether Karl Holz delivered the goat hairs directly to Frau Halm, as suggested by the conversation book entry, or through a "third party" as indicated in Halm's account, Halm's account nonetheless contests Schindler's assertion that his wife received the goat hairs directly from Beethoven. Schindler's assertion that Beethoven "never afterwards received a visit" from the "instigator", undoubtedly Karl Holz, is incorrect; numerous letters documenting their continued friendship and correspondence following this incident refute this, in particular a letter from Beethoven to Holz dating from April 26th, 1826, the day after Beethoven's meeting with Halm, which reads:

Beloved Friend!

You may rest assured that I have completely forgotten the recent incident and that it will never alter my feelings of gratitude to you. Please, therefore, do not show anything of this in your behaviour. You will always be welcome to me. I hope that next Sunday you will not despise my dinner table. [...] If you have time to visit me this week I shall be delighted if you do so. You will find me quite unchanged, the same as usual. I shall expect you on Sunday for certain.

Ever your friend

Beethoven

¹ [translation Emily Anderson]

Schindler's re-telling of the story, from 1860, omits several earlier inconsistencies. As well as providing a more specific date, Schindler clarifies that Karl Holz was the "instigator" alluded to in his first account. Consistent with his first account, Schindler again implicates Beethoven personally in the prank, while still laying the blame on Holz:

A Juvenile Trick

We promised our readers an example of our master's disposition, despite his misfortunes and frequent ill-humor, towards buf-foonery and practical joking. The wife of Anton Halm, the pianist and composer, wanted a lock of Beethoven's hair. The request was made through Karl Holz, who persuaded the master to send his ardent admirer some hairs from the beard of a goat, actu-ally not too different from Beethoven's own coarse gray hair. The lady, delighted with the memento of her musical idol, boasted far and wide of the gift, but before long she learned how she had been duped. Her husband was still deeply sensitive of his honour as a military officer, and in an aggrieved letter to our master related what he had heard. When Beethoven realized that his prank had been taken as an insult, he atoned for it by cutting off a lock of his own hair and sending it to the lady with a note begging for forgiveness. This incident occurred in 1826.¹⁴ [translation Constance S. Jolly]

Halm's account states that it was Beethoven who informed Halm personally of the deception, who atoned for it with a lock of his own hair. As Schindler was not acting as Beethoven's secretary at the time of this incident, his accounts must be interpreted with caution. In addition, considerable animosity existed between Schindler and Holz, and Schindler's three biographies are replete with slanders against his personal enemies, including Holz, which likely colored Schindler's accounts. It remains unclear from all of the surviving accounts and the surviving documentary evidence whether Beethoven was personally involved in or aware of the prank, a detail to which Anton Halm may not have been privy, or which he may not have wanted to disclose.

Alexander Wheelock Thayer received the Halm-Thayer Lock from Anton Halm on October 12th, 1859, while interviewing Halm during his biographical research on Beethoven. Thayer's pencil inscription accompanying the hairs reads, "Hair from Beethoven's head received from himself by Anton Halm, on 25th April, 1826. Given me by Ant. Halm this 12th Oct. 1859. A. W. Thayer," (Methods S1C).

In addition to the hairs Thayer received from Halm, a second lock of hair drawn from the original Halm Lock, the Halm-Epstein Lock, is also known to exist. The Halm-Epstein Lock was given to Halm's pupil, the pianist Julius Epstein (1832-1926). The Halm-Epstein Lock, along with the Erdödy, Hiller and Bernard Locks, is one of only four locks of Beethoven's hair for which results from scientific analyses have yet been published.³,⁴ It was last sold at the Sotheby's auction house on June 11th, 2019. A third lock of hair possibly originating from the Halm Lock, belonging to a "Herr Oberstleutnant August Halm," is described as part of an exhibit of Beethoven memorabilia displayed in Vienna during the 1927 centenary of Beethoven's death. The exhibition's description of the lock states: "Beethoven's hair, enclosed with a letter proving its authenticity." The whereabouts of this lock are currently unknown.

Two DNA extractions, each consisting of 25cm of hair, were performed on the Halm-Thayer Lock, for a total of 50cm of hairs sampled. The Halm-Thayer Lock, purchased together with the Bermann Lock in the summer of 2017 from Thayer's living
descendants for analysis in The Beethoven Genome Project, is currently in the collection of American Beethoven Society member Kevin Brown.

## Moscheles Lock

The Moscheles Lock has a well-documented provenance, with a first-hand account of its initial acquisition and only a single break in its chain of custody between ca. 1917 and ca. 1940. Although it was separated from its original provenance letter in 1911, it nonetheless remains affixed to a provenance note bearing the signature of its namesake recipient, Ignaz Moscheles. The Moscheles Lock was cut from Beethoven's head by his friend and secretary, Anton Schindler, on March 24th, 1827, two days before Beethoven's death, but while Beethoven was "still fully conscious"¹¹² and mailed to Ignaz Moscheles. The Moscheles Lock was retained in the Moscheles family until ca. 1917 when Moscheles' son, Felix, died childless. The Moscheles Lock resurfaced in 1940 in the possession of the Ukrainian-American violin virtuoso, Louis Krasner. The lock then remained in the Krasner family until 2010, when it was acquired by the American Beethoven Society. Genetic testing has demonstrated that the Moscheles Lock is almost certainly authentic.

On March 24th, 1827, Beethoven's amanuensis, Anton Schindler, mailed the Moscheles Lock, along with a detailed letter and newspaper clipping, to a prominent musician and friend of Beethoven in London, Ignaz Moscheles (1794-1870). The letter, which arrived on April 5th, is now on permanent loan to the Beethoven-Haus; a portion of it was published in Volume 5 of the *Harmonicon* magazine in an article announcing Beethoven's terminal illness and death in May 1827:

> I have just come from Beethoven. He is already dying, and before this letter is beyond the walls of the capital, the great light will have been extinguished forever. He is still fully conscious, however. I hasten to dispatch this letter, in order to run to him. I have just cut these hairs from his head and am sending them to you. God be with you!¹¹² [translation Theodore Albrecht]

Anton Schindler's reputation suffered a serious blow in the 1970s, when it was discovered that he had inserted more than 150 entries in his own hand (writing as himself) into Beethoven's conversation books many years after the composer's death.¹¹⁸ Though this was apparently done primarily to enhance his own reputation,¹¹⁹ each of these entries must be confirmed by another source before it can be trusted as accurate. While any claims Schindler made must be examined with great care, existing customs stamps on the provenance letter, as well as subsequent accounts by the Moscheles family, support the veracity of Schindler's account.

Following Ignaz Moscheles' death in 1870, his widow, Charlotte (1805-1889), stated in her two-volume biography of Ignaz Moscheles that all of the Beethoven memorabilia were given to their son, Felix Moscheles (1833-1917):

> The lock of Beethoven's hair, the sketches in his own hand, the metronome tempi of the 9th Symphony, and the sketch-book which Schindler sent him, were always kept and regarded as the most sacred relics, and are now in the possession of his son Felix.¹²⁰ [translation A. D. Coleridge]

Most of these items were sold as the "Moscheles Collection" before Felix's death by the famous Berlin antiquariat, Leo Liepmannssohn, on November 17th-18th, 1911. The collection consisted mainly of music manuscripts, but listed on p. 3 are items concerning "Beethoven's last illness and death, contained in letters of Beethoven and Schindler to Moscheles." A detailed inventory appears on pp. 7-12; Schindler's letter is auction number 5/section II/letter 6. However, neither the lock of hair nor the periodical clipping were sold along with the letter (https://digi.ub.uni-heidelberg.de/diglit/liepmannssohn1911_11_17/0031).

Schindler's letter came to the Beethoven-Haus on permanent loan in 1998 as part of the Wegeler Collection (Karl Wegeler had purchased the Beethoveniana of the Moscheles collection at the Liepmannssohn auction), but it did not come with the accompanying lock of hair or newspaper clipping. The intact chain of custody of the Moscheles Lock thus ends ca. 1917.

In 1940, a lock of hair described as the Moscheles Lock, separated from the original provenance letter, was exhibited in Boston. The lock was affixed to a piece of paper bearing the signature of Ignaz Moscheles and quoting from the original Schindler letter, and was described in some detail in the Boston Symphony Orchestra's Concert Bulletin of their 60th season (1940-41) by the Beethoven biographer John N. Burk. By this time, this lock was part of the collection of the Ukrainian-American violinist Louis Krasner (1905-1993). Burk described the lock in a segment titled "Rare Beethoven Relics":

> In a case in the First Balcony Gallery are several items of Beethoven memorabilia, lent by Mr. Louis Krasner, which are of unusual interest because of their initial showing in Symphony Hall. [...] There is also a copy made by Moscheles for Robert Schumann of a sketch of Beethoven drawn from life by Hornemann.

> In the same frame with the sketch are a flower picked on Beethoven's grave in 1852 by the English musician, George Doane, and a lock of Beethoven's hair, sent to Moscheles by Schindler in 1827. Moscheles has written an accompanying note stating: "L. v. Beethoven died on 26th March, 1827. A. Schindler wrote me on the 24th of March: 'This hair I have today cut from his [Beethoven's] head and I am sending this to you.'"¹²¹

Over his lifetime, Krasner amassed a large collection of musical manuscripts and memorabilia, most of which were donated to the Houghton Library at Harvard University by members of the Krasner family between 1986-2001. The circumstances by which Louis Krasner acquired the lock are not currently known. The lock of hair, exactly as arranged in the 1940 description, was acquired by the
American Beethoven Society for genetic testing from the New York based antiquarian dealers J & J Lubrano in the fall of 2010 (Methods S1D). The Society gifted the lock to the Ira F. Brilliant Center for Beethoven Studies, San Jose State University, San Jose, California that same year.

On July 15th, 2016, then director of the Ira F. Brilliant Center for Beethoven Studies, William Meredith, engaged two conservators to open the frame at the conservation laboratory of the Legion of Honor Museum, San Francisco, extracting 24 hairs, amounting to approximately 130 cm of hair. During this process, conservators noted that more exposed portions of the hairs appeared to be discolored, consistent with a process known as 'photo-aging' during which melanin breaks down when exposed to light.¹²² Two DNA extractions, each consisting of 40cm of hair, were performed on the Moscheles Lock, for a total of 80cm of hairs sampled. The remaining hairs that were not sampled remain in the collections of the Ira F. Brilliant Center for Beethoven Studies.

## Cramolini-Brown Lock

The Cramolini-Brown Lock has a relatively poor historical provenance, with no documentation tracing it to the original Cramolini Lock, for which a detailed first-hand account of acquisition does exist. There currently are two locks of hair purported to be from the original Cramolini Lock, and a third which may yet exist and whose provenance may be traceable to the original. The Cramolini-Brown Lock which we analyzed was acquired from RR Auction. Another lock of hair is in the collections of the Beethoven-Haus in Bonn, as item V 10 of the Hans Conrad Bodmer Collection, with a note of provenance tracing it to Darmstadt, Germany, the town in which Ludwig Cramolini eventually settled to pursue his career as an operatic tenor singer and theater director, and start a family. The third lock, whose existence and possible whereabouts are unknown, is recorded as being gifted in 1827 to Ludwig Cramolini's then fiancée, Nanette Schechner (1804-1860), who had performed with high praise in a rendition of Beethoven's opera, *Fidelio*, in 1826.¹¹⁵ Genetic testing has demonstrated that the Cramolini-Brown Lock is almost certainly inauthentic.

The original Cramolini Lock is documented as being removed from Beethoven's corpse on March 27th, 1827, on the day following his death, by the renowned operatic tenor, Ludwig Cramolini (1805-1884). Cramolini had removed the lock without formal permission, resulting in a confrontation between himself and Beethoven's friend and secretary, Anton Schindler. Ludwig Cramolini recorded his account of the acquisition of the hairs in a diary, excerpts of which would eventually be published a 1907 *Frankfurter Zeitung* article entitled "An Beethovens Leiche" (On Beethoven's Corpse) and, in an abridged English translation, in a 1907 *New York Times* article entitled "Beethoven as Seen Through Diary of Man Who Knew Him." Cramolini's full account of his visits to Beethoven during the final months of his life, as well as his participation in commemorations during Beethoven's funeral, would be published in the second volume of Friedrich Kerst's 1913 collection of Beethoven reminiscences.¹²³ Cramolini's account of the acquisition of the hairs reads:

> On the 27th, after the rehearsal for A. Müller's operetta 'Die erste Zusammenkunft', I drove to Beethoven's apartment, a small pair of scissors in my pocket. There I found Schindler, who had already fended off a great number of people curious to see Beethoven, but he let me pass. And so I stood before the covered corpse, which rested on long wooden boards upon chairs, as was customary in those days. In the presence of an old woman (Beethoven's housekeeper, I believe), I lifted the shroud, quickly clipped off a ringlet of hair and wanted to depart immediately, when Schindler entered. I embraced him, wept, and admitted that I had cut some hair from Beethoven's head as an eternal memento for myself and Nanette Schechner (singer at the Vienna Opera). Schindler behaved like a lunatic, demanded that I return the hair, said it was an insult, and all this before the body of the great Beethoven, which angered me so that I asked him to follow me into the antechamber, so that I might answer him outside the presence of the divine master; for here, I thought, it was a crime. I waited for Schindler quite a while - in vain. He failed to come, and thus I returned home and later gave Nanette Schechner some of the hair, for which she was exceedingly grateful. I still have my share of the booty, as Schindler called it.¹²⁴ [translation William Meredith]

As a result of splitting the hair between himself and Nanette Schechner, the original Cramolini Lock is documented as two locks of hair. It is currently unknown what has become of the Cramolini-Schechner Lock. There is no doubt that Ludwig Cramolini was in Vienna at the time of Beethoven's death as Cramolini's own account,¹²³ as well as records in Beethoven's conversation books,¹²⁵ document him and Nanette Schechner visiting Beethoven and singing to him during the course of Beethoven's final illness. Ludwig Cramolini was also among the singers at Beethoven's funeral.

The Cramolini-Brown Lock is folded in a small slip of paper with what may be preliminary notes for a funeral procession: "1. leader with staff. 2. 8 children 2 girls with candles 2 girls w/ flower baskets wherein flws. & fruit / 2 boys w/ candles 2 children with pitchforks, scythes, flowers...Soprano, Alto & Basso"¹²⁴ (Methods S1E). As Beethoven's funeral was being planned on March 27th, this detail may be consistent with the circumstances of the hair's removal and concealment in the immediate aftermath of Beethoven's death. This document has been interpreted as preliminary arrangements for Beethoven's funeral.¹²⁴ On the reverse is twice penciled in the name "Beethoven" (Methods S1E).

The Cramolini-Brown Lock was first offered for sale in October 2012 by the respected antiquarian dealer Thomas Kotte in Roßhaupten, Germany, for €35,000 (approximately $45,600 at the time).¹²⁴ Either because of the high price, the lack of authentication, or both factors, the lock did not sell. It reappeared at an auction in Amherst, Massachusetts, from the auction house RR Auction. At the Kottke auction in 2012, a large number of items related to the Cramolini family were additionally advertised. These include what was claimed to be the dried placenta of Ludwig Cramolini's grandson (also named Ludwig Cramolini), numerous letters, and a family tree tracing the ancestry of the Cramolini family to Ludwig Cramolini's grandson.
The Cramolini-Brown Lock was acquired at auction on March 11, 2015 from RR Auction, by three members of the American Beethoven Society for analysis in The Beethoven Genome Project.<sup>126</sup> The entire Cramolini-Brown Lock consists of approximately 250 hairs, an average of 3" in length, of blond, brown, gray and black hairs (<u>Methods S1E</u>). In total, 109 hairs of varying lengths with at least 15 bulbs adhering were sent to the University of Tübingen's Paleogenetics Department. The majority of hairs with bulbs were long, black curly hairs. Four DNA extractions of 25cm each, and three DNA extractions of 2 bulbs with approximately 1.5cm adhering to them, were destructively sampled in seven separate extractions, for approximately 109 cm of hair ultimately sampled.

Stumpff Lock

The Stumpff Lock (Figure 3) is the second of the locks of hair tested which boasts an intact chain of custody (Figure 1). While a first-hand account of its cutting does not survive, its provenance in the immediate aftermath of its cutting, between March 28th and May 7th, 1827 is well documented. The Stumpff Lock furthermore remains affixed to a document bearing the signature of its original, namesake owner, Johann Andreas Stumpff (<u>Methods S1F</u>). The Stumpff Lock is stated by Sotheby's to have remained within the family of its eventual owner, Patrick Stirling, until its acquisition at Sotheby's in London in November of 2016 by a member of the American Beethoven Society. Genetic testing has demonstrated that the Stumpff Lock is almost certainly authentic.

The lock was originally sent to London-based Thuringian-born harp maker Johann Andreas Stumpff (1769-1846) on March 28th, 1827, in a letter written by a mutual friend of Stumpff and Beethoven, Johann Baptist Streicher (1796-1871), the son of Beethoven's close friend Nannette Streicher (see Müller Lock). Also included in this letter was a small sheet of music manuscript. Both items were sent to Stumpff by Streicher on behalf of another mutual friend of Beethoven, the prominent art and culture journalist Johann Valentin Schickh (1770-1835), who was taking responsibility for Beethoven's funeral at that time and was too busy to write. The hairs were therefore first acquired at some point between Beethoven's death on March 26th and Streicher's sending of the letter on March 28th, 1827. Below is the text of Streicher's letter to Stumpff of March 28th, 1827, relating to the hair and music manuscript:

> Since Herr Schickh has eagerly taken responsibility for Beethoven's funeral, he is prevented at the moment from writing to you and Herr Schultz himself. Meanwhile, he sends you the enclosed lock of Beethoven's hair, cut after his death, as well as a little piece of manuscript; a larger will follow.<sup>112</sup> [translation Theodore Albrecht]

This correspondence was, along with the letter of Schindler to Moscheles from four days before, reproduced in the same volume of *Harmonicon* that announced Beethoven's death (see *Moscheles Lock*). Stumpff then acknowledges receipt of the hair and music shortly after receiving them in a letter sent to Streicher on April 16th, 1827:

> The passing of that irreplaceable great German man, our friend Beethoven, pierced me deeply. Here I sit, bent over your dear letter that confirmed the news of it for me, and stare at the lock that adorned the head from which flowed the immortal works, which are and shall remain the admiration of all cultivated nations. [...] Now, my dear friend, I thank you most sincerely for the lock of hair and music of our departed friend that you sent, with the request that you give Herr von Schickh my many regards, and extend my thanks for the tender proof of his friendly sentiments toward me.<sup>112</sup> [translation Theodore Albrecht]

The lock next appears affixed to a letter from Stumpff to the thirteen-year-old orphan and inheritor of the Keir branch of the Scottish Stirling clan estates, Patrick Stirling (1813-1839) (Figure 3; Methods S1F).<sup>127</sup> The letter contains two notable features idiosyncratic to Stumpff's correspondence, one of which is a short poem, and the other Stumpff's exceedingly fine handwriting. The text of this letter is reproduced below:

For Master P. Stirling of

Brighton

The *headinkel*, these hair's have grac'd lies low

But what it wrought - will ever grow. }

with J. A. Stumpff Compts -

May 7th, 1827 of Lud. v. Beethoven.

44 Great Portland Street

While it is not yet clear what prompted Stumpff to send these hairs to the orphan Patrick Stirling, it is notable that the Stirling family patronized several prominent Romantic era musicians. Patrick's sister, Mary Wedderburn, was godmother to Edvard Grieg (1843-1907),<sup>128</sup> while Patrick's aunt, Jane Wilhelmina Stirling, famously patronized Frédéric Chopin (1810-1849). An as yet unexplained detail worthy of mention, and consistent with an origin for the hairs in early nineteenth-century Vienna, can be found on the accompanying note in which the Stumpff Lock was originally folded (<u>Methods S1F</u>). On the reverse of this note is penned in dark ink
‘Beethovens hair,’ as well as the lightly penciled name ‘Schuppanzigh,’ in a separate hand (Methods S1F). Ignaz Schuppanzigh (1776-1830) was a close associate of Beethoven’s, having premiered many of his string quartets as first violinist of the Schuppanzigh Quartet, as well as the Ninth Symphony, Op. 125.

The Stumpff Lock was sold at auction to American Beethoven Society member Kevin Brown at auction in late November 2016 for analysis in The Beethoven Genome Project.¹²⁹ Sotheby’s description of the Stumpff Lock stated that “This lock of hair has come down to the present owners by direct descent,” (https://www.ssothebys.com/en/auctions/ecatalogue/2016/music-continental-books-manuscripts-116406/lot.5.html). Two DNA extractions, each consisting of 25cm, or four hairs, were initially performed for authentication purposes, amounting to a total of 50cm, or 8 hairs, removed from the lock. Subsequently, the Stumpff Lock was chosen, owing to marginally superior DNA preservation (Methods S1I) for further extractions to generate libraries, for production sequencing of a high-coverage genome. An additional 56 hairs, amounting to 275cm, were removed and destructively sampled in 11 additional DNA extractions, for a total of 325cm of hair removed altogether. The Stumpff Lock at the time of writing resides in the collection of American Beethoven Society member Kevin Brown.

## Hiller Lock

The Hiller Lock, elsewhere referred to as the Guevara Lock,² has a comparatively poor provenance history, lacking a first-hand account of its acquisition, as well as having an incomplete chain of custody (Figure 1). It is first mentioned as being inherited as a birthday present in 1883 by Paul Hiller. Paul Hiller claims that the lock was initially acquired by his father, Ferdinand Hiller, on March 27th, 1827. However, Ferdinand Hiller recorded no account of the lock’s acquisition, either in his diary at the time, or in his subsequent memoirs. The lock remained in Paul Hiller’s possession until at least 1911. The lock’s whereabouts between 1911 and 1943 are not known with certainty. In 1943, the lock was acquired by a Danish doctor aiding in the escape of Danish Jews to neutral Sweden. In 1994, the Hiller Lock was acquired by members of the American Beethoven Society. Genetic testing has demonstrated that the Hiller Lock is definitely inauthentic.

Ferdinand Hiller (1811-1885) was a composition pupil of Johann Nepomuk Hummel (1778-1837), and later became a prominent Romantic era composer who succeeded Felix Mendelssohn (1809-1847) as director of the Leipzig Gewandhaus Orchestra. In his 1871 reminiscences, Hiller records four visits to the dying Beethoven with Hummel, between March 8th and March 23rd, 1827.¹³⁰ During one of these visits, Hummel’s wife, Elisabeth (“Betty”), would acquire a lock of hair, which was acquired as part of the Yvonne Hummel Collection by the American Beethoven Society from the Hummels’ descendants.¹³¹ An additional lock of Beethoven’s hair attributed to Elisabeth Hummel resides in the collections of the Beethoven-Haus Bonn as item R 1 e. Despite this, Hiller makes no mention of either viewing Beethoven’s corpse, or removing a lock of hair following Beethoven’s death.²,¹³⁰

The earliest known mention of the Hiller Lock, believed to have been penned by Ferdinand Hiller’s son, Paul Hiller, consists of a fragmentary inscription that was discovered when the locket containing the hairs was opened following its acquisition by the American Beethoven Society. Upon this damaged inscription can be discerned the words “Beethoven”, “abgeschnitten”, and “Ferdinand Hiller” (Methods S1G). On the reverse of the inscription is a portion of a page from a 44-volume periodical, *Exposé de la Situation Générale de L’Algérie*, published in 1881 (Methods S1G).

The inscription found on the locket today is a replacement of an unknown date, though likely added around the time of the locket’s refurbishment by Cologne-based art dealer, Hermann Großhennig, on December 18th, 1911. The inscription reads, “This hair was cut off of Beethoven’s corpse by my father, Ferdinand v. Hiller, on the day after Ludwig van Beethoven’s death, that is 27 March 1827, and was given over to me as a birthday present in Cologne on 1 May 1883. Paul Hiller” (Methods S1G). On the reverse of the current inscription is a note documenting the locket’s refurbishment in 1911: “Newly pasted to make it dust-free. Original condition improved”² [translation Russel Martin].

Between 1911 and 1943, nothing is known with certainty about the lock’s whereabouts. As attested by Thomas Wassard Larsen, one of the owners of the lock prior to its acquisition by the American Beethoven Society, the lock was received by a Danish doctor, Kay Alexander Fremming, in the port town of Gilleleje, Denmark, from an unknown Jewish refugee seeking the safety of neutral Sweden in October of 1943:

> My name is Thomas Wassard Larsen, and I am writing to you about a lock of Beethovens hair, sold by Sotheby's auctions i London. I hope you understand the meaning with this letter, because I'm not very good at writing in English.

The lock was owned by my mother, who had to sell it due to her economical situation. My mother Michele was born in France a cupple of years before 2.nd world war. During w.w.2 my grandmother had 8 kids including my mother, and she could not feed them all so therefore my mother was adopted by a nice family in Denmark. She was no in the age of 8 years.

My mothers new parents were a Doctor and a nurse who lived in a little town in North Sealand called Gilleleje. This little town was one of the closest to Sweden, to witch many judes fled during 2.nd w.w. Many of these judes were wery poor and some of them had som awful deceases.

My mothers new father who was a doctor helped many of these judes, in the start only with medicin, but later he worked together with the local fishermen, in the night to smuggl judes to Sweden. It was one of these judes who gave the lock of Beethovens hair to him for his help. My grandfather kept this medallion until his dead in 1969, the same year that i was born.²
A historically well known community of approximately 7,800 Ashkenazi Jews in Copenhagen had survived in relative safety in Nazi-occupied Denmark until the local Gestapo were ordered to detain them on October 1st, 1943. This prompted a mass exodus of Copenhagen's Jews to neutral Sweden, many of whom used the port town of Gilleleje, 40 miles north of Copenhagen and allowing a short sea voyage to Sweden. Ultimately 7,220 Jews and 686 non-Jewish spouses escaped with the help of the Danish Resistance movement.<sup>132</sup>

The Hiller Lock of hair would remain within the Fremming family between 1943 and 1994, until being acquired by members of the American Beethoven Society at auction at Sotheby's in London. At auction, the lock consisted of 582 hairs, approximately 4.5" in length, after which 160 hairs were given to the principal investor, Dr. Alfredo Guevara, and 422 were kept by the Ira F. Brilliant Center for Beethoven Studies (Methods S1G). The hairs are blond, brown, gray and black.

Accepted by many as authentic, the hairs have been subjected to a number of scientific tests, which have led some of Beethoven's medical biographers to conclude that Beethoven's health problems, hearing loss, and death may have been caused or compounded by plumbism.<sup>3,4,26,27,133</sup> It has additionally been concluded from these analyses that Beethoven did not receive mercury treatment for a hypothesized infection with syphilis, and that he did not receive opiates during the treatment of his final illness. The Hiller Lock is the only lock of hair putatively originating from Beethoven for which data from a DNA analysis exists, consisting of a partial PCR and Sanger Sequencing based analysis of several variants within hypervariable regions I and II carried out by LabCorp in 1999.

Five hairs, totaling approximately 25cm, were sent to the University of Tübingen's Paleogenetics Department by Dr. Alfredo Guevara, and subsequently sampled in a single DNA extraction.

## Kessler Lock

The Kessler Lock is of enigmatic provenance, with only a single known statement from 1948 attesting to its acquisition. The Kessler Lock nonetheless appears to have a complete chain of custody (Figure 1). The Kessler Lock was reputedly recovered during one of Beethoven's two exhumations, in 1863 or 1888, in Vienna's Währinger Ostfriedhof, by the father of musicologist Hubert Kessler (1898-1985). In 1948, Hubert Kessler received the lock in a letter from his uncle, approximating 50 strands of hair, and accompanied by several fragments of textile (Methods S1H). The relics were contained within an envelope with the inscription, "Aus Beethoven's Sarg [From Beethoven's coffin]" (Methods S1H). Owing to poor DNA preservation, the authenticity of the Kessler Lock could not be determined.

"Dearest Hubert!

Having received your pleasant letter from the third of this month, let me inform you that, according to what I remember, my honored father (that is, your grandfather) was present in person at the exhumation. Beethoven's remains were examined by P. Also as the representative of his superior and friend Domböck and brought home the relic (Beethoven's hair), which was always greatly esteemed by us." [translation Birgit Lodes]

Neither of Beethoven's exhumation reports from 1863 or 1888 describe hairs as being preserved in Beethoven's grave.<sup>7,134</sup> However, Schubert's entire head of hair was recovered during the 1863 exhumation, supporting the notion that the conditions in the Währinger Ostfriedhof may have been conducive to the survival of hair. Schubert, who died 20 months after Beethoven, was separated from Beethoven by two grave plots, and exhumed simultaneously. The exhumation report notes, however, that the soil in Schubert's grave was 'damp' and the coffin much better preserved, whereas the soil in Beethoven's grave was 'dry' and 'loamy,' presumably responsible for the poorer preservation of organic materials.<sup>134</sup> The 1863 exhumation report goes on to state:

> [...] the members of the administration took individual parts of the remnants of clothing and the wood of the coffin of Beethoven as well as of Schubert; parts were given over to the few persons present at this serious act who were visibly moved by strong emotions. Most of these remnants, however, were put aside and for the time being looked over by Dr. v. Breuning<sup>134</sup> [translation William Meredith]

However, the report later states that the remnants of clothing kept by Dr. v. Breuning were placed in a large tin box, soldered shut, and reinterred with Beethoven's remains.<sup>134</sup> It is unclear from the 1863 exhumation report whether all of these recovered materials were reinterred, or if some were still retained by those present.

The individual referred to as 'P' in the letter to Dr. Kessler is likely Dr. Carl von Patruban, who, along with Dr. Standthartner, examined Beethoven's remains as they were being exhumed on October 13th, 1863. Of the 32 people counted as present during Beethoven's first exhumation, only 13 are named in the exhumation report. None by the name of Domböck can be identified, although a 'Dobyhak' is mentioned as being present on October 22nd.<sup>134</sup>

A contemporary newspaper account published on September 1st, 1888 in the *Evening Post* states that, apart from the three anthropologists present during Beethoven's second and final exhumation in 1888, "very few persons witnessed the exhumation, and most of these were officials," rendering the recovery of the hairs during the 1888 exhumation less likely.

A single DNA extraction on what appeared to be a bulb was carried out, followed by a second extraction on four hairs, amounting to approximately 17cm. The Kessler Lock and associated textile fragments are currently owned by the University of Illinois Music Department.
## Identification of living patrilineal descendants of Aert van Beethoven

The paternal lineage of Ludwig van Beethoven has been the subject of much research and has been reconstructed with reasonable certainty at least as far back as 1535<sup>23</sup> (Methods S1P). In almost all respects, this reconstruction is widely accepted, but two areas worthy of discussion remain. First, the correct identification of Beethoven's putative great-great-grandfather, Kornelius, has been met with some reservations. Of the two men bearing this name and living within the vicinity of Beethoven's known ancestors, genealogists overwhelmingly favor Kornelius van Beethoven, born on October 20th, 1641, at Bertem near Louvain, in present-day Belgium.<sup>115</sup> However, another Kornelius van Beethoven, born in 1630, has been considered, with reservations, as a candidate.<sup>135</sup> Nonetheless, both of these candidates are believed to be closely related, and are not expected to disrupt the Y chromosome pedigree. Second, anomalously, no baptismal record has yet been found for Beethoven's father, Johann van Beethoven.<sup>23,115</sup>

Extra-pair paternity (EPP) is a known danger to the reconstruction of genetic patrilineages, necessitating the adoption of candidate selection strategies designed to minimize the probability of EPP. EPP rates are known to have ranged from 1-2% in Western Europe across the last 400 years, and, historically, have varied from as low as 0.31% to as high as 6% in regions comprising present-day Belgium, depending on rural versus urban setting, as well as socio-economic status.<sup>136</sup> In order to best mitigate the possibility of EPP, these five research participants were selected among numerous candidates to maximize the number of independent lineages, additionally taking into account genealogical documentation of urban vs. rural settlement and the socioeconomic status of each patrilineage. These potential donors were always separated by at least twelve meioses from each other in the direct paternal line.

Five individuals were selected, who met all of the above criteria and represented independent lineages sharing a common ancestor with Aert van Beethoven (1535-1609), Beethoven's great-great-great-great-great grandfather. A genealogy of these five individuals is presented in Methods S1P; living individuals are not named for privacy reasons. All five consented to provide saliva samples for Y chromosome testing. Saliva samples were collected using the Oragene OG-500 kit. Lysing of cells took place immediately after the collection of the saliva. DNA was extracted and purified in accordance with the manufacturer's recommended instructions (pre-pIT L2P PD-PR-006 https://www.dnagenotek.com/us/pdf/PD-PR-006.pdf). One participant requested that any remaining DNA extract be incinerated following sequencing, which was carried out.

## Identification of living descendants of Karl van Beethoven

We identified, from a published genealogy of Ludwig van Beethoven,<sup>23</sup> three living individuals descending from Karl van Beethoven (1806-1858), the son of Beethoven's younger brother Kaspar Anton Karl van Beethoven (1774-1815) and his wife, Johanna van Beethoven (née Reiß; 1786-1869). We received ethical approval to test for IBD-segment sharing between them and the Beethoven genome (Human Biology Research Ethics Committee, University of Cambridge, application HBREC.2020.48, December 18th, 2020; confirmed by the Director of the Research Ethics Commission, Austrian Academy of Sciences, as raising no concerns, September 16th, 2020). All three of these individuals are documented as 7th-degree genetic relatives of Beethoven.

## Permits

Ethical approval was granted (Medical Ethical Committee UZ Leuven/KU Leuven, procedure number S61715; Department of Archaeology, University of Cambridge, approval date May 1st, 2019), allowing that up to five relatives from different branches of the Van Beethoven patriline may be approached.

Ethical approval was granted to test for IBD-segment sharing between three genealogically documented descendants of Karl van Beethoven and the Beethoven genome (Human Biology Research Ethics Committee, University of Cambridge, application HBREC.2020.48, December 18th, 2020; confirmed by the Director of the Research Ethics Commission, Austrian Academy of Sciences, as raising no concerns, September 16th, 2020).

## METHOD DETAILS

Hair sample decontamination, DNA extractions, initial double-stranded library preparation and indexing amplifications were performed in the dedicated ancient DNA cleanroom facilities in the University of Tübingen's Paleogenetics Department in Tübingen, Germany (sample ID prefixes JK & TU) and the Max Planck Institute for the Science of Human History's Department of Archaeogenetics in Jena, Germany (sample ID prefix HEB) (Data S1A).

## Decontamination, extraction and purification

For each hair sample, hair shafts were decontaminated either in four immersions in sterile, UV-irradiated water prior to extraction, or a single 10 s immersion in 0.5% final concentration bleach followed by four immersions in sterile, UV-irradiated water (Data S1A). The hairs were then placed in 1000µl of active extraction buffer comprising an inactive extraction buffer containing 10mM final concentration Tris buffer (pH 8.0), 10mM NaCl, 5mM CaCl₂, 2.5mM EDTA (pH 8.0), and 2% SDS. To create the active extraction buffer, 40 µl of 1M DTT and 100µl of Proteinase K were mixed with 860 µl of inactive extraction buffer, per extraction.<sup>16</sup> The DNA extract was subsequently incubated overnight at 37°C while rotating at 15 rpm.<sup>15</sup>

After centrifuging the digested hairs into a pellet, the resulting DNA extract was purified into 100µl of TET buffer. Samples processed in Tübingen were purified using a silica-based Qiagen MinElute column affixed to a Hi-Pure Extender Assembly, whereas samples purified in Jena utilized a Hi-Pure Viral DNA silica-column with included extender assembly.<sup>137</sup> The volume and composition of the binding buffer was optimized for the retention of ultra-short DNA fragments and low copy number DNA templates such as those
expected in historical hair samples.¹⁸ Standard Qiagen and Hi-Pure washing buffers¹³⁷ were used following manufacturer recommended protocols.

## Library preparation, indexing and sequencing

Following purification, either 10µl or 20µl of purified extract from the Cramolini-Brown, Hiller, Kessler, Moscheles, Stumpff, Halm-Thayer and Bermann Lock samples underwent initial double-stranded library preparation and double-indexing protocols compatible with Illumina sequencing technologies, incorporating amplification strategies and polymerases intended to reduce PCR biases or artifacts during indexing amplification of ancient DNA libraries.¹⁷,¹³⁸,¹³⁹ Negative controls were incorporated during extraction and library preparation stages in order to gauge the presence of background levels of DNA during lab work prior to double-indexing.¹⁴⁰ Quality control checks included library-based real-time qPCRs with the Roche LightCycler96 following both initial library-preparation and subsequent indexing amplification, as well as the generation of two fragment size profiles to assess molarity prior to sequencing, using an Agilent Bioanalyzer 2100.¹⁴¹ A mitochondrial DNA capture was additionally performed on a library prepared from the Kessler Lock.¹⁴² Single-stranded libraries using 30µl of purified extract from the Stumpff, Bermann, Halm-Thayer and Müller Locks were additionally prepared at the Max Planck Institute for Evolutionary Anthropology's Department of Paleogenetics.¹⁹ Sequencing was performed on Illumina HiSeq2500, HiSeq4000 and NextSeq500 platforms (Illumina, San Diego, CA), using a variety of sequencing chemistries (Data S1A). Three libraries (TU50.BH6.1U, TU50.BH6.2U, TU50.BH6.3U) from the Cramolini-Brown Lock were prepared using UDG-treatment.¹⁴³

## QUANTIFICATION AND STATISTICAL ANALYSIS

### Bioinformatics processing of initial low-coverage data

Initial processing of reads for assessments of DNA preservation and authentication were performed using EAGER version 1.92.38,⁹⁷ including quality checking of FASTQ files with FastQC, and clipping and merging of paired-end reads with Clip&Merge. Autosomal and sex chromosomal alignments were performed against hg19 using BWA version 0.7.12, with a lower read-length cut-off of 30 base pairs with MAPQ ≥ 30 and disabling of seeding with -l 1000 being specified.⁸² The mapping algorithm CircularMapper was used to perform mitochondrial alignments, employing BWA while extending both ends of chrMT of hg19 by 500 bases to avoid spurious low coverage calls at either end of the chrMT reference. Duplicate removal, sorting and indexing of BAM files were performed using samtools version 1.9.⁸³ Library complexity extrapolations were performed using default settings with the preseq tool lc_extrap⁹⁶ in order to assess relative sample preservation (Methods S1) and to estimate the number of additional extractions and libraries required to attain a high coverage genome. Genotyping was performed using GATK version 3.5.⁸⁴

### Mitochondrial contamination estimation, haplogroup assignment, and regional haplogroup frequency analysis

Wherever possible, consensus mitochondrial genomes were called from non-UDG treated, double-stranded libraries, and paired-end data preferred over single-end data. Only the Müller Lock consensus genome was called from shallow shotgun data sequenced from single-stranded libraries.

Final endogenous mitochondrial contamination rates were assessed with Schmutzi, using the share/schmutzi/alleleFreqMT/197/freqs/panel of putative contaminants included in the program.⁸⁷ With the exception of the Kessler Lock, which lacked sufficient preservation for contamination estimation to be performed, all samples were found to contain between 0-3% contamination, with an average of 1% contamination (Data S1A).

Endogenous consensus mitochondrial genomes were called using the Schmutzi tool endocaller using default parameters.⁸⁷ Endogenous consensus FASTA files were uploaded into Haplogrep2.0 version 2.2,⁸⁵ querying Phylotree build 17,⁷⁹ in order to assign mitochondrial haplogroups and to detect local private mutations (Table S2). The Kessler Lock was found to lack sufficient coverage for a full endogenous mitochondrial consensus genome to be called, despite undergoing two separate extractions and an mtDNA capture (Data S1A).

Regional frequencies of the H1b1+16,362C mtDNA haplogroup, shared among the Müller, Bermann, Halm-Thayer, Moscheles and Stumpff Locks, were assessed by comparison against FamilyTreeDNA's mtFull database, comprising 203,514 full mitochondrial genomes at the time of analysis. No individuals completely matching this mitochondrial genome were identified. However, 219 individuals within the H1b1+16,362C haplogroup, differing only by the lack of the private mutation, were found. The geographic distribution of these individuals was found to be broadly in Western and Central Europe, as well as countries harboring recent European diasporas (Data S1D). No specific clustering within or among countries was observed.

### High-coverage Beethoven autosomal genome sequencing and genotype calling

Nuclear DNA in hair has an extremely low average fragment length owing to the activity of endonucleases expressed during hair formation.¹⁴⁴,¹⁴⁵ To make use of the overwhelming fraction of ultra-short reads recovered during single-stranded library preparation (Data S1A and S1B), a lower read-length cut-off of 20bp was introduced after adapter trimming (leehom 1.1.5),¹⁰⁰ mapping (bwa 0.7.12, parameters: -n 0.01 -o 2 -l 16500)⁸² and indel realignment (GATK 3.5).⁸⁴

An accessibility mask was created to account for the greater chance of misalignments due to the short read length. For this, reads were first filtered for a mapping quality of at least 25. Reads shorter than 35bp were required to contain a genome-wide unique k-mer while permitting for up to one mismatch MapL procedure in de Filippo et al.²¹ Sites were only included which overlapped with the
map35_100 mappability regions developed for the Altai Neanderthal genome, and which did not overlap with tandem repeat regions or indels; see supplementary section 5b in Prüfer et al.<sup>146</sup> Any regions falling within the upper- and lowermost 2.5% of the coverage distribution were excluded, after correcting for local GC content. This accessibility mask reduced the length of the autosomal genome to approximately 1.64Gb, while raising average coverage from 19.68-fold coverage across the full autosomal reference to 24.06-fold within accessible regions (Table S4).

Genotypes were called using snpAD (version 0.3.4), which employs an error model that takes the type of substitution and the location within a given read into account.<sup>20</sup> SnpAD was run with 10 position dependent error matrices on either end of reads. Approximately 7.5 sites per 10,000 were called as heterozygous on the autosomes within accessible regions. The reference allele was supported on average by 57% of reads at heterozygous sites with one reference and one non-reference allele. Autosomal contamination was estimated by testing of the X-chromosome with ANGSD (version 0.910) to be approximately 2.9%.<sup>93</sup>

In order to recover any common variants removed during filtering steps, and to provide genotype phasing information, imputation and phasing were performed using Beagle version 5.1<sup>95</sup> using the full 1000 Genomes Project Phase 3 release as a reference panel,<sup>147</sup> retaining positions with a Genotype Probability (GP) ≥ 0.99.

## HLA genotyping

We applied a development version of OptiType<sup>78</sup> to sequence data from FASTQ files merged from all available libraries, mapped against OptiType's custom Human leukocyte antigen (HLA) reference panel containing 1025 alleles with "common" or "intermediate" CIWD 3.0 designation (https://github.com/FRED-2/OptiType, tag DER). We obtained the top 3 HLA class I and class II genotypes for the loci HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1. Haplotypes were assigned based on previously reported frequencies and properties such as linkage disequilibrium.<sup>148,149</sup> Alleles were genotyped and both haplotypes could be unambiguously called. The resulting haplotypes are:

HLA-A*01:01~B*08:01~C*07:01~DQA1*03:03~DQB1*03:01~DRB1*04:01~DRB4*01:03~DPA1*01:03~DPB1*04:01

HLA-A*11:01~B*56:01~C*01:02~DQA1*01:01~DQB1*05:01~DRB1*01:01~NULL~DPA1*01:03~DPB1*57:01

These haplotypes are consistent (according to frequencies observed in modern European populations) with those haplotypes commonly found in central/western Europe. The second HLA-B allele besides HLA-B*08:01 could not be resolved between HLA-B*54:01, B*55:01 and B*56:01 due to a lack of unique read support in the regions distinguishing them. All reads covering the SNPs distinguishing these three alleles are either co-mapping to HLA-B*08:01 or the subject's HLA-C alleles. In any case, since all three belong to the HLA-B22 serological family, the allele's exact identity bears no additional clinical significance. Considering the frequency of the three alleles and their haplotypes in modern populations, the most plausible allele is HLA-B*56:01.

A shorter version of the first haplotype, HLA-A*01:01~B*08:01~C*07:01~DRB1*04:01 has been previously reported in populations from central and western Europe such as the Netherlands, England and Austria,<sup>150,151</sup> among other populations. A shorter version of the second haplotype, HLA-A*11:01~B*56:01~C*01:02~DRB1*01:01, has been reported for the Canary Islands, Croatia, Poland, France<sup>151-153</sup> and Russia (Khamaganova, unpublished, reported in Allele Frequency DataNet), among other populations.

## Sex chromosomal karyotyping

Sex chromosomal karyotyping was performed on shallow sequence data for all eight purported Beethoven hair samples using an R script developed for low coverage shotgun sequence data, which compares the X chromosome read count (Rx) against the average read count across the autosomes.<sup>99</sup>

## Relatedness testing of autosomal and X chromosome DNA among locks of hair

In order to explicitly test if the five samples with matching mitochondrial genomes derived from a single individual, we assessed levels of autosomal relatedness among all eight samples using READ<sup>94</sup> (Figure 2; Table S5). We maximized the number of genotype positions included in each pairwise comparison by comparing the high-coverage Stumpff Lock genome against the low coverage Müller, Bermann, Halm-Thayer, Moscheles, Cramolini-Brown, Hiller and Kessler Lock genomes. We selected a publicly available and ancestry-matched dataset of 41 low- to medium-coverage medieval Bavarian genomes as an external reference panel.<sup>80</sup> We used this reference panel to calculate an appropriate average proportion of non-matching alleles (P0) among all independent pairwise comparisons to assess degrees of relatedness.

READ is able to identify up to second-degree relatives with a false-positive rate of 3% using as few as 1,000 shared single nucleotide variants (SNVs) among pairwise comparisons between samples, after filtering for minor allele frequency (MAF) ≥ 0.10.<sup>94</sup> We generated pseudo-haploid autosomal genotype calls from the BAM files of all samples, including the medieval Bavarian reference panel, using ANGSD version 0.921.<sup>93</sup> We then filtered genotypes according to an intersection of the accessible genome, and a list of sites with MAF ≥ 0.10 among the EUR superpopulation of the 1000 Genomes Project Phase 3 release,<sup>147</sup> retaining 3,060,820 positions. In order to provide an additional axis to graphically represent autosomal relatedness, we repeated the READ analysis exclusively using pseudo-haploid calls from the X-chromosome, again using the intersection of our accessibility mask and sites on the X-chromosome with MAF ≥ 0.10.

## Damage rate assessment

DNA accumulates characteristic damage patterns over time, whereby cytosines deaminate into uracils, in increasing frequencies towards the 5' end, which are subsequently misread by polymerases as thymines. This is termed 5' C-T deamination.<sup>154</sup> We determined
damage rates for all samples using mapDamage2.0, reporting nuclear and mitochondrial damage rates separately (Data S1A). All samples, including non-matching samples, displayed damage patterns characteristic of ancient DNA, and consistent with their documented or presumed antiquity.

## Principal components analyses

PCA's were initially performed for the high-coverage Stumpff Lock genome and low-coverage Cramolini-Brown and Hiller Lock genomes using global reference panels from the Phase 3 release of the 1000 Genomes Project¹⁴⁷ for analyses of ancestry on a global scale (Figure S1). A second set of PCA's were performed on the Cramolini-Brown and Hiller Lock low-coverage genomes using reference panels of 21 and 50 modern and historical West Eurasian populations, respectively, drawn from the Allen dataset (https://reich.hms.harvard.edu/allen-ancient-dna-resource-aadr-downloadable-genotypes-present-day-and-ancient-dna-data) (Methods S1J and S1L). Each genome was merged with its respective reference panel using the mergeit tool from the EIGENSOFT package.⁹⁸,¹⁵⁵ PCA's were performed using smartpca with default settings.

Higher resolution PCA's for the high-coverage Beethoven genome were performed using individuals from the FamilyTreeDNA customer database. We used the PCA function in PLINK,⁸⁶ which uses the implementation from the program GCTA¹⁵⁶ (Figures S2 and S3). Individuals were queried from each European country in the FamilyTreeDNA database, and closely related individuals were removed using the software KING.¹⁵⁷ Up to 150 individuals per country were randomly selected for analysis to keep sample sizes balanced. For the marker set, we took the intersection of the 574,384 SNVs genotyped in Beethoven's genome, and the 132,268 SNVs found on all of the Illumina genotyping arrays used. SNVs were pruned for linkage disequilibrium using the flag -in-dep-pairphase 100kb 1.0.9, and only SNVs with a genotyping rate of 95% were kept, resulting in a final count of 45,329 SNVs.

## ADMIXTURE analyses

We used the full 1000 Genomes Project phase 3 reference panel¹⁴⁷ to assess Beethoven's ancestry composition among a diverse panel of five global populations, containing 26 subpopulations, with ADMIXTURE.²² Beethoven's genome was filtered to include only accessible regions (see section High-coverage Beethoven autosomal genome sequencing and genotype calling), followed by filtering for DP ≥ 14 and QC ≥ 30. The reference panel was first filtered by MAF ≥ 0.05, and to only include filtered SNV sites from Beethoven's genome. The two datasets were merged and converted into PLINK using -geno 0.01, -mind 0.01 and -hwe 1e-50, resulting in 157,634 converging SNV positions among 2,505 individuals. ADMIXTURE was then run using K = 1 to K = 12 using five-fold cross-validation. We found that the cross-validation errors for K = 5 to K = 12 were roughly comparable (CV error = 0.51997-0.52114). Results are plotted for K = 5 (Figure S5), broadly representing European (EUR), East Asian (EAS), South Asian (SAS), African (ASW & AFR) and Native American (AMR) ancestry, and K = 12 (Figure S4) for a more detailed breakdown of ancestries within these global populations. All filtering and merging steps were performed using bcftools version 1.10.2⁸¹ and PLINK version 1.90.⁸⁶

## Geo-genetic triangulation with IBD-segments

We estimated the spatial distribution of Beethoven's likely ancestors using a novel method we call Geo-Genetic Triangulation (GGT) (Figure 4; Methods S1L-S1O). GGT ensures that only locations with a high likelihood of being ancestral to Beethoven are selected. Broadly, there are three steps, which we detail below: (1) segment matches between the subject and other genetic testers are identified; (2) genetic triangulation between three or more individuals identifies segments inherited identical-by-descent (IBD) from a recent common ancestor; (3) each triangulated segment is screened for coinciding ancestor locations which increases the confidence of recent shared ancestry. When an IBD-segment is inherited by two individuals, only one of many ancestral lines is typically shared between the two. The GGT method substantially reduces the noise of irrelevant ancestor locations.

First, we generated a match list between the subject and FamilyTreeDNA database. We considered matches to be those with the longest segment ≥ 6cM, and we removed any “micro-segments” < 2cM. We repeated this procedure pairwise between each match and the other matches.

Next, segments shared between the subject and the subject's matches were triangulated by looking for groups of matches where two or more members shared a segment with the subject and also an overlapping segment between each other. If numerous matches had segments overlapping the same genomic range, we applied a threshold for the maximum number of checked permutations of triangulation group member candidates to reduce the execution time. We found 720 permutations to be a good compromise between execution time and the number of identified triangulating segments. If segments were clustered into separate match groups but had nearly identical start and end points (within 0.5 cM) and shared members, we de-duplicated them into the same match group. Any segments with a total overlap between group members of < 2cM were removed.

Lastly, we queried available genealogies for all matches sharing triangulated segments with the subject and removed any dubious relationships (older than 10 generations). We used the Google Geocoding API (https://developers.google.com/maps/documentation/geocoding) to convert free text locations into coordinates. Locations that were simply country names were removed, and for any provincial locations we randomly chose a point within that province. We derived administrative boundaries using the Level 1 polygons from the Database of Global Administrative Areas (GADM; https://gadm.org/), and sampled points using the spsample function of the sp package in R.⁹² For each triangulated segment group in the previous step, we identified ancestor locations shared by more than one group member. These coincident locations are the most likely ancestral origins of the IBD segment shared by the subject and his matches. In order to determine whether ancestor coordinates were proximate enough to be considered coincident, we grouped points into equally sized spatial units. We created a hexagonal sampling grid using the spsample function with cell-size of