{"paper_id":"4def6d9e-ab19-45d8-8b63-e12742c4301d","body_text":"The oral microbiome of King Richard III of England | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return\"[object Function]\"==o.call(a)}function e(a){return\"string\"==typeof a}function f(){}function g(a){return!a||\"loaded\"==a||\"complete\"==a||\"uninitialized\"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){(\"c\"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){\"img\"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),\"object\"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height=\"0\",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),\"img\"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||\"j\",e(a)?i(\"c\"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName(\"script\")[0],o={}.toString,p=[],q=0,r=\"MozAppearance\"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&\"[object Opera]\"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?\"object\":l?\"script\":\"img\",v=l?\"script\":u,w=Array.isArray||function(a){return\"[object Array]\"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split(\"!\"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split(\"=\"),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(\".\").pop().split(\"?\").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split(\"/\").pop().split(\"?\")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&\"css\"==i.url.split(\".\").pop().split(\"?\").shift()?\"c\":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The oral microbiome of King Richard III of England View ORCID Profile Irina M. Velsko , View ORCID Profile Alexander Hübner , View ORCID Profile Zandra Fagernäs , View ORCID Profile James Fellows Yates , View ORCID Profile Allison E. Mann , View ORCID Profile Courtney Hofman , View ORCID Profile Andrew T. Ozga , View ORCID Profile Cecil M. Lewis Jr. , View ORCID Profile Camilla Speller , View ORCID Profile Sarah Fiddyment , View ORCID Profile Michael Francken , Joachim Wahl , View ORCID Profile Johannes Krause , View ORCID Profile Anita Radini , View ORCID Profile Turi King , View ORCID Profile Christina Warinner doi: https://doi.org/10.1101/2025.09.21.677585 Irina M. Velsko 1 Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology , Leipzig, Germany 07745 2 Archaeogenetics Research Unit, Leibniz Institute for Natural Products Research and Infection Biology Hans Knöll Institute , Jena, Germany , 07745 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Irina M. Velsko For correspondence: irina_marie_velsko{at}eva.mpg.de tk932{at}bath.ac.uk warinner{at}fas.harvard.edu Alexander Hübner 1 Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology , Leipzig, Germany 07745 2 Archaeogenetics Research Unit, Leibniz Institute for Natural Products Research and Infection Biology Hans Knöll Institute , Jena, Germany , 07745 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexander Hübner Zandra Fagernäs 3 Globe Institute, University of Copenhagen , Copenhagen, Denmark 1353 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zandra Fagernäs James Fellows Yates 1 Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology , Leipzig, Germany 07745 2 Archaeogenetics Research Unit, Leibniz Institute for Natural Products Research and Infection Biology Hans Knöll Institute , Jena, Germany , 07745 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James Fellows Yates Allison E. Mann 4 Department of Anthropology, University of Wyoming , Wyoming, USA 82071 5 Department of Anthropology, University of Oklahoma , Oklahoma, USA 73019 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Allison E. Mann Courtney Hofman 5 Department of Anthropology, University of Oklahoma , Oklahoma, USA 73019 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Courtney Hofman Andrew T. Ozga 5 Department of Anthropology, University of Oklahoma , Oklahoma, USA 73019 6 Department of Biological Sciences, Nova Southeastern University , Fort Lauderdale, Florida, USA , 33328 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrew T. Ozga Cecil M. Lewis Jr. 5 Department of Anthropology, University of Oklahoma , Oklahoma, USA 73019 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cecil M. Lewis Jr. Camilla Speller 7 Department of Anthropology, University of British Columbia , Vancouver, Canada , V6T 1Z4 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Camilla Speller Sarah Fiddyment 8 Department of Archaeology, University of York , York, YO10 5DD, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sarah Fiddyment Michael Francken 9 Institute for Archaeological Sciences, University of Tübingen , Tübingen, 72070, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Francken Joachim Wahl 10 Institute for Archaeological Sciences, Section Paleoanthropology, University of Tuebingen , 72070, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Johannes Krause 1 Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology , Leipzig, Germany 07745 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Johannes Krause Anita Radini 11 School of Archaeology, University College Dublin , Dublin, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anita Radini Turi King 12 Genetics and Genome Biology, University of Leicester , Leicester, UK LE1 7RH 13 Milner Centre for Evolution, University of Bath , Bath, BA2 7AY Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Turi King For correspondence: irina_marie_velsko{at}eva.mpg.de tk932{at}bath.ac.uk warinner{at}fas.harvard.edu Christina Warinner 1 Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology , Leipzig, Germany 07745 2 Archaeogenetics Research Unit, Leibniz Institute for Natural Products Research and Infection Biology Hans Knöll Institute , Jena, Germany , 07745 14 Faculty of Biological Sciences, Friedrich Schiller University , Jena, Germany 07743 15 Department of Human Evolutionary Biology, Harvard University , Cambridge, MA 02138 16 Department of Anthropology, Harvard University , Cambridge, MA, USA 02138 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christina Warinner For correspondence: irina_marie_velsko{at}eva.mpg.de tk932{at}bath.ac.uk warinner{at}fas.harvard.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Metagenomic investigation of archaeological dental calculus has provided insights into the changing oral health, disease, and diet of past human societies, but little is known about the oral microbiota of exceptionally high-status individuals, whose diet and lifestyle sharply differed from the general population. Here we analyze the dental calculus metagenome of King Richard III of England (1452-1485) and compare it to new and previously published dental calculus metagenomes from predominantly non-elite contexts in England, Ireland, the Netherlands, and Germany spanning the Neolithic to the present. Deep sequencing and de novo assembly enabled the investigation of metagenome-assembled genomes (MAGs) from periopathogens within the genus Tannerella . DNA preservation within the dental calculus of King Richard III was found to be exceptional, yielding an extraordinarily well-preserved oral microbiota. Oral microbiome species diversity fell within the range previously observed among other northern European populations over the past 7,000 years, suggesting that a royal lifestyle and a rich diet did not substantially shift his oral microbiome composition. Reconstructed Tannerella genomes contained many virulence factors found today among periopathogens, including Tannerella forsythia . Insufficient plant and animal DNA was recovered to investigate diet, suggesting that dental calculus may not be a sufficient source of dietary DNA for dietary reconstruction, even when well-preserved. The dental calculus of King Richard III has produced one of the richest and best preserved ancient oral metagenomes studied to date and contributes to understanding the ecology and evolution of the human oral microbiome. Introduction In 2012, the skeletal remains of King Richard III of England were discovered beneath a car park on the grounds of the former Grey Friars church in the city of Leicester, UK [ 1 ]. Subsequent osteological [ 1 – 3 ] and genetic and statistical [ 4 ] analyses of the evidence confirmed the identity of the remains of the long lost king. Richard III became king of England in 1483 during a period of civil conflict between the Plantagenet Houses of York and Lancaster later known as the Wars of the Roses (1455-1487). Remembered as a usurper, Richard III was accused by chronicler John Rous [ 5 ] and Sir Thomas More [ 6 ] of plotting the murder of his nephews to seize the throne. Crowned king at age 30, he reigned for two tumultuous years and was killed at the Battle of Bosworth in 1485, after which his body was hastily buried at the Grey Friars Church in Leicester. His life and brief reign became the subject of an eponymous tragic play by William Shakespeare in 1593 in which Richard III was portrayed as a ruthless villain [ 7 ]. Although this literary legacy has made him one of England’s most well-known medieval kings, relatively little is known about his life other than its political outline, and the details of his rise to power following his brother’s death remain sharply disputed [ 8 ]. Archaeology provides an opportunity to augment his scant historical record. The discovery of Richard III’s remains has provided an unusual chance to not only confirm specific historical details of his death but also to investigate less well-understood aspects of his life. Analysis of his remains has confirmed his death in battle, as his skeleton bore the marks of at least eleven perimortem wounds, including two likely fatal wounds to the back of the head and several humiliation injuries that may have been inflicted after he was stripped of armor [ 2 ]. Historical accounts of a hasty burial have also been supported by the absence of evidence for a coffin, or even shrouding, with his corpse having been placed within a cramped grave, possibly with his hands bound [ 1 ]. Accounts of his uneven shoulder height are supported by the identification of idiopathic adolescent-onset scoliosis in his skeleton [ 3 ], which likely began during his training as a knight in Yorkshire. Well-preserved DNA within his skeleton has confirmed his sex as male, and mitochondrial genome sequencing has conclusively linked him to two female-line relatives [ 4 ]. Isotopic analysis of his enamel bioapatite (Sr, Pb, O) and collagen from dentine and bone (O, C, N) has provided further insights into aspects of his life history [ 9 ]. His oxygen and strontium isotopic values are consistent with his childhood movements between eastern and western England, and his carbon and nitrogen isotopic values reflect a changing diet in childhood, followed by a likely increase in meat and high trophic level fish consumption from his teenage years onwards, and later a likely further increase in the regular consumption of luxury foods, such as wine, fish, and wildfowl, after being crowned king. The excellent biomolecular preservation of the remains of Richard III suggests that an analysis of the DNA present in his dental calculus might yield further insights into his life, and specifically on his oral health and diet. Dental calculus, a mineralized form of dental plaque also known as tooth tartar, forms incrementally during life, entrapping the oral microbiota and serving as a long-term biomolecular archive of the oral microbiome [ 10 , 11 ]. Food biomolecules may also become entrapped in dental calculus and can further aid in dietary reconstruction [ 12 , 13 ]. However, while microbial metagenomics has been widely applied to reconstruct the microbial structure and diversity of the oral microbiome through time [ 14 , 15 ] and to track the prevalence and virulence of periodontal pathogens [ 16 – 19 ], the use of dental calculus metagenomics to study diet has been more limited [ 20 ], and many challenges remain for authentication of DNA from dietary sources [ 21 ]. Studies of the impact of diet on oral microbiome composition in living populations suggest that although individual microbial species may differ in abundance between populations with different diets, the overall species composition remains consistent across a variety of diets [ 14 , 22 – 25 ]. To date, studies of ancient dental calculus have likewise found relatively little difference in the microbial composition of calculus from populations practicing different subsistence strategies [ 20 , 26 – 28 ]. This leaves us with an unclear understanding of whether and how social status may have historically impacted oral microbiota. To date, the majority of published metagenomic data from ancient dental calculus come from individuals who were not of high social status [ 29 – 31 ] or whose social status is unknown. Analyzing the dental calculus metagenome of King Richard III offers the opportunity to investigate the oral microbiota and diet of a high-status individual for which there are both historical records and archaeological data for comparison. Here, we metagenomically characterize dental calculus from King Richard III and assess the presence of dietary DNA, as well as compare the microbial species profile to new and published dental calculus of predominantly non-elite individuals from locations in northern Europe spanning the last 7,000 years. We found that the dental calculus of Richard III contains an extraordinary amount of well-preserved DNA, and that the microbial species composition is indistinguishable from that of other historic-era calculus from across northern Europe, suggesting that a royal lifestyle and a rich diet did not substantially alter his oral microbiota. We reconstructed metagenomically-assembled genomes (MAGs) within the genus Tannerella , and found that the periopathogen Tannerella forsythia of Richard III falls within the known diversity and virulence of other modern and ancient T. forsythia in European populations. Putative dietary sequences could not be authenticated. Although deeper sequencing may provide additional dietary DNA sequences, the very low proportion of putative dietary sequences suggests that not all dental calculus may be a sufficient source of dietary DNA for dietary reconstruction, even when exceptionally well-preserved. Results Exceptional DNA recovery from the dental calculus of Richard III We extracted DNA from four calculus samples collected from three teeth of Richard III (Table S1), and during laboratory processing we observed very high recovery of DNA, ranging from 122.4-506.7 ng/mg ( Table 1 ). We compared this DNA recovery to 143 other archaeological dental calculus samples previously measured in our laboratories (Table S2), which ranged from 0.1 ng/mg to 437 ng/mg, with a median of 36 ng/mg ( Figure 1A ). We found that the amount of DNA recovered from the calculus of King Richard III is among the highest we have ever measured from an archaeological context, and is comparable to that at other recent archaeological sites with exceptional preservation in northern Europe (Dalheim, Tickhill, Middenbeemster) and Oceania (Rapa Nui). Such high DNA concentrations are similar to those measured from modern calculus (83.4-346 ng/mg) and suggest an excellent state of DNA preservation. To take advantage of this preservation and allow de novo genome assembly, we sequenced each dental calculus library from Richard III to a high depth totaling more than 400 million sequences ( Figure 1B ). To facilitate comparative analysis, we also performed DNA extraction and deep sequencing of dental calculus from three individuals from the Neolithic Linearbandkeramik (LBK, 8th millennium BP) site of Stuttgart-Mühlhausen (Table S1) in Baden-Württemberg, Germany [ 32 ], and performed deeper sequencing of dental calculus libraries (Table S1) from the well-preserved G12 individual at the site of Dalheim, Germany (medieval, 750-1000 BP) [ 12 ]. We then selected and downloaded published dental calculus metagenomic datasets within the AncientMetagenomeDir (61) dating to 200-1000 BP from England, Ireland, the Netherlands, and Germany (Table S3) for comparative microbiome analysis. View this table: View inline View popup Download powerpoint Table 1. DNA extraction results Download figure Open in new tab Figure 1. Quantification of extracted DNA and sequenced reads. A . Normalized DNA recovery reported in ng DNA / mg dental calculus, with the dental calculus of Richard III (RIII) indicated in purple, other ancient dental calculus in gray, and modern dental calculus in red. B . The number of DNA sequences per individual analyzed in this study (except the four libraries from Richard III which are reported separately), including both newly generated and published sequencing data after read processing and quality filtering; note use of log scale. Well-preserved oral microbiota but no confirmed dietary DNA Taxonomic classification of newly sequenced dental calculus from Richard III, Stuttgart-Mühlhausen, and the medieval site of Dalheim with kraken2 [ 33 , 34 ] using the GTDB [ 35 ] indicated that all samples were exceptionally well-preserved, with > 90% of DNA sequences attributed to an oral source (dental calculus or dental plaque) based on SourceTracker analysis ( Figure 2 ; Table S4). Additional preservation assessment using cuperdec [ 14 ] confirmed a high proportion of oral taxa in these samples, as well as in the comparative dental calculus datasets (Figure S1). Download figure Open in new tab Figure 2. SourceTracker analysis of dental calculus preservation. All newly sequenced DNA libraries in this study exhibit a high preservation of oral microbial species with minimal contamination from other sources. Published data for sample G12 (G12_pub) and B61, from the same site, are shown for comparison. Dotted line indicates 90%. Within the dental calculus of Richard III, however, no dietary sequences could be authenticated or confirmed. No plant sequences were identified following taxonomic classification with MALT [ 36 , 37 ] using the NCBI nt database, and non-human animal sequences were limited to two species known to have contaminated and unreliable reference genomes [ 21 ]: Cyprinus carpio (common carp, 4,112 reads) and Erinaceus europaeus (European hedgehog, 42,683 reads) (Table S5). These two taxa were also identified in negative and environmental controls, further supporting their identification as false positives. Modern dental plaque likewise contained few dietary reads, with the largest number of reads assigned to contaminated genomes ( Cyprinus carpio ), model organisms (e.g., Danio rerio ), and wheat ( Triticum aestivum ) (Table S5). This suggests that dietary DNA incorporation and preservation within oral biofilms may be limited. In contrast to dietary DNA, recovery of DNA from the oral microbiota was excellent. We identified nearly four hundred microbial species in the dental calculus of Richard III, which is similar to the number of species detected in other well-preserved archaeological dental calculus samples from England, Ireland, the Netherlands, and Germany ( Figure 3A ). Species evenness as measured by the Shannon index was similar across the dental calculus data sets ( Figure 3B ). Analysis of between-sample diversity places the dental calculus of Richard III within the known microbial diversity of northern Europe ( Figure 3C ), indicating that the oral microbial profile of the king was not notably different from that of other individuals living in a broadly similar geographic region and time period. The main difference between the king’s calculus and other archaeological dental calculus from northern Europe appears to be primarily the amount of microbial DNA preserved and not which microbial species were present or how abundant they were. Download figure Open in new tab Figure 3. Microbiome metagenomic diversity analysis comparing calculus from King Richard III to other archaeological individuals in northern Europe. A . Number of microbial species detected. B . Shannon index of species evenness. C . Principal component analysis (PCA) of between-sample microbial diversity. Phylogenetic and virulence factor diversity of Tannerella forsythia One species found in high abundance and with good preservation in the dental calculus of Richard III was Tannerella forsythia. T. forsythia is a common oral species that is today associated with periodontal disease [ 38 ], but which was historically highly abundant in calculus on teeth with no evidence of disease [ 30 ]. We performed de novo assembly and binning of the dental calculus metagenomes, and recovered one metagenome-assembled genome (MAG) of T. forsythia from Richard III, four from the Radcliffe Infirmary in Oxford, England, and three from Dutch site of Middenbeemster. We also recovered four genomes of an unnamed Tannerella species from the Radcliffe Infirmary, and one genome each from the sites of Stuttgart-Mühlhausen and Dalheim in Germany. All fourteen Tannerella genomes meet the minimum quality thresholds for medium and high quality MAGs [ 39 ] used for modern-day genome reconstruction (Table S6, Table S7). To confirm an ancient origin for the reconstructed Tannerella genomes, we mapped the reads from each individual (except the four libraries from Richard III which were each analyzed separately) that produced a Tannerella MAG against the T. forsythia reference genome and assessed the 5’ C-to-T damage patterns ( Figure 4A ). DNA sequences from Richard III and the Radcliffe Infirmary produced expected age-related damage patterns, indicating that the Tannerella sequences in these samples originate from an ancient source [ 40 ]. Libraries from the Dutch site of Middenbeemster were prepared with partial-USER treatment and showed an expected ancient DNA damage pattern of reduced damage only on the first base [ 41 ]. Libraries from the German sites were prepared with full-USER treatment (Stuttgart-Mühlhausen) or a proofreading enzyme (Dalheim) and consequently show no DNA damage [ 42 , 43 ]. Notably, only the samples from the Netherlands show a baseline damage frequency of 0, while the others range between 0.025 and 0.075, suggesting that other Tannerella species may be present in these samples. As only a single MAG was recovered from each assembly, it is likely that additional Tannerella species are present at lower abundance, and hence did not produce a MAG during assembly due to insufficient read counts. At present, four Tannerella species are recorded as inhabiting the human oral cavity in the Human Oral Microbiome Database (eHOMD) v.4 [ 44 , 45 ], but reference genomes are available for only two: T. forsythia and T. serpentiformis . Further work is needed to investigate the diversity of this genus to better understand its ecology in ancient dental calculus. Download figure Open in new tab Figure 4. Analysis of ancient Tannerella forsythia phylogeny and virulence. A . 5’ C-to-T damage patterns for each dental calculus sample (each dental calculus library for Richard III) mapped against the T. forsythia reference genome, grouped by country of origin. Richard III and other samples from England show expected damage patterns, while samples from the Netherlands were partial-USER-treated and show correspondingly low levels of damage on the first base. Damage was not observed for the German samples because they were processed using full-USER treatment or a proofreading enzyme during library construction. B . Phylophlan single-marker gene phylogeny of reference and ancient de novo reconstructed Tannerella forsythia genomes, mid-point rooted. RAxML bootstrap values of deep nodes are shown. C . Presence/absence matrix of virulence factors for T. forsythia , as well as genome completeness and contamination, aligned with the tree tips in panel B. Genome labels are provided in Figure S2. Phylogenetic analysis of the de novo assembled genomes, along with reference genomes of T. forsythia from NCBI and the outgroup genome of T. serpentiformis , placed the ancient T. forsythi a genomes from Richard III, England, and the Netherlands in a clade along with most of the reference genomes ( Figure 4B ). Two reference genomes and three of the unnamed Tannerella genomes (two from England and one genome from Neolithic Germany) fall outside of the main T. forsythi a clade, suggesting these belong to different strains, and possibly different but currently unnamed species. Patristic distance calculations support this designation, as values within the T. forsythia clade are 0.053 when excluding these genomes, which is at the boundary of species-level cut-offs (0.05) [ 46 ], while the average patristic distance within the clade when including these genomes is 1.7, far higher than species-level designations (Table S8). The final de novo assembled ancient genome from Germany falls in a clade with the T. serpentiformis genome. High bootstrap values on deep nodes in the tree ( Figure 4B ) support the separation of these Tannerella genomes into distinct clades. The Tannerella genome from Germany that falls near T. serpentiformis is from the medieval G12 individual published by [ 12 ]. This study reported the presence of multiple Tannerella species, further resolution was not possible due to the genome availability of only one species, T. forsythia , within the Tannerella genus at the time. To investigate whether the assembled Tannerella MAG from this sample is T. serpentiformis , we further mapped all G12 sample libraries against the T. serpentiformis reference genome. We observed similar damage patterns when mapping to either the T. forsythia or the T. serpentiformis genome (Figure S3), with slightly more mismatches detected when reads are mapped against the T. serpentiformis genome. This suggests that T. serpentiformis is not necessarily a closer match to the Tannerella species found in sample G12, and it is possible that this Tannerella genome represents another novel, as-yet-undescribed species in the genus. To assess the pathogenic potential of the reconstructed genomes in the broader Tannerella clade, we examined a selection of eight known virulence factors for T. forsythia ( Figure 4C ) using a previously curated list [ 14 ]. The T. forsythia genome from Richard III is the only reconstructed MAG to include both surface layer (S-layer) glycoprotein genes tfsA and tfsB . While these virulence factor genes are nearly ubiquitous in the reference genomes, several could not be detected in the de novo reconstructed genomes ( Figure 4B ), which may be due to the lower completeness of these genomes rather than a true absence. For genomes that fall outside the main T. forsythia clade, a third or more of the virulence factor genes could not be detected. While this also may be due to a lower completeness of the reconstructed MAGs, it may also reflect a true absence given that these genes were not consistently detected in reference genomes and may not be ubiquitous in Tannerella species other than T. forsythia . Overall, additional exploration of the genomic and genetic diversity of oral Tannerella is warranted to better understand the phylogenetic relationships and gene content observed in this study. Discussion We report the oral microbiome profile of four dental calculus samples from King Richard III of England, as well as new comparative dental calculus data from Neolithic and medieval Germany. DNA preservation within the dental calculus of Richard III was exceptional, but the reconstructed oral microbiota did not stand out from dental calculus of similar age and geographic origin regarding the number of microbial species detected, nor the abundance and distribution of microbial species. Despite the high DNA recovery and deep sequencing of the dental calculus metagenomes, no authentic dietary DNA was identified. Phylogenetic assessment of the abundant periopathogen Tannerella forsythia revealed that the T. forsythia genome reconstructed from Richard III is highly similar to genomes found in present-day living populations, as well as in other archaeological dental calculus from Northern Europe. A wide range of dietary microfossils, proteins, and metabolites have been previously reported in modern and archaeological dental calculus [ 13 , 47 – 49 ], but dietary DNA is less well studied. Nevertheless, detection and authentication of dietary DNA in ancient dental calculus remains a potential source of information about diet at individual level. While DNA sequences derived from particular meat and vegetable sources have been reported in ancient dental calculus [ 12 , 50 ], few studies have reported on the presence or abundance of potential dietary reads, while those that have investigated dietary reads have generally been unsuccessful in authenticating them [ 20 , 21 , 51 ]. In some cases, dietary DNA may be present, but the overwhelming abundance of microbial reads reduces the chances of dietary reads being incorporated into DNA libraries and sequenced. It is also possible that dietary DNA is mostly degraded by enzymes in saliva [ 52 ] or in the microbial biofilm [ 53 , 54 ], and very little is incorporated into dental calculus. Despite the deep sequencing of the dental calculus from Richard III, totaling nearly 400 million DNA sequences from three teeth, no authentic dietary DNA sequences were identified. Poor DNA preservation is unlikely to be the reason given the very high DNA recovery, which was similar to that from present-day dental calculus, and the excellent preservation of the oral microbiome. Additional or deeper sequencing may eventually produce enough additional DNA sequences to identify potential dietary components, but this approach is expensive and not guaranteed to produce results. The low number of putative dietary sequences observed in present-day dental plaque suggests that the incorporation and preservation of dietary DNA may be limited in oral biofilms. It is unclear why other dietary remains, such as plant microfossils and dietary proteins, are more robustly recovered from dental calculus, but at present it appears that dental calculus may not be a sufficient source of dietary DNA even when exceptionally well-preserved and deeply sequenced. Isotopic analysis of femur and rib fragments from the skeleton of king Richard III suggest that he consumed a diet rich in non-local water sources, such as wine, and high trophic level game animals, such as fish and waterfowl, during the last 2-3 years of his life [ 9 ]. However, this aristocratic and unusually rich diet does not appear to have resulted in detectable microbial community changes compared to the dental calculus of lesser elites and commoners from across northern Europe, as all dental calculus analyzed in this study exhibited shared and overlapping species diversity. Whether the king’s diet may have promoted the growth of specific individual oral microbial species, rather than affecting the entire microbial community, is unclear. Microbial databases remain incomplete and insufficient to fully resolve many oral taxa at the species level, as evidenced by our findings for the genus Tanerella . To date, a direct relationship between diet and dental plaque biofilm species composition remains weakly and inconsistently supported in the literature [ 22 , 28 ]. Population-level dental calculus data collected over time in a single region are needed to more finely track species and strain-level microbial changes following a dietary change. Further advances in laboratory techniques that enable DNA extraction from distinct layers of microbial accumulation on dental calculus may also open the field to exploring oral microbiome changes throughout the lifetime of an individual. Tannerella forsythia is an oral bacterial species strongly associated with periodontal disease in living populations [ 55 , 56 ], and it is grouped together with Porphyromonas gingivalis and Treponema denticola within the “red complex” consortium of oral species with high pathogenic potential [ 38 ]. Our analysis of virulence genes within reconstructed T. forsythia genomes from ancient dental calculus found that these genes have been present in the species for at least several centuries, confirming earlier mapping-based assessments [ 17 – 19 ]. Although we did not detect all eight genes in each reconstructed genome, this absence could in part be an artefact of de novo metagenome assembly. Current approaches rely on sufficient read length and coverage depth for genome reconstruction, and ancient DNA libraries may fall short of these lower limits [ 57 ]. Oral species diversity within the genus Tannerella has not been extensively explored. At present, only two named Tannerella species, T. forsythia and T. serpentiformis , are available in NCBI databases, but two additional unnamed species are recognized in the extended Human Oral Microbiome Database (eHOMD) [ 44 , 45 ]. These two unnamed species, designated HMT-808 and HMT-916, do not have available genomes. As we downloaded only named Tannerella genomes from NCBI for our analysis, we do not expect our tree to include these additional species. However, our Tannerella phylogeny and virulence factor analysis hint that historic dental calculus samples contain Tannerella species other than T. forsythia and T. serpentiformis . In our phylogeny, 29 Tannerella genomes–including 3 historic Dutch genomes, 4 historic English genomes, and the genome from King Richard III–fall into a large clade that appears to represent true T. forsythia genomes. Only a single Tannerella genome from medieval Germany appears to belong to T. serpentiformis (or a closely related species). However, two historic English and 1 Neolithic German Tannerella genomes, as well as 2 NCBI genomes designated T. forsythia , fall basal to the main T. forsythia clade, suggesting that these genomes may represent a distinct Tannerella species that is more similar to T. forsythia than to T. serpentiformis . While patristic distance calculations support this finding, further genomic investigation is necessary to confirm these observations. In addition to discovering additional Tannerella species diversity, our data suggest higher Tannerella diversity within the dental calculus of specific individuals. The elevated baseline damage frequency observed when mapping Tannerella DNA sequences from Richard III and other ancient individuals from England and Germany against the reference T. forsythia genome supports that these calculus samples may contain more than one Tannerella species. Because only a single Tannerella genome was reconstructed from each sample, however, it is likely that one species is dominant and present at an abundance that enabled de novo genome assembly, while the other species are less abundant and did not have sufficient sequences for de novo assembly [ 12 , 57 ]. While several studies have performed genomic and genetic investigations of T. forsythia in ancient dental calculus [ 12 , 17 – 19 , 58 ], they did not consider how the presence of other unnamed or unknown Tannerella species may have affected their analyses. Further investigation may reveal that some of the reported patterns of T. forsythia virulence gene prevalence in past populations can be attributed to these additional species. 5 Conclusion The dental calculus of King Richard III is exceptionally well-preserved and ancient DNA analysis has revealed an oral microbiome species composition that is highly similar to that of other human dental calculus from northern Europe. Despite excellent DNA preservation and deep sequencing of the DNA libraries, we were unable to identify authentic dietary DNA. We identified a higher level of taxonomic diversity in de novo reconstructed Tannerella genomes than has been reported to date in ancient dental calculus, indicating that further investigation of the diversity of the periodontal disease-associated genus Tannerella is warranted to better understand how the oral microbiome has changed through human history. Methods Dental calculus sampling Dental calculus samples of King Richard III were collected from lingual surfaces of three teeth: left lower first premolar, left lower canine, and right lower second premolar. Prior to analysis, the dental calculus from the canine was split into two sub-fractions, resulting in a total of 4 samples: R3-1 (GRF001.A), R3-2A (GRF001.B), R3-2B (GRF001.C), and R3-3 (GRF001.D) ( Table 1 ; Table S1A). Six swabs from the archaeological storage facility were collected to monitor for possible contamination: sample bag (R3-Bag), sample box (R3-Box), storage refrigerator (R3-Fr), laboratory gloves (R3-Gl), osteologist hands (R3-HS), and a laboratory water sample (R3MODBL) (Table S1A). Samples and swabs were transferred to the ancient DNA cleanroom facilities at the Laboratories of Molecular Anthropology and Microbiome Research (LMAMR) at the University of Oklahoma for DNA extraction. Dental calculus of three additional individuals from the Neolithic Linearbandkeramik (LBK, 6th millennium BCE) site of Stuttgart-Mühlhausen (Viesenhäuser Hof) in Baden-Württemberg, Germany [ 32 , 59 – 61 ] were also collected for comparative analysis (Table S1A): gr. I-064/335 (SMH005.A), gr. I-056/528 (SMH007.A), gr. II-027/274 (SMH008.A). Samples were transferred to the ancient DNA cleanroom facilities at the University of Tübingen, Germany for DNA extraction. DNA extraction DNA from the dental calculus of Richard III was extracted in a dedicated ancient DNA facility at LMAMR in accordance with established contamination control precautions and workflows. Two cleanroom non-template extraction controls (R3-AncBL1, R3-AncBL2) were processed alongside the experimental samples during all analytical steps to monitor for possible contamination. Prior to decalcification, dental calculus samples were UV-irradiated for 2 min using a Stratagene Stratalinker UV 1800 Crosslinker. To remove remaining surface debris and contaminants, they were agitated in 1 ml of 0.5 M EDTA solution for 15 min and decanted. The dental calculus samples were then processed using the DNA extraction protocol described in [ 62 ], with minor modifications. In brief, the samples were crushed to powder using a sterile steel spatula and resuspended in 1 ml 0.5M EDTA (Sigma) and incubated overnight at room temperature. A 100 μl proteinase K solution (>600 mAU ml -1 ; Qiagen) was then added and incubated at 37°C for 8 hours, followed by continued digestion under agitation at room temperature until decalcification was complete. The DNA was then purified using a MinElute column (Qiagen) connected to a Zymo reservoir (Zymo Research) following modified manufacturer instructions in which the amount of PB binding buffer was increased to 13 ml [ 62 ]. The DNA was eluted in 60 μl EB buffer (Qiagen), and the concentration of the eluate was quantified using 1 μl with a Qubit HS assay (Life Technologies). DNA from the Stuttgart-Mühlhausen dental calculus samples was extracted in a dedicated ancient DNA facility at the University of Tübingen in accordance with established contamination control precautions and workflows, and following the DNA extraction protocol described in [ 62 ] without modification. Illumina library construction Shotgun Illumina libraries (Table S1B) were constructed from 60-100 ng DNA from Richard III calculus samples and 30 μl of extract for non-template extraction controls using a NEBNext DNA Library Prep Master set for 454 (New England Biolabs, E6070) according to manufacturer instructions. End repair was performed in 50 μl reactions with 100 ng of DNA. The end repair cocktail was incubated for 20 min at 12°C and 15 min at 37°C and then purified using a MinElute column (Qiagen) and eluted in 30 μl EB buffer. Blunt end adapters (IS1/IS3 and IS2/IS3) were prepared following Meyer and Kircher (2010) and ligated to the end-repaired DNA in 50μl reactions. The reaction was incubated for 15 min at 20°C and then purified using QiaQuick columns (Qiagen) and eluted in 30 μl EB. The adapter fill-in reaction was performed in a final volume of 50 μl and incubated for 20 min at 37°C followed by 20 min at 80°C to inactivate the Bst polymerase. Libraries were amplified and indexed in a 50 μl PCR reaction, using 26.3 μl H 2 O, 5 μl 10x Platinum Taq MasterMix, 3 μl 2mM dNTPs, 1 μl BSA (2.5 mg/ml), 1.5 μl 50 mM MgCl2, 1.5 μl i5 indexing primer (10 μM), 1.5 μl i7 indexing primer (10 μM), 0.2 μl PlatinumTaq, and 10μl of library template. Thermocycling conditions were 2 min at 95°C, followed by 12 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C, and a final 7 min elongation step at 72°C. The optimal number of PCR cycles for each sample was first confirmed by qPCR, and all negative controls were amplified for 20 cycles. All libraries were purified using a MinElute PCR Purification kit (Qiagen) following manufacturer instructions and quantified using a Bioanalyzer 2100 High Sensitivity DNA assay (Agilent). Shotgun Illumina libraries (Table S1B) were prepared similarly for the Stuttgart-Mühlhausen dental calculus samples, with minor modifications as described in the Non-UDG Treated Double-Stranded Ancient DNA Library Preparation for Illumina Sequencing protocol available on protocols.io ( dx.doi.org/10.17504/protocols.io.bakricv6 ). A second aliquot of extracted DNA was then additionally prepared following USER enzyme treatment in order to remove DNA damage, as described in the Full-UDG Treated Double-Stranded Ancient DNA Library Preparation for Illumina Sequencing protocol available on protocols.io ( dx.doi.org/10.17504/protocols.io.bqbpmsmn ). DNA sequencing Richard III DNA libraries (Table S1B) were pooled in equimolar amounts and sequenced on an Illumina HiSeq 2000 instrument with PE 2x100bp chemistry in RapidRun mode at the Yale Center for Genome Analysis (YCGA). A total of 393,365,842 sequencing reads were generated: R3-1, 73,678,628; R3-2A, 104,804,438; R3-2B, 125,374,688; R3-3, 89,508,088. At the MPI-EVA, the Stuttgart-Mühlhausen non-UDG treated libraries (SMH005.A0101, SMH007.A0101, SMH008.A0101; Table S1B) were sequenced on an Illumina NextSeq 500 with PE 2x75bp chemistry, and the full-UDG libraries (SMH005.A0102, SMH007.A0102, SMH008.A0102; Table S1B) were sequenced on an Illumina HiSeq 4000 with PE 2x75bp chemistry. A total of 724,359,015 sequencing reads were generated: SMH005.A, 213,889,522; SMH007.A, 164,169,600; SMH008.A, 346,299,893. To generate more medieval dental calculus sequencing data for comparative analysis, we also deeply sequenced four previously generated DNA libraries (Table S1B) of individual G12 (S5, S6, S7, S8) from the site of Dalheim, Germany [ 12 ] on two flow cells of an Illumina HiSeq 2000 using PE 2x150bp chemistry at the Yale Center for Genome Analysis (YCGA). A total of 240,767,032 sequencing reads were generated: S5, 86,797,120; S6, 55,998,288; S7, 50,206,795; S8, 47,764,829. Data processing Raw data was processed using the nf-core/eager v2.4.4 pipeline [ 63 ], including adapter trimming, read quality-filtering (min length 30bp, quality 40), and merging with adapter-removal, mapping against human genome hg19 with bwa aln v0.7.17-r1188 [ 64 ], and selecting all reads that did not map to the human genome into a single fasta file with samtools v1.17 [ 65 ]. These fasta files of non-human reads were used for taxonomic profiling. Published comparative samples Normalized DNA extraction yields (ng DNA/mg calculus) measured in our laboratory from 143 archaeological dental calculus samples from diverse global sites and 3 present-day individuals ( Figure 1A ) were obtained from the literature [ 11 , 12 , 29 , 31 , 51 , 66 – 68 ] and are provided in Table S2. Published dental calculus metagenomic datasets from northern Europe dating 200-1000 BP were selected using AncientMetagenomeDir (accessed 2024/01) for [ 69 ] (Table S3). These included samples from England [ 30 , 70 , 71 ], Ireland [ 29 ], the Netherlands [ 31 ], and Germany [ 12 ]. All sequencing data for these samples were downloaded from ENA and processed with the nf-core/eager pipeline as described above for the new genetic data generated in this study, but datasets with fewer than 1M sequenced reads were excluded due to high stochasticity in identified species obfuscating signals of contamination or false-positive identification vs. true presence. This approach excluded all environmental swabs (R3-AncBL1, R3-AncBL2, R3Bag, R3Box, R3FR, R3GL, R3HS, R3MODBL) collected during sampling of the four calculus samples from Richard III. Taxonomic profiling and diversity analysis Newly sequenced and published metagenomic datasets ranged from <3M to >300M reads ( Figure 1B ), with most datasets between 5M and 30M reads. Notable exceptions were the newly sequenced libraries from King Richard III and from Germany (Stuttgart-Mühlhausen and Dalheim), which were intentionally deeply sequenced to facilitate de novo genome assembly. To improve data quality and consistency, samples with fewer than 5M reads were excluded from all downstream analysis. All samples were taxonomically profiled with kraken2 v2.1.3 [ 33 , 34 ] using the GTDB v202 [ 35 ] database downloaded from the Struo2 [ 72 ] ftp server ( http://ftp.tue.mpg.de/ebio/projects/struo2/ ). Taxonomic tables were filtered in R v4.3.2 to remove potential contamination by removing all species with fewer than 5,000 reads across all samples and then filtering out all species that were present at less than 0.01% relative abundance. The number of species and Shannon index were calculated in R using the package vegan v2.6-8 [ 73 ]. A principal component analysis (PCA) was run in R with the package mixOmics v6.26.0 [ 74 ]. After adding a value of +1 to all entries in the table to remove 0 values, a centered-log ratio transformation [ 75 ] was run on the dataset and a PCA was performed on the transformed datatable. All diversity analysis results were plotted in R with ggplot2 [ 76 ]. Microbiome preservation assessment Preservation of the samples was assessed using two methods. First, SourceTracker v1.0 [ 77 ] was applied to the newly generated data to determine whether the species profile was predominantly attributable to an oral source or to potential contamination sources (Table S4). Comparative sources included modern human dental calculus [ 14 , 30 ], dental plaque [ 78 ], stool [ 78 – 80 ], and skin [ 78 , 81 ], as well as archaeological bone [ 14 ] and sediment [ 82 ]. Second, the R package cuperdec [ 14 ] was used to assess the proportion of taxa from an oral source within all samples, including both newly generated sequences and published comparative data. Negative controls and one published dental calculus sample failed to meet the threshold for high oral content (Table S2; Table S3) and were excluded. Dietary analysis For identification of putative dietary DNA sequences, we used the high-throughput aligner MALT [ 36 , 37 ] together with the NCBI nt database (October 2017; uploaded to Zenodo under DOI: 10.5281/zenodo.4382154) to taxonomically profile: the Richard III dental calculus samples (R3-1, R3-2A, R3-2B, and R3-3), negative controls (R3-AncBL1, R3-AncBL2), and environmental controls (R3Bag, R3Box, R3FR, R3GL, R3HS, R3MODBL); and the dental plaque [ 78 ], stool [ 78 – 80 ], skin [ 78 , 81 ], archaeological bone [ 14 ] and archaeological sediment [ 82 ] datasets also used for preservation assessment. We employed a relaxed percent identity parameter of 85% and a base tail cut off (“minimum support”) of 0.01%. Resulting RMA6 files were loaded into MEGAN6 CE [ 83 ] and Operational Taxonomic Unit (OTU) tables were exported for Spermatophyta (seed plants) and Euteleostomi (major clade of vertebrates) (Table S5). De novo genome reconstruction All metagenome samples were de novo assembled and binned using a Snakemake [ 84 ] pipeline previously described in [ 85 ], and which is available on GitHub under https://github.com/alexhbnr/ancient_metagenome_assembly . Assemblies were performed on an individual level, such that datasets from all libraries of a single individual were assembled together, resulting in a single output of metagenome-assembled genomes from all four libraries of Richard III. In brief, unmerged paired-end sequencing datasets were assembled using MEGAHIT v1.2.9 [ 86 ]. After correcting the consensus contig sequences to remove miscoding lesions due to the presence of ancient DNA [ 85 ], the contigs were binned using metaBAT v2.15 [ 87 ], MaxBin v2.2.7 [ 88 ], and CONCOCT v1.1.0 [ 89 ] and subsequently refined using metaWRAP v1.3.2 [ 90 ]. The resulting metagenome-assembled genomes (MAGs) (Table S6) were further refined and validated and annotated with bakta v1.9.4 [ 91 ] using a Snakemake pipeline previously described in [ 85 ] and available on GitHub under https://github.com/alexhbnr/automatic_MAG_refinement to remove potentially chimeric contigs. Phylogenetic analysis and authentication All genomes designated as Tannerella forsythia in NCBI databases were downloaded for phylogenetic comparison to the de novo reconstructed ancient genomes. The completeness and contamination of all reference genomes from NCBI were assessed with checkM v1.2.2 [ 92 ] using the program dRep v3.4.3 [ 93 ] (Table S6). A genome of Tannerella serpentiformis (GCA_003033925.1) was downloaded for use as an outgroup to root the tree. All NCBI genomes and all de novo reconstructed ancient genomes were used as input for the program PhyloPhlAn v3.1 [ 94 , 95 ] to reconstruct a tree based on selected marker genes specific for the species T. forsythia . Bootstrapping (200 replicates) was performed by RAxML using RAxML-NG v1.2.2 [ 96 ] on the protein marker gene alignment file produced by Phylophlan. The resulting tree was loaded into R and visualized with ggtree [ 97 ] with a midpoint root. Patristic distances were calculated from the tree using cophenetic.phylo in the R package ape v5.8 [ 98 ] (Table S8). The reconstructed genomes were confirmed to be of ancient origin by assessing the damage patterns on reads from each sample mapped against the T. forsythia reference genome (NC_016610.1). All libraries were mapped using bwa aln v0.7.17-r1188 with the flags -n 0.01 -o 2 -l 16500. Mapped reads were filtered with samtools v1.17, and only reads with a mapping quality of ≥ 25 and a length ≥ 30bp were considered suitable for damage analysis (Table S7). DamageProfiler v1.1 [ 99 ] was used to quantify DNA damage on mapped reads. Libraries of the sample G12 were additionally mapped against the T. serpentiformis reference genome, and the mapped reads were also filtered and analyzed using DamageProfiler. Virulence gene content assessment The Tanerella genomes downloaded from NCBI were annotated with bakta [ 91 ] using the same settings as in the de novo assembly pipeline above. The annotated gene names were cross-referenced against the known T. forsythia virulence factors listed in [ 14 ], and marked as 1 if present and 0 if absent (Table S6). The binary table was loaded into R and visualized as a matrix using tidyverse v1.3.0 [ 100 ] and ggplot2 [ 76 ], and then combined with the phylogenetic tree using patchwork v1.2.0 [ 101 ]. The completeness and contamination of all genomes were likewise visualized in the same way using R. Dietary DNA assessment Potential dietary DNA sequences were assessed using MALT [ 36 , 37 ]. The metagenomic datasets were aligned with MALT v 0.4.0 against the NCBI nt database (October 2016). Reads matching eukaryotes were evaluated for the number of reads mapped and damage patterns to identify genuine ancient reads. No potential dietary eukaryotes could be confirmed as authentic due to insufficient numbers of reads or a lack of DNA damage patterns. Author Contributions T.K., A.R., and C.W. conceived the study. T.K., E.R., C.M.L., M.F., J.W., J.K., and C.W. provided samples and resources. A.M., C.H., C.S., S.F., and A.O. performed lab work. I.M.V., A.H., Z.F., and J.F.Y., performed data analysis and interpretation. I.V. and C.W. wrote the manuscript with input from all authors. Ethics statement All required permissions were obtained to perform genetic data generation from newly analyzed archaeological dental calculus in this study. The excavation and analysis of the remains of Richard III was carried out by the University of Leicester Archaeological Services as part of the excavation of the Grey Friars Friary under a licence granted by the U.K Ministry of Justice. Permission to analyze remains from the site of Stuttgart-Mühlhausen was provided by the State Office for Monument Preservation in Konstanz, Germany. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement All new sequencing data generated for this study was deposited in the European Nucleotide Archive under accession PRJEB84038 at https://www.ebi.ac.uk/ena/browser/view/PRJEB84038 . Code used to perform analyses and generate the figures is provided in a GitHub repository at https://github.com/ivelsko/RIII_oral_micro . Acknowledgments We thank Joanna Drath, Eva Rosenstock, Alisa Hujic, and Michal Feldman for assistance with the Stuttgart-Mühlhausen remains. Funding for this study was provided by the Werner Siemens Stiftung (“Paleobiotechnology” to C.W.), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (EXC 2051 Project-ID 390713860, “Balance of the Microverse”), the Max Planck Harvard Research Center for the Archaeoscience of the Ancient Mediterranean (MHAAM), and the Max Planck Society. Funder Information Declared Werner Siemens Foundation , Paleobiotechnology Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64 , EXC 2051 Project-ID 390713860, “Balance of the Microverse” Max Planck Harvard Research Center for the Archaeoscience of the Ancient Mediterranean (MHAAM) Max Planck Society, https://ror.org/01hhn8329 Footnotes https://www.ebi.ac.uk/ena/browser/view/PRJEB84038 https://github.com/ivelsko/RIII_oral_micro References 1. ↵ Buckley R , Morris M , Appleby J , King T , O’Sullivan D , Foxhall L. 2013 ‘The king in the car park’: new light on the death and burial of Richard III in the Grey Friars church, Leicester, in 1485 . Antiquity 87 , 519 – 538 . OpenUrl CrossRef 2. ↵ Appleby J et al. 2015 Perimortem trauma in King Richard III: a skeletal analysis . Lancet 385 , 253 – 259 . 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