Genetic history of Cambridgeshire before and after the Black Death

The extent of the devastation of the Black Death pandemic (1346–1353) on European populations is known from documentary sources and its bacterial source illuminated by studies of ancient pathogen DNA. What has remained less understood is the effect of the pandemic on human mobility and genetic diversity at the local scale. Here, we report 275 ancient genomes, including 109 with coverage >0.1×, from later medieval and postmedieval Cambridgeshire of individuals buried before and after the Black Death. Consistent with the function of the institutions, we found a lack of close relatives among the friars and the inmates of the hospital in contrast to their abundance in general urban and rural parish communities. While we detect long-term shifts in local genetic ancestry in Cambridgeshire, we find no evidence of major changes in genetic ancestry nor higher differentiation of immune loci between cohorts living before and after the Black Death.

The Augustinian Friary (Friary) was established in Cambridge between 1279/80 and 1289, when it was first mentioned in a royal pittance.The Friary grew rapidly and thrived, becoming a studium generale or national study house with internal connections in 1318 and having 70 friars present in 1328.It continued until the Dissolution 1538, as one of the most important Augustinian friaries in England and one of the largest institutions in Cambridge.
The skeletons reported in this study were recovered during archaeological excavations in advance of development of the site undertaken by the Cambridge Archaeological Unit in 2016-2017 in accordance with the appropriate planning regulations.The burials have been dated by a combination of stratigraphy, documentary sources, typological dating of associated artefacts particularly pottery, radiocarbon dating of a sample of the skeletons and ancient DNA evidence for Y. pestis..The skeletons were initially studied by Benjamin Neil of the Cambridge Archaeological Unit and were reexamined for the After the Plague project by Sarah Inskip.The human skeletal remains are held by the Historic Environment Team of Cambridgeshire County Council (archaeology@cambridgeshire.gov.uk), to whom any requests for access should be made.
Human remains have been recovered from three locations at the Friary: an early cemetery and the later chapter house and cloister.Twenty-eight burials (26 male and 2 female) included in this study (Table S1) form a subset of 72 burials that were excavated by the Cambridge Archaeological Unit in 2016-2017 and come from the cemetery and chapter house (100)(101)(102)(103).The friary appears to have acquired the right to burial of individuals in 1290.Based on radiocarbon dating, stratigraphy, artefact typology and architectural criteria the cemetery burials are dated to c. 1290-1400/20 and those from the chapter house to c. 1330/50-1538.Some of the burials were accompanied by single buckles located near the pelvis, indicating that the bodies were buried in a clothed state, with surviving evidence for associated leather girdles and some evidence for textiles.Some of the skeletons definitely lacked buckles and these were probably buried in shrouds.It appears that members of the Augustinian order received clothed burial, while shrouded burials are of lay individuals (104).The lay individuals would include patrons and benefactors, as well as lay servants of the Friary and corrodians.The 72 burials recovered from the Friary represent only a portion of the estimated 200-700 individuals likely to have been interred there between 1290 and 1538.Three of the 28 individuals examined here have been directly radiocarbon dated and four individuals had tested positive for Y. pestis (10)

Bene't Street
St Bene't's (a contraction of Benedict's) parochial church in Cambridge was established in c. 1000-1050 CE and remains in use, with burial continuing until the 1850s.A strip of land along the western side of the churchyard was transferred to Corpus Christi College between 1352 and 1377, to form an entrance route between Bene't Street and the College.Part of this strip was excavated by the Cambridge Archaeological Unit in 2006, revealing highly truncated individual burials and part of one mass burial (105).The skeletons reported in this study were recovered during archaeological excavations undertaken in advance of development of the site by the Cambridge Archaeological Unit in 2005-2006 in accordance with the appropriate planning regulations.The burials have been dated by a combination of stratigraphy, documentary sources, typological dating of associated artefacts particularly pottery and ancient DNA evidence for Y. pestis.The skeletons were initially studied by Natasha Dodwell of the Cambridge Archaeological Unit and were reexamined for the After the Plague project by Sarah Inskip.The human skeletal remains are currently held by the Cambridge Archaeological Unit and will be transferred to the Historic Environment Team of Cambridgeshire County Council (archaeology@cambridgeshire.gov.uk), to whom any requests for access should be made.
Among four skeletons from the mass burial whose teeth were tested for DNA, two yielded positive and one tentative identifications of Y. pestis (10).It is probable that this mass burial relates to the Black Death as it is likely to predate the construction of the College buildings, which took place between 1352 and 1377, and this has recently been confirmed (35).These individuals, although none of them have been directly radiocarbon dated, are interpreted as parishioners of St Bene't's, who died during the Black Death in 1349.Three of the tested individuals were male and one was female (Table S1).

Baptist Chapel
The Providence Calvinistic Baptist Chapel (Baptist Chapel) in Cambridge was in use for just four years 1833-1837 CE.Part of the associated cemetery was excavated in 2012, by Oxford Archaeology East in advance of development of the site in accordance with the appropriate planning regulations (106).The burials have been dated by a combination of documentary sources, stratigraphy and typological dating of associated artefacts.The skeletons were initially studied by Mark Gibson and Louise Loe of Oxford Archaeology East and were reexamined for the After the Plague project by Sarah Inskip.The human skeletal remains were initially held by Oxford Archaeology East, but will be transferred to the Historic Environment Team of Cambridgeshire County Council (archaeology@cambridgeshire.gov.uk), to whom any requests for access should be made.
There originally may have been a maximum of 20 graves in the investigated area, with at least 16 graves in five rows 11 of which were recorded.The graves were aligned north-northwest to southsoutheast and were mainly earth cut, but two were brick-lined and there is evidence for coffins and shrouds.The majority of the individuals recovered died at a young age and are interpreted as members of non-conformist families in Cambridge, documentary evidence indicates that the individuals who formed the community came from a range of social backgrounds.DNA was extracted from teeth and auditory ossicles of six individuals, including 4 females and 2 males (Table S1).

Holy Trinity
Holy Trinity Church in Cambridge (Holy Trinity) is first mentioned in 1174, when it is said to have been burnt down, and was probably established c. 1050-1150 CE.Excavations by the Cambridge Archaeological Unit in 2016-17 in advance of development of the site in accordance with the appropriate planning regulations revealed seventeen articulated burials (107).The burials have been dated by a combination of documentary sources, stratigraphy and typological dating of associated artefacts.The skeletons were studied by Benjamin Neil of the Cambridge Archaeological Unit and Sarah Inskip and Jenna Dittmar of the After the Plague project by Sarah Inskip.In accordance with the terms of the diocesan faculty under which the excavations took place and as is standard practice for still active churches the human skeletal remains were reburied at Holy Trinity Church, Cambridge.
Although the human skeletal remains were reburied, they were subject to thorough osteological analysis and a subset of 10 individuals subject to DNA analyses (Table S1).The 10 skeletons (6 males and 4 females) included in this study were placed in coffins in the vaults constructed after a vestry building of 1833/4 and prior to the end of burial in the cemetery in 1855.The individuals are interpreted as parishioners of Holy Trinity, burial in a vault indicates that they were relatively wealthy, and it is likely that the individuals in specific vaults were related to each other, although not necessarily genetically.

Midsummer Common
The circumstances of the discovery of these human skeletal remains is not recorded, but they were probably recovered during documented drainage works in the area.The human skeletal remains have been dated by a combination of documentary sources and radiocarbon dating.The human skeletal remains are held by the Duckworth Laboratory of the Department of Archaeology, University of Cambridge, to whom any requests for access should be made via their website: https://www.arch.cam.ac.uk/institutes-and-facilities-overview/duckworth-laboratory/contactduckworth.
From 1574 until the last outbreak in Cambridge in 1665/6, some individuals infected with the plague were isolated and moved to pest houses, located some distance from the town in its surrounding fields.Although primarily about quarantine and isolation of the living, parish registers record that from at least 1603 onwards some individuals who died at pest houses were buried there rather than returned to their parish cemetery.The location of the pest houses changed over time, with at least four locations known.One of these sites was Midsummer Common, where pest houses are mentioned in 1593 and 1630.Two skulls discovered in the late 19th or early 20th century at Midsummer Common were presented to the Duckworth Collection by the town clerk, John Edleston Ledsam Whitehead (1853-1923, town clerk 1887-1923).A tooth from one of the skulls was dated to 1450-1630 cal ad (10).Both tested individuals were genetically determined to be male and neither of them had tested positive for Y. pestis.

Clopton
Clopton was a village in west Cambridgeshire, about 19 kilometers southwest of Cambridge.The village was established by the 10th-11th centuries and appears to have thrived until forcible enclosure for sheep grazing in c. 1480-1520 CE.The church is documented as having gone out of use in 1561.There is good evidence that the village fell primarily within the hinterland of Cambridge, although its location meant that it also had links to urban centers in Hertfordshire.The church of St Mary had been established at Clopton by the late twelfth century, but is probably substantially older.A new church was dedicated in 1352 and this continued to stand after the village was deserted.
John Alexander excavated c. 70 skeletons from the church and cemetery between 1960 and 1964, out of over 120 burials with records (108).These investigations were undertaken as a research excavation.The burials have been dated by a combination of documentary sources, stratigraphy, typological dating of associated artefacts particularly pottery, radiocarbon dating of a sample of the skeletons and ancient DNA evidence for Y. pestis.The skeletons were initially studied by Charles Bernard Denston of the Duckworth Laboratory and were reexamined for the After the Plague project by Sarah Inskip.Some of the human skeletal remains were reburied in the churchyard of Hatley St George, Cambridgeshire, in 1967.The remaining human skeletal remains involved in this study are held by the Duckworth Laboratory of the Department of Archaeology, University of Cambridge, to whom any requests for access should be made via their website: https://www.arch.cam.ac.uk/institutes-and-facilities-overview/duckworth-laboratory/contactduckworth.
The excavations focused primarily on the cemetery to the south of the church, which would have been where the bulk of the parishioners were buried.The burials follow a highly uniform rite, so there are no archaeological indications of the status of particular individuals.The bulk of the parish population were primarily engaged in agricultural activities, with others engaged in a range of trades and occupations.The limited excavations in and around the church means that the individuals who were buried there, which would have included clerics such as John Thorney as well as wealthy parishioners, are largely absent from the studied human remains.Radiocarbon dating and other evidence is consistent with a date of c. 1200-1561 CE for the burials.
DNA was extracted from the tooth roots of 17 individuals, including 13 males and 4 females (Table S1).Three individuals tested positive for Y. pestis, two more individuals possibly were positive but not confirmed (Table S1).

Hemingford Grey
Excavations at Meadow Lane, north of Hemingford Grey, 19km northwest of Cambridge were undertaken by Oxford Archaeology in advance of development of the site in accordance with the appropriate planning regulations in 2006, revealing sixteen burials in a late 17th-early 18thcentury nonconformist cemetery that has been linked to the Society of Friends or Quakers (109).The burials have been dated by a combination of documentary sources, stratigraphy and typological dating of associated artefacts.The skeletons were initially studied by Sharon Clough and Louise Loe of Oxford Archaeology East and were reexamined for the After the Plague project by Sarah Inskip.The human skeletal remains were initially held by Oxford Archaeology East, but will be transferred to the Historic Environment Team of Cambridgeshire County Council (archaeology@cambridgeshire.gov.uk), to whom any requests for access should be made.
Textual evidence for the cemetery comes from the vicar at Hemingford Grey Parish noting in the Parish register burials that took place away from the Parish cemetery at a site referred to as Wobourn or Oubourne.This lists 17 burials between 1681 and 1721, although it is unclear if the list is complete and earlier and later burials are possible.The individuals buried came from Hemingford Grey and the adjacent Parishes of Fenstanton, St Ives and Hemingford Abbot and there are several examples of multiple members of a single family.The individuals are interpreted as non-conformists, documentary evidence indicates that the individuals who formed this community came from a range of social backgrounds.The burials follow a relatively uniform rite, so there are few archaeological indications of the status of particular individuals.
DNA was extracted from teeth and ribs of 7 individuals, including one female and 6 males (Table S1).

Supplementary Tables
Table S1.Sequence data summary for the newly reported later medieval and post-medieval genomes.
Table S2.Mitochondrial DNA haplogroup frequencies in Cambridge(shire) through historic time in context of modern and prehistoric data from across Britain.Table S3.Average connectedness (PiC7cM) between medieval and postmedieval residents Cambridgeshire and modern genomes of the UK Biobank.Table S4.Allele frequency of the phenotype informative SNPs by time period and, for medieval samples in this study, by relative time to the Second Pandemic (before or after).Table S5.Sample-by-sample genotypes for the SNPs involved in the pigmentation prediction (eye, hair and skin colour) from the HIrisPlex-S set.Genotypes are reported in the form of counts of effective alleles.

Table S6. Sample-by-sample genotypes for the other SNPs involved diet and disease.
Genotypes are reported in the form of counts of effective alleles.Table S7.Kinship among medieval, post-medieval and modern genomes from the UK Biobank.

Table S9. Overlap of highly differentiated immunity gene variants between London and Cambridge cohorts.
Table S10.Enrichment of highly differentiated immunity variants in the Cambridge preand post-plague cohorts.Table S11.Imputation accuracy evaluated by downsampling PSN31.

Fig. S3 :
Fig. S3: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from All Saints by the Castle.Only genomes > 0.05x are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S4 :
Fig. S4: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Clopton.Only genomes > 0.05× are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S5 :
Fig. S5: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from the Hospital of St John.Only genomes > 0.05x are included; error bars represent one standard deviation estimated from 20-fold block jackknife; outliers labelled in bold.

Fig. S6 :
Fig. S6: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Augustinian Friary.Only genomes > 0.05× are included; error bars represent one standard deviation estimated from 20-fold block jackknife; outliers labelled in bold.

Fig. S7 :
Fig. S7: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Bene't Street.Only genomes > 0.05× are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S8 :
Fig. S8: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Midsummer Common.Only genomes > 0.05x are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S9 :
Fig. S9: PCA of West Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Hemingford Gray Quakers.Only genomes > 0.05× are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S10 :
Fig. S10: PCA of west Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Providence Calvinistic Baptist Chapel.Only genomes > 0.05x are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S11 :
Fig. S11: PCA of west Europe individuals from UK Biobank, showing lsq-projection of historical genomes from Holy Trinity Church.Only genomes > 0.05× are included; error bars represent one standard deviation estimated from 20-fold block jackknife.

Fig. S12 .
Fig. S12.Heterozygosity of the medieval Cambridge genomes from before and after the Black Death.Average heterozygosity estimates for 5.4 million variants with MAF > 0.05 were obtained from imputed genotypes of 50 genomes with coverage > 0.1× from 4 sites in Cambridge.

Fig. S13 .
Fig. S13.Histograms comparing nucleotide diversity of the medieval Cambridge genomes from before and after the Black Death.

Fig. S14 .
Fig. S14.The power to observe FST values greater than 0.0089, the 95 percentile reported by Klunk et al. (16) among neutral variants, based on the sample size of the Cambridge dataset for each combination of allele frequencies before and after the Black Death.The populations are assumed to be in Hardy-Weinberg equilibrium.The four variants highlighted by Klunk et al. are labeled according to the frequencies reported in their London cohort before and after the Black Death.

Fig. S15 .
Fig. S15.Relationship between heterozygosity and coverage in imputed ancient genomes.A. Scatter plot of individual heterozygosity and coverage values of imputed genomes.Three individuals (PSN357, PSN412, PSN870) with long runs of homozygosity were excluded.B. Variance of heterozygosity estimates in genomes grouped by their coverage.