1 
 
Supplementary Information 1 
Mycobacterium leprae diversity and population dynamics in 2 
medieval Europe from novel ancient genomes 3 
  4 
Saskia Pfrengle, Judith Neukamm, Meriam Guellil, Marcel Keller, Martyna Molak, Charlotte 5 
Avanzi, Alena Kushniarevich, Núria Montes, Gunnar U. Neumann, Rezeda I. Tukhbatova, 6 
Nataliya Y. Berezina, Alexandra P. Buzhilova, Dmitry S. Korobov, Stian Suppersberger 7 
Hamre, Vitor M.J. Matos, Maria T. Ferreira, Laura González-Garrido, Sofia N. Wasterlain, 8 
Célia Lopes, Ana Luisa Santos, Nathalie Antunes-Ferreira, Vitória Duarte, Ana Maria Silva, 9 
Linda Melo, Natasa Sarkic, Lehti Saag, Kristiina Tambets, Ella Reiter, Philippe Busso, 10 
Stewart T. Cole, Alexei Avlasovich, Charlotte A. Roberts, Alison Sheridan, Craig Cessford, 11 
John Robb, Johannes Krause, Christiana L. Scheib, Sarah A. Inskip and Verena J. 12 
Schuenemann. 13 
 14 
 15 
 16 
 17 
 18 
 19 
 20 
2 
 
Table of Contents 21 
Supplementary Note 1: Archaeological information of the samples 4 22 
1.1 Nonneseter site, Bergen, Norway 4 23 
1.2 Saint Petersburg, Russia 5 24 
1.3 Kirk Hill (also published as Kirkhill), St Andrews, Scotland, U.K. 6 25 
1.4 Lagos, Portugal 7 26 
1.5 Cordiñanes de Valdeón, León, Spain - COR_XVIII 9 27 
1.6 Belarus - Studenka necropolis 10 28 
1.7 Edix Hill (Barrington A), Cambridgeshire, UK. (BAEH89/90/91) 12 29 
1.8 Church End, Cherry Hinton, Cambridgeshire (HAT358/1). 13 30 
1.9 The Hospital of St John the Evangelist, Cambridge, Cambridgeshire (JDS10) 14 31 
1.10 The Hospital of Sant Llàtzer or Santa Margarida, Barcelona, Spain 15 32 
1.11 Blokhuizen, The Netherlands. 18 33 
1.12 Santarém, Portugal 19 34 
1.13 Beja, Portugal 20 35 
1.14 Travanca, Portugal 21 36 
1.15 Dryburn-Bridge, East Lothian, Scotland 21 37 
1.16 Santa Lucia, Spain 22 38 
1.17 Kich Malka 23 39 
Supplementary Note 2: Sample Processing and Genome-wide analyses 25 40 
2.1 Sampling 26 41 
2.2 Library Preparation 27 42 
2.2.1 Double-stranded DNA Libraries 27 43 
3 
 
2.2.2 UDG-treated DNA Libraries 28 44 
2.3 Enrichment strategies 29 45 
2.3.1 Human mitochondrial capture 29 46 
2.3.2 Mycobacterium leprae gene screening 29 47 
2.3.3 Mycobacterium leprae genome-wide enrichment 29 48 
2.4 DNA sequencing 30 49 
2.5 Genome-wide analysis - Read processing, mapping, and variant calling 31 50 
2.5.1 Processing of published samples 32 51 
2.5.2 SNP typing 32 52 
2.5.3 SNP alignment and SNP Effect analysis 32 53 
2.5.4 Phylogeny 33 54 
2.5.5 Estimation of divergence time (BEAST analysis) 34 55 
2.5.6 Temporal signal 35 56 
2.6 Human mitochondrial DNA analyses and molecular sex determination 35 57 
Supplementary Figures 37 58 
Supplementary Tables 44 59 
References 56 60 
 61 
  62 
4 
 
Supplementary Note 1: Archaeological information 63 
for the sites and samples 64 
1.1 Nonneseter site, Bergen, Norway 65 
Stian Suppersberger Hamre 66 
The tooth (left first upper molar) from Bergen, Norway, comes from the Nonneseter site. The 67 
site was excavated in 1872 and 1891 [52] but the recovered skeletons were never analysed 68 
and the remains were not collected according to modern standards. All the skeletal remains 69 
were lumped together and when the osteological material was first analysed in 2006 [53], the 70 
sample consisted of several large wooden crates with commingled material. Skeletons were 71 
excavated from the graveyard on the north and the south side of the convent as well as 72 
inside the convent church. A large number of graves were found but the exact number is 73 
unknown. The estimated minimum number of individuals is 84 adults and 27 sub-adults. The 74 
Nonneseter convent was established around 1150 and secularised in 1528. Thus, it is 75 
reasonable to assume that the burials took place during this period. There are very few 76 
documented cases of leprosy from this period and this partial facial skeleton was the first 77 
described case of lepromatous leprosy from medieval Bergen. Due to too little being 78 
preserved of this individual, a reliable determination of sex is not possible and the person’s 79 
age at death cannot be estimated more accurately than adult. The signs of lepromatous 80 
leprosy, however, are there. This individual shows resorption of both the anterior and 81 
posterior walls of the alveoli for the incisors and canines and pitting which has spread all 82 
across the hard palate and perforated the posterior-sagittal portion of it. Pitting is also 83 
present on the nasal floor.  84 
 85 
5 
 
1.2 Saint Petersburg, Russia 86 
Nataliya Y. Berezina (l), Alexandra P. Buzhilova  87 
 88 
The sample from the individual from St. Petersburg, Russia (collection ID 7546-671) is a part 89 
of the collection of the radiologist and one of the founders of paleopathology in Russia, D.G. 90 
Rokhlin. His collection consisted of archaeological (from Bronze Age) and modern (till the 91 
mid-20th century) individuals with various bone manifestations of pathologies, including 92 
infectious diseases. Unfortunately, after his death in 1981, the collection was improperly 93 
stored resulting in the loss of many of the labels with sample identification information. 94 
Sample #7546-671 belongs to this collection, with the radiocarbon date of the sample shows 95 
that it is part of the modern material (19th-20th century).  96 
 97 
Sample #7546-671 is a skull of an adult female with clear bone manifestations of leprosy: 98 
rhinomaxillary remodeling, rounding of the margins of the piriform aperture, alveolar 99 
recession intravital tooth loss and cribra orbitalia which may result from insufficient nutrition 100 
and/or chronic inflammation [54]. This woman may be from a medical collection obtained 101 
from a ‘leper’ colony cemetery. A scientific study was undertaken to establish the situation of 102 
lepers in the Russian Empire in 1894 [55]. There was only one ‘leper’ colony near St-103 
Petersburg, named Krutye ruchii. This was a colony with barracks for singles, houses for 104 
families, doctors, staff and services. There were 85 people in the colony (47 men and 38 105 
women) in 1911, and 88 people (52 men and 36 women) were treated in the colony in 1912. 106 
The size of the colony did not increase much over time, remaining approximately at the 107 
same level. In the 1930s, many famous local doctors worked and conducted research on 108 
Hansen’s Disease here. It can be assumed that the examined skull could have been brought 109 
to St. Petersburg for a scientific medical collection. 110 
 111 
 112 
6 
 
1.3 Kirk Hill (also published as Kirkhill), St Andrews, Scotland 113 
Alison Sheridan, Charlotte A. Roberts, Philippe Busso, Charlotte Avanzi and Stewart T. Cole 114 
The sampled individual, an adult female (SK 275A), comes from an early medieval 115 
inhumation cemetery, associated with St Mary’s Church (formerly known as the Church of 116 
the Blessed Mary of the Rock) on the east Scottish coast at St Andrews. The sample, both 117 
for DNA analysis and for radiocarbon dating, was taken from the left petrous temporal bone. 118 
A rescue excavation undertaken in 1980‒81 in response to coastal erosion of the cemetery 119 
by the sea, uncovered the remains of over 300 individuals [30]. Skull SK 275A was found 120 
resting on the torso of an unrelated individual, SK 275, and it had clearly been redeposited 121 
among the earliest layers of graves, which had previously been radiocarbon-dated to as 122 
early as the 7th century CE (ibid.). The radiocarbon date of cal CE 1030–1155 (95.4%; 123 
SUERC-91431 [GU54472], 940±25 BP, d13C‰ -20.0, d15N‰ 11.3, C/N ratio 3.4) that was 124 
obtained for SK 275A suggests a high level of disturbance from centuries of intercutting 125 
graves. 126 
The earliest reference to the existence of a hospital in St Andrews dates to the second 127 
quarter of the 12th century (ibid., 310), but from 1178 there are references to a leprosy 128 
hospital at St Nicholas, to the south of the medieval city. The occurrence of other chronic 129 
diseases in the people buried within the Kirk Hill cemetery may suggest that this could have 130 
been associated with an early hospital site, but the mixed population of males, females, and 131 
children is otherwise that of a general lay burial ground. This new dating evidence indicates 132 
that even as late as the 12th century, people with leprosy were not yet segregated from the 133 
general population, at least in death. This has been noted in the majority of instances where 134 
skeletons with leprosy have been found, including in the UK [1]. 135 
7 
 
The skull had previously attracted attention as constituting rare early evidence for leprosy in 136 
Scotland, the relevant features having been identified osteologically by Dr Dorothy Lunt [31]. 137 
There are very few skeletons identified with leprosy that have been identified in Scotland. 138 
The skeleton was probably female and at death was a young adult aged between 25 and 35 139 
years, but this was based on dental wear patterns [56], which as a sole age estimation 140 
method is not reliable. There was inflammatory pitting of the nasal and oral surfaces of the 141 
palatal bones and the alveolar bone around the maxillary incisor tooth sockets. The anterior 142 
nasal spine was absorbed. The posterior aspect of the palatal and maxillary bones showed 143 
bone loss. 144 
DNA was extracted from sample taken from the petrous portion of the temporal bone and 145 
screened for the presence of M. leprae and M. lepromatosis as described elsewhere in this 146 
work. Only M. leprae was detected and the genome sequence is reported here.  147 
 148 
1.4 Lagos, Portugal 149 
Maria Teresa Ferreira, Sofia Wasterlain and Vitor M.J. Matos 150 
The buildings of the Lagos leprosarium and a modern urban dump dated from the 15th – 17th 151 
centuries were identified during the archaeological survey previous to the construction of an 152 
underground car park located in Valle de Gafaria (Lagos, Portugal) [57, 58]. The area was 153 
located outside the line of medieval walls that protected the city of Lagos. It was necessary 154 
to carry out a meticulous archaeological excavation that, in addition to exposing the 155 
leprosarium buildings, allowed the identification of a cemetery associated with it, as well as 156 
recovering the skeletal remains of 158 African enslaved individuals from the nearby dump 157 
[57–59]. 158 
At the cemetery associated with the leprosarium, eleven adult individuals were exhumed: 159 
two females, two males, and one individual of unknown sex [21]. Five of these individuals 160 
8 
 
showed several bony lesions. After the differential diagnosis (based both on macroscopic 161 
and radiological analyses) leprosy was the most probable diagnosis for two individuals 162 
(PAVd’09_I.5, adult female, and PAVd’09_I.34, adult male). As for the remaining three 163 
individuals, one was diagnosed with treponematosis, another one with brucellosis, and one 164 
with Legg-Calvé-Perthes disease [21]. 165 
Historical sources reveal that medieval leprosaria admitted not only leprosy patients but also 166 
the very poor, mentally disabled, and people suffering from other potentially stigmatizing 167 
diseases (such as syphilis, tuberculosis, among others). In fact, the leprosarium location, 168 
outside the city walls and nearby the urban dump where the corpses of slaves were 169 
discarded testifies the social exclusion of these individuals in this period and location [21, 170 
58]. 171 
For the present study, the young adult female skeleton, with the reference PAVd’09_I.5, was 172 
analysed. This skeleton is relatively well preserved, but with both femurs, right tibia and 173 
fibula fragmented, and left tibia and fibula, and feet bones absent [21]. Several pathological 174 
signs compatible with a diagnosis of probable Hansen’s Disease were observed: 175 
rhinomaxillary lesions, including rounding of the pyriform margins, complete resorption of the 176 
nasal spine, and perforated palate; woven bone deposits at the humerus, radius, ulnae and 177 
hand phalanges; porosity and osteolytic lesions at carpal and metacarpal bones; hand 178 
phalanges with erosive/destructive lesions on distal extremities [21]. That is, this female 179 
skeleton presents abnormal bone formation, abnormal bone destruction, sclerosis on lytic 180 
margins, bilateral and symmetrical lesions, and both axial and appendicular involvement 181 
[21]. Radiocarbon dating was performed on a tibial bone fragment while the maxillary bone 182 
fragment provided possible results for our ancient DNA study. The 14C dates the sample to 183 
the 13th to 14th (Table 1, Additional file: Supplementary Note 3, Table S1) century. Hence, 184 
radiocarbon dating dates the individual 100 - 200 years further back in time than the Lagos 185 
leprosaria. 186 
9 
 
1.5 Cordiñanes de Valdeón, León, Spain (COR_XVIII) 187 
Laura González-Garrido, Sofia N. Wasterlain, Célia Lopes 188 
According to historical documentary sources, leprosy was a relatively common disease in 189 
the medieval Iberian Peninsula [60]. In the 13th century, leprosy was widespread in the north 190 
of Spain [61]. This is documented by the presence of 24 leprosy hospitals established in 191 
Asturias on the main pilgrim routes to Santiago de Compostela (Galicia) and Santo Toribio 192 
de Liébana (Cantabria). 193 
The Barrejo medieval necropolis (12th - early 13th century) is located in the valley of Valdeón, 194 
National Park of Picos de Europa, corresponding to the locality of Cordiñanes de Valdeón 195 
(COR), in the province of León (northwestern Spain). This location is delimited and relatively 196 
isolated by the Cantabrian Mountains. From Barrejo necropolis 27 individuals have been 197 
recovered: 25 adults (18 males and seven females) and two non-adults (5-8 years old). For 198 
the present study, the adult male skeleton, with the reference COR_XVIII was analysed. This 199 
individual was inhumed in a supine position with upper limbs at the sides of the torso on a 200 
west-east axis, in a stone-lined grave and lacking grave goods. The skeleton COR_XVIII is 201 
relatively well preserved although its structure was affected by chemical diagenesis due to 202 
the necropolis proximity to the river Cares. There were also some osteological elements 203 
missing, namely the right side of the mandible, the proximal epiphyses of both fibulae, two 204 
vertebrae, feet and hand bones (two tarsals, three carpals, two metacarpals and, two hand 205 
and 21 feet phalanges). 206 
 COR_XVIII shows destructive rhino-maxillary alterations, complete resorption of the nasal 207 
spine and perforated palate; bilateral and symmetrical periostitis on tibiae and fibulae and 208 
cortical periosteal reaction, and right fibulae subperiosteal bone reaction. There are no 209 
destructive lesions on distal phalanges of the hands or feet. The radiographic analysis of the 210 
tibiae and fibulae shows the reduction in the size of the medullary cavities, particularly in 211 
tibiae, and maintenance of cortical thickness. Differential diagnosis based both on 212 
10 
 
macroscopic and radiological analyses of the lesions point to an early stage of leprosy for 213 
COR_XVIII [62]. 214 
In the medieval period, a small commercial exchange [63] and different pilgrim routes (Tolivar, 215 
1966) could bring infirmed people to the small village of Cordiñanes de Valdeón. The burial 216 
ritual of COR_XVIII was equivalent to that of other individuals buried in this necropolis, which 217 
suggests that people suffering Hansen’s Disease were not necessarily stigmatised in death. 218 
1.6 Belarus - Studenka necropolis  219 
Alena Kushniarevich 220 
 221 
Sample BEL024 originates from the burial mound N96 of the Studenka necropolis dated 10-222 
12th century CE, near Studzenka Village, Byhau region, Mahileu distr (Mahileu Dniepr river 223 
region). The site was excavated in 2015 by Alexei Avlasovich. The archaeological dating of 224 
the mound is hindered by the absence of ceramic vessel’s crowns. However, the funeral rite 225 
and presence of the circular ceramics suggest that the mound was erased not earlier than 226 
the end of 10th or beginning of 11th century CE. 227 
 228 
The skeletal material was investigated by Vladimir Shipillo. A skull (with mandible) belonged 229 
to a male individual 25-30 years old. The skull is characterized by undeveloped relief with a 230 
relatively inclined forehead; large values of longitudinal and small values of transverse head 231 
diameters; pronounced dolichocranic (cranial index=72 mm); average values of nose index 232 
(57.8 mm); average values of orbital index (77.5 mm). The skull had an ovoid shape. The 233 
face was orthogonal (face protrusion index=91.9 mm). The stature of the individual was 234 
calculated using Pearson and Li formulae applied to the right femur and estimated to 162.09 235 
cm. 236 
  237 
11 
 
The Studenka necropolis belongs to the Ancient Rus epoch. The mound burial is located on 238 
the left bank of Greza River, the right inflow of Drut River, 1.5 km north-east from Studenka 239 
Village (Glukhsk sub-region of Bykhau region, Mahileu District). The necropolis consists of 240 
107 hemispherical mounds with a rounded shape. The mounds’ height ranged from 0.4–241 
2.8m, and 5–16m in diameter. Nearly half of the mounds have marks of disturbances due to 242 
different extents, as for example vandal digging or the exploitation of the road that crosses 243 
the necropolis. 244 
 245 
Mound 96 is located in the south-eastern part of the necropolis. Its height is 1.24 m, length 246 
along the north-south line 8.09 m, along the east-west – 6.31 m. The edge width is 0.7 m. 247 
The mound is hemispherical in shape being elongated along the north-south line. The 248 
mound’s body consists of yellow sand with ash and coal increments; the lower ashbin was 249 
found 1.15 m from the top of the mound and had a thickness of 2-9 cm. 250 
  251 
The mound contained the inhumation burial at the level of the lower ashbin. Although the 252 
skeleton was disturbed by the root system of the trees, it was possible to see that the skull 253 
was at the east end of the burial, the vertebrae and ribs in the centre, and the leg bones in 254 
western part indicating that the body was oriented with the head towards the east. 255 
Seventeen fragments of the ceramic pot were found on both sides of the skull. Analysis of 256 
the ashbin structure allows the reconstruction of the funeral rite: The body was inhumed at 257 
the horizon level; before internment, the burial place was ritually cleansed by fire, the dead 258 
body with head oriented eastward was placed in the centre of the place; a mound of 60-65 259 
cm was created above the dead body; at this level the funeral feast was performed and the 260 
mound was increased to its final height. 261 
 262 
  263 
12 
 
1.7 Edix Hill (Barrington A), Cambridgeshire, England (BAEH89/90/91) 264 
Sarah Inskip 265 
 266 
The cemetery at Edix Hill, also known as Barrington A and distinct from Barrington B, dates 267 
from the 5th to 7th century CE. It lies approximately 8 miles south-west of the modern city of 268 
Cambridge. Excavations have taken place there since the nineteenth century, but the 269 
individual assessed here comes from rescue excavations undertaken by the Cambridgeshire 270 
County Council from 1989-1991. In these excavations 115 graves were excavated 271 
containing the remains of at least 149 individuals, however, it is thought that over 300 graves 272 
could have been originally present. It is archaeologically dated from stratigraphy and grave 273 
good typologies supplemented by later radiocarbon dating [64].  274 
 275 
The individual sampled was a young middle adult (25-36 years old) female (sk42b) from 276 
grave 18B. Sex was confirmed by genetic testing. She was the earliest of three burials in 277 
grave17/18, but lay largely undisturbed (Malim and Hines 1998:52). This particular individual 278 
is noteworthy for her burial. She is one of two bed burials at the site (the other being Grave 279 
60) and just a handful of bed burials from Western Europe. The burial practice is thought to 280 
be reserved for those of higher status. She was buried with multiple grave goods including a 281 
glass bead, silver ring (necklace), a key, 2 knives, a bucket, a weaving batten, iron brackets 282 
and rod (probably from a wooden box), a comb, a spindle whorl, copper possibly from a 283 
pendant, a fossil sea urchin, a sheep astragalus and piece of glass. She was supine 284 
extended with her right arm extended and the left flexed over the abdomen. Originally 285 
identified as having Hansen’s Disease by Corinne Duhig (Malim and Hines 1998), the 286 
woman had extensive remodelling to the nasal aperture and loss of the nasal spine. She has 287 
periostitis on the tibiae, fibulae and first metatarsal. There was little obvious change to the 288 
hands and feet, although it is possible that there was some loss of bone density. One left 289 
13 
 
manual digit showed evidence for volar grooving. The positive sample was obtained from the 290 
upper right canine tooth. 291 
1.8 Church End, Cherry Hinton, Cambridgeshire, England (HAT358/1). 292 
Craig Cessford and Sarah Inskip 293 
 294 
The cemetery at Church End dates from the 10th to the 12th century and lies approximately 6 295 
km southeast of the modern city of Cambridge [65]. It was excavated by the Hertfordshire 296 
Archaeological Trust (now Archaeological Solutions) and is situated on land at 69-115 297 
Church End, Cherry Hinton. In the late 9th–mid 10th century a large thengly (aristocratic) or 298 
manorial centre was established. Associated with this was a timber chapel/church and 299 
graveyard which was in use between the 10th and 12th centuries. Only part of the cemetery 300 
was investigated, but over 670 graves were excavated and including disarticulated remains 301 
c. 980 individuals were identified. The graves were mostly East-West aligned simple earth 302 
cut graves. Most individuals were buried in extended supine. Very few grave goods were 303 
recovered. As part of the ‘After the Plague’ project, two individuals were identified as having 304 
Hansen’s Disease based on skeletal lesions. The burials were typical for the cemetery 305 
(West-East extended).  306 
 307 
One was an adolescent male (sk2012) who was between 13-17 years in grave 1. Sex was 308 
confirmed genetically. He had porosity on the medial surface of the frontal process of the 309 
maxilla. There is some evidence of resorption on the frontal process and remodelling of the 310 
nasal aperture. There is woven new bone in maxillary sinuses and nasal aperture. Woven 311 
new bone is present on the periosteal surfaces of the metatarsals, pedal phalanges, the 312 
distal fibulae and tibiae. The positive sample was obtained from an upper right canine.  313 
 314 
The second individual (sk 2529) was a young middle (25-36 year old) female in grave 155. 315 
Sex was confirmed by genetic testing. She had extensive resorption of the anterior nasal 316 
14 
 
spine, rounding of nasal aperture and destructive changes penetrating into the left maxillary 317 
sinus with porosity and lamellar new bone growth (NBG) in the maxillary sinus. Woven bone 318 
was present on the periosteal surfaces of the distal humeri, proximal ulnae, distal left radius, 319 
distal tibiae and distal fibulae (resulting in thickened appearance of distal tibiae). It was also 320 
observable on the right third metacarpal, the right calcaneus, both first metatarsals and 321 
hallucial phalanges. There was no clear concentric remodelling of the phalanges, but these 322 
elements are poorly preserved. The positive sample came from an upper right canine. 323 
 324 
1.9 The Hospital of St John the Evangelist, Cambridge, Cambridgeshire, 325 
England (JDS10) 326 
Craig Cessford and Sarah Inskip 327 
 328 
The individual assessed here, genetically sexed as female, was from disarticulated material 329 
associated with the Hospital. The hospital was founded in the town of Cambridge at the very 330 
end of the 12th century by the townsfolk, with burial rights acquired in the early 13th century, 331 
and was in use until the early 16th century when it was dissolved to create St John’s College, 332 
Cambridge. The hospital was principally founded for the care (in a social and spiritual sense, 333 
rather than medically) for the ‘poor and infirm’ and for the ‘maintenance of poor scholars and 334 
other sick people’; with pregnant women, ‘lepers’, the wounded, ‘cripples’ and the insane all 335 
specifically excluded. The cemetery of the hospital was located below the Old Divinity 336 
School on St John’s Street. Over 400 complete or partial articulated skeletons were 337 
recovered by the Cambridge Archaeological Unit in 2010/2011 [66]. The woman is solely 338 
represented by her skull, which does not show any distinct lesions associated with Hansen’s 339 
Disease, although the preservation precludes detailed analysis. The positive sample came 340 
from an upper left first molar. Radiocarbon dating of stratigraphically associated material 341 
indicates that this individual died during the 13th century. The finding of this individual in the 342 
15 
 
hospital cemetery raises interesting questions since individuals identified with ‘leprosy’ 343 
(leprosis) are specifically named as being excluded. It is possible that this woman did not 344 
manifest typical or extreme external lesions and was in the hospital for other reasons, or for 345 
some reason, perhaps relating to status or social relationships, was able to get into the 346 
hospital despite her condition. It is also possible that if the woman only developed 347 
identifiable signs after she was admitted to the hospital she might have been allowed to 348 
remain.  349 
 350 
1.10 The Hospital of Sant Llàtzer or Santa Margarida, Barcelona, Spain 351 
Núria Montes Salas 352 
 353 
The Hospital of Sant Llàtzer or Santa Margarida (Saint Lazarus or Saint Margaret), also known 354 
as hospital dels Messells (the hospital of the ill), was probably founded in the 11th century 355 
whose purpose was to shelter all those who suffered from leprosy in Barcelona [67]. The first 356 
documents related to the hospital that are preserved are from the end of the 12th century, 357 
although its founding date may be earlier [67]. 358 
 359 
The hospital was established on the edge of the city of Barcelona at the intersection of two 360 
main paths that led directly to the city gates (El Portal de la Boqueria and Porta Ferrissa). The 361 
Hospital of Colom, founded in 1219, and the Hospital d’en Vilar, from 1311, were also located 362 
along the same path. In the 14th century, a third wall was built, and these hospitals were 363 
enclosed inside.  364 
According to 12th and 13th century documentary sources, the Hospital of Sant Llàtzer might 365 
have sheltered both poor people and people suffering from leprosy simultaneously [67]. 366 
However, in the 14th century, once the health assistance network of Barcelona became more 367 
complex, only people considered as ‘lepers’ would have been accepted in the hospital [67]. In 368 
16 
 
1401, the six hospitals of Barcelona merged into one General Hospital, l’Hospital de la Santa 369 
Creu, although the leprosarium remained at the same place until 1906, when it was moved to 370 
a new location outside the city [67]. 371 
 372 
The account books for 1379 to 1395 have been preserved, which include detailed information 373 
about daily life at the hospital, the diet and the origin of the patients [67]. The ill from the city 374 
of Barcelona and its surroundings were supposed to have priority for being accepted into the 375 
hospital. However, according to the account books, at the end of the 14th century many of the 376 
patients were foreigners [67]. Moreover, it seems that travelling and pilgrimage were usual 377 
among them [67].  378 
 379 
Between 2007 and 2009, archaeological works in the surrounding area of the Romanesque 380 
church of Sant Llàtzer located part of the cemetery and buildings of the leprosarium [68]. The 381 
archaeological remains were discovered during the restoration tasks of several buildings of 382 
Carme Street and Hospital Street and hence the graves that were located outside the working 383 
area remained unexcavated. The excavation was promoted by the city council and the 384 
construction company Teyco SL and it was carried out by the archaeological company Atics 385 
SL. The remains recovered were housed in the Barcelona History Museum (MUHBA). 386 
 387 
The cemetery of the hospital of Sant Llàtzer is the only one directly related to a leprosarium 388 
that has been excavated in Spain. The cemetery was probably used between the 11th and the 389 
18th centuries CE. A total of 79 skeletons were recovered from the site during the excavations, 390 
corresponding to different chronological periods: 11th-13th centuries (14 individuals), late 13th-391 
14th centuries (50 individuals), 15th-16th centuries (5 individuals) and 17th-18th centuries (10 392 
individuals). The burials were dated according to associated pottery [68]. Generally, all burials 393 
were simple oval shaped structures excavated directly into the soil, except for a single tomb 394 
built with flat stones which dated to the 11th – early 13th century [68]. Some collective burials, 395 
17 
 
all of them corresponding to the 14th century, were also located. These burials had different 396 
levels and were also oval shaped. 397 
 398 
Between 1989 and 1991, another extensive archaeological intervention was carried out in the 399 
interior of the chapel and several burials corresponding to the 15th – 16th centuries and the 18th 400 
century were located [69]. A previous study of the human remains recovered from this 401 
excavation concluded that none of the skeletons showed lesions related to leprosy and hence 402 
these graves may be related to monks, nuns and wealthy families [69]. Moreover, some burials 403 
from the 12th century –early 13th century were also located under the Chapel of Saint 404 
Sepulchre, which may correspond to clergymen or workers of the hospital [69]. 405 
 406 
A total of 35 samples from 18 human skeletons recovered from the cemetery of Sant Llàtzer 407 
were used for DNA extraction (Additional file 1: Table S1). The sex of the skeletons was 408 
estimated following the standard methods based on the morphology of the cranium and the 409 
pelvis [70, 71]. Age at death was estimated from the changes in the auricular surface of the 410 
ilium [72] and the pubic symphysis [73]. Regarding non-adult individuals, age estimations were 411 
made on the basis of epiphyseal fusion [74] and dental development [75]. 412 
 413 
For the palaeopathological analysis, the remains were examined macroscopically under white 414 
light in the laboratories of the Autonomous University of Barcelona. For those individuals 415 
displaying lesions that could be related to Hansen’s Disease [76–79], a tooth and a bone 416 
sample from an active lesion were selected for DNA extraction whenever possible. The 417 
samples were photographed and, in those cases where the bone sample showed an active 418 
lesion, an x-ray and a CT scan were also carried out. The radiological analyses and CT scans 419 
were performed at the facilities of the Hospital General de Catalunya (General Hospital of 420 
Catalonia) by a specialized technician. The samples were handled at all times with nitrile or 421 
latex gloves. 422 
 423 
18 
 
Several of the tombs were cut by modern constructions or by other graves and hence some 424 
skeletons were incomplete. However, rhinomaxillary changes could be observed in seven of 425 
the individuals selected for DNA extraction. A sample from the maxillary bone was taken for 426 
the skeletons UF701_UE7016, UF103_UE49, UF11_UE1069 and UF101_UE43, which 427 
showed active lesions. For the skeleton UF21_UE1137, the sample was taken from the 428 
ethmoid. The skeletons UF703_UE7027 and UF803_UE8020 also showed rhinomaxillary 429 
changes, but taking a sample from the maxillary bone would have been too destructive and 430 
therefore a hand bone sample was taken instead. 431 
For the remainder of individuals, most of whom did not have a preserved skull, bones from the 432 
hands and feet were selected for DNA analyses. Most of them displayed active lesions in the 433 
hand bones and/or the feet, except for the skeletons UF800_UE 8008, UF801_UE8011, 434 
UF102_UE46 and UF18_UE1123, where the hands and feet were poorly preserved. In those 435 
four cases, hand or foot bones without any evident lesions were selected as samples. 436 
The only individuals sampled that did not show evident lesions that could be related to 437 
Hansen’s Disease were UF102_UE46, UF801_UE8011 and UF100_UE40, which were only 438 
partially preserved. Skeleton UF100_UE40 showed periostitis on the internal side of the 439 
second, third and fourth left ribs as well as on the proximal end of the diaphysis of the left tibia. 440 
Moreover, an osteolytic lesion could be observed in the medial phalanx of the 4th ray of their 441 
right hand. In this case, a sample of the proximal end of the second left rib was taken in order 442 
to test for an infection by Mycobacterium tuberculosis. 443 
1.11 Blokhuizen, The Netherlands. 444 
Sarah Inskip  445 
Archaeological skeletal material from Blokhuizen, located in North Holland, can be broadly 446 
dated from the 10th to the 12th century and may relate to a village known as Geddingmore [80]. 447 
One hundred and thirty individuals were excavated by the Archeologisch Werkgemeenschap 448 
voor Nederlands in 1983 and at the time of research 119 of these were stored at the Laboratory 449 
19 
 
for Osteoarchaeology and Funerary Archaeology at the University of Leiden. No articulated 450 
individuals had evidence for Hansen’s Disease, however one disarticulated adult (over 14 451 
years) metatarsal displayed concentric remodelling often associated with the disease. Due to 452 
the scarcity of Hansen’s Disease cases for the country, in fact there are no published cases 453 
at all for the region [1], it was decided that the metatarsal should be sampled and then 454 
radiocarbon dated if proving positive. Unfortunately, we were unable to obtain M.leprae DNA 455 
from the bone.  456 
1.12 Santarém, Portugal 457 
Vitória Duarte, Vitor M. J. Matos, Ana Maria Silva 458 
Between 2007 and 2008 an archaeological campaign took place in the context of the 459 
construction of several residential buildings (Villa Rosa Palace, Avenida 5 de Outubro n. 5-8) 460 
in the historical centre of Santarém, a city located in the central region of Portugal. A total of 461 
137 burials and 22 ossuaries were exhumed. Among these, 44 primary burials and 7 ossuaries 462 
are possibly associated with the Late Roman and Visigothic periods. One of the individuals 463 
(skeleton 2385), a young adult male dated from the 3rd-4th centuries CE (172-383 cal CE; Beta-464 
524726), found in decubitus dorsalis (SW-NE orientation), was diagnosed as a possible case 465 
of leprosy. Although this diagnosis is uncertain, it was based on the presence of several post-466 
cranial lesions, which among others included hand and feet proliferative and destructive 467 
lesions, such as acroosteolysis in two proximal phalanges of the right foot. The poor 468 
preservation of the rhinomaxillary area precluded the observations of eventual leprosy related 469 
bone changes in this region [81]. Eight bone samples from skeleton 2385 (S2385 / VRP2385) 470 
were collected and analyzed under the scope of this work (Additional file 1: Table S1). 471 
 472 
 473 
20 
 
1.13 Beja, Portugal 474 
Nathalie Antunes-Ferreira, Vitor M. J. Matos, Ana Luisa Santos 475 
The Santo André hermitage (ermida) in Beja, south Portugal, was probably founded during 476 
the 12th century CE. Documentary sources also indicated the existence of a leprosarium 477 
(gafaria) between the 14th to 16th centuries, in the surrounding area of this hermitage [82]. The 478 
leprosarium necropolis, dated from the Medieval to Early Modern periods, was found during 479 
the archaeological excavation that took place in 2003 during hermitage rehabilitation works 480 
[35, 83]. The ten graves associated with the leprosarium necropolis were uncovered in survey 481 
number 6 (area of 18.6 m2). The graves were oval pits opened on the ground without any 482 
delimitation structure. The seven individuals exhumed were found according to Christian 483 
canons, in a supine position and with a NE–SW orientation, aligned with the hermitage wall 484 
[83]). Three additional skeletons remained in place because they were outside the 485 
rehabilitation working area [83]. The first radiocarbon dating (skeleton 3; Beja_3) performed 486 
by Oxford University “failed due to very low yield” [35]. The second attempt (skeleton 6) 487 
revealed a conventional radiocarbon age of 1265-1313 cal CE (Beta-517063). The 488 
preservation of the skeletons was affected by constructions in the area and a drainage pipe 489 
[83]. Skeleton 4 (Beja_8) was diagnosed as a probable case of leprosy. This individual 490 
presented destructive remodeling in the rhinomaxillary region and leprosy related lesions on 491 
metacarpals, metatarsals, and hand and foot phalanges. These lesions were bilateral in both 492 
hands and feet, and symmetrical in the feet. Skeletons 1, 3 (Beja_3), 6, and 8 (Beja_8) 493 
displayed lesions that are considered possibly – but not probably – related to leprosy [35]. The 494 
incompleteness of skeletons 5 and 7 due to anthropic taphonomic factors precluded the 495 
observation of the areas of interest. Eleven bone samples from three individuals (Beja_3, 496 
Beja_4, Beja_8) were collected and analyzed under the scope of this work (Additional file 2: 497 
Table S1). 498 
21 
 
1.14 Travanca, Portugal 499 
Linda Melo, Vitor M.J. Matos, Ana Luisa Santos, Ana Maria Silva 500 
An archaeological intervention in the parish church of Saint Mamede in Travanca, a village 501 
belonging to the Santa Maria da Feira Municipality, Aveiro district, was carried out between 502 
2016 and 2017. Despite the long use of this Christian space, from the Medieval period (5th-503 
15th century CE) to the beginning of the 20th century, only Post-Medieval graves preserved 504 
human bone remains. A total of 266 primary burials and 47 ossuaries were recovered from 505 
the 412 graves excavated. Among these, individual number 403 (complete reference: 506 
IPT_17/K_8/9/UE_2855/Skeleton_403), a poorly preserved and very fragmented skeleton 507 
belonging to an adult male, presented several leprosy related bony lesions, namely in the 508 
rhinomaxillary area and feet (Melo et al., 2021). This skeleton, radiocarbon dated from the 509 
17th-19th century CE (Beta 514831), was buried in the churchyard, within a wooden coffin and 510 
oriented West-East. Forty-seven rosary beads and a cross with a crucified Jesus Christ were 511 
found in the abdominal region and close to the left forearm. This individual represents the first 512 
probable evidence of leprosy in Northern Portugal and its funerary context, namely being 513 
found with a rosary in a regular cemetery (i.e. not associated to a leprosarium) and without 514 
evidence of atypical funerary treatment in death, seems to indicate that during this period, in 515 
this geographic region, leprosy patients were not stigmatized and segregated as reported for 516 
the Medieval period [84] or he managed to hide his illness. One bone sample (IPT_17) was 517 
collected and analyzed under the scope of this work (Additional file 1: Table S1). 518 
1.15 Dryburn-Bridge, East Lothian, Scotland 519 
Alison Sheridan, Charlotte A. Roberts, Philippe Busso, Charlotte Avanzi and Stewart T. Cole 520 
This skeleton (Burial 11) was excavated from the site of Dryburn Bridge, near Innerwick in 521 
East Lothian, Scotland, with a site date of 2300–2000 cal BC. Two Late Neolithic/Early Bronze 522 
Age burial cists were identified. Cist 2 contained two skeletons (10 and 11). One was 523 
22 
 
disarticulated (11: child aged 6-8 years) and one articulated (10: older adult male). Roberts J. 524 
describes Burial 11 as in a fair condition and 40% complete, and showing ‘Resorption of the 525 
nasal spine and the region around and above the central incisors, remodelling of the bone and 526 
widening of the nasal aperture and slight pitting of the palatal surface. There was evidence of 527 
slight new bone growth on the inner surfaces of the nasal bones, and when the face was 528 
looked at in profile it had a dished appearance around the nose and mouth area’ [85]. All these 529 
bone changes confirmed in 2016 by C. A. Roberts as potentially illustrating rhinomaxillary 530 
syndrome. However, tuberculosis and treponemal disease (congenital syphilis) should be 531 
considered as differential diagnoses. Nevertheless, while M. tuberculosis complex DNA was 532 
identified by GM Taylor, it could not be replicated [85]. Samples from the individual also have 533 
been screened previously for M. leprae by PCR (G.M. Taylor, personal communication) but 534 
none was found. However, the primers and protocol used then would not have detected the 535 
presence of M. lepromatosis, which has been shown to cause leprosy in red squirrels in 536 
Scotland and elsewhere [50]. Consequently, DNA was again extracted from this skeleton and 537 
screened for both M. leprae and M. lepromatosis but DNA from neither mycobacterium was 538 
detected. 539 
 540 
1.16 Santa Lucia, Spain 541 
Natasa Sarkic 542 
The archaeological site of Santa Lucia belongs to the municipality of Aguilafuente, located 35 543 
km NW of Segovia (Spain). There were noticed two main phases of occupation: Roman villa 544 
from lower imperial period, 4th century CE and the Visigoth necropolis from the 6th-7th centuries 545 
CE [86]. The individual that is the object of our study belongs to the posterior phase, to Visigoth 546 
necropolis of Santa Lucía. The skeletal remains were discovered during the excavations 547 
carried out in the 2018. The grave of this individual, marked as UE 542, disturbed the previous 548 
burial, UE 348. Given the pit of the 542 cuts the pit of 348, it is clear that the 542 was buried 549 
23 
 
posteriorly. It was noted that the individual 542 did not present the same burial position as the 550 
rest of the individuals excavated in the necropolis. All the individuals were in supine position 551 
with extended lower extremities and arms extended or folding one or both arms across the 552 
chest, with E-W orientation (typical Cristian burial). However, 542 was buried in the flexed left 553 
lateral decubitus position, in such forced position that he suffered a postmortem dislocation of 554 
the right coxofemoral joint. It was an adult male individual, between 25-30 years of age, and 555 
his height was 165 ± 1,98 cm according to Pearson [87]. 556 
Pathological changes observed in the skull include the disappearance of the anterior nasal 557 
spine, rounding and widening of the nasal opening, destructive remodeling or partial 558 
reabsorption of the alveolar process of the anterior maxilla without loss of the upper incisors. 559 
Periostitis in the form of isolated plaques was noted in all of the long bones and foot. The 560 
alterations present in the hands are very striking and significant, especially in the right hand. 561 
Some of the proximal phalanges show periostitis and four of the five metacarpals of the right 562 
hand have an abnormal thickening in the diaphysis. Some of them also show signs of porotics 563 
changes and bone resorption of the distal epiphysis. The first metatarsal of the right foot shows 564 
porotic lesions with osteolysis in its distal third and bone resorption of the distal epiphysis. The 565 
third metatarsals and fourth show osteolysis in their distal thirds and the fourth in turn shows 566 
porosis. The right fifth metatarsal is pen-shaped with bone retraction in the distal epiphysis 567 
and a sequestration and cloaca in the proximal epiphysis. The lesions on the left foot are 568 
similar, osteolytic processes are documented in the distal epiphyses of most metatarsals. The 569 
fifth metatarsal has lost the distal epiphysis as a result of bone retraction and the phalanx 570 
proximal hallux is conical in shape, also due to resorption. 571 
 572 
1.17 Kich Malka, Russia 573 
Natalia Berezina, Dimitry Korbov 574 
 575 
24 
 
Kich-Malka catacomb burial ground was created by the Alans, and is located in the Kislovodsk 576 
Basin, North Caucasus, Russia. A female skull from the Kich-Malka catacomb burial ground 577 
was discovered during the investigation of the destroyed catacomb. At least nine people were 578 
buried in the catacomb: two adults and seven children. The rich complex of grave goods allows 579 
to date the burial to the border of the 7-8 centuries CE - the first half of the 8th century CE 580 
[88].  581 
25 
 
Supplementary Note 2: Radiocarbon Dating 582 
Saskia Pfrengle 583 
 584 
Since our study focuses on the genetic diversity of ancient M. leprae genomes and the link 585 
to historical population dynamic events, it is essential to put the samples into the correct 586 
archaeological time. Therefore, it is indispensable to perform radiocarbon dating of the 587 
samples of which the genome coverage was suitable to include them to estimate the 588 
divergence time by BEAST. In total, 14 samples were directly dated (Table 1, Additional file 589 
1: Table S1, Fig. S1).  590 
For the dating, collagen was extracted from bone and tooth samples according to the 591 
established protocols applied in the dating laboratories [89–93]. Finally, the age of the 592 
samples was determined by measuring the 14C/12C ratio using the MICADAS accelerator 593 
mass spectrometry (AMS). Since the dating of the samples was performed at four different 594 
laboratories, at the Scottish Universities Environmental Research Center in Glasgow (Kirk 595 
Hill and JDS097), the Curt-Engelhorn-Zentrum Archaeometry in Mannheim (Bergen, 596 
R7456_671, and PAVd’09_I.5), at the Chrono Center of the Queen's University in Belfast 597 
(EDI006, BEL024, CHRY023, and CHRY044), and at the Laboratory of Ion Beam Physics at 598 
the ETH Zurich (UF_21, UF_25, UF_101, UF_700, UF_703, and UF_803) the 14C data were 599 
all calibrated using OxCal v4.4.4 [94, 95]. 600 
Our dated samples cover the entire medieval period and range between the 6th century AD 601 
and the 16th century AD (Additional file 1: Fig. S1), except the sample R7456_671. This 602 
sample is historic and too young to perform exact radiocarbon dating. The calibrated age of 603 
the sample was estimated to 18th – 20th century (Additional file 1: Fig. S1).  604 
26 
 
Supplementary Note 3: Sample Processing and 605 
Genome-wide analyses 606 
Saskia Pfrengle, Judith Neukamm, Martyna Molak, Meriam Guelli, Marcel Keller, Gunnar U. 607 
Neumann 608 
3.1 Sampling 609 
Tübingen and Zurich: To minimize the risk of potential contamination with modern DNA, the 610 
surface of all bone and tooth samples were initially UV irradiated from all sides at least for 30 611 
minutes. For DNA extractions, we applied a well-established guanidine-silica based 612 
extraction protocol developed for ancient DNA work [151]. For the DNA extraction, we used 613 
30-120 mg of bone powder. For the DNA-extraction step, positive and negative controls 614 
were produced; positive controls to determine whether the DNA was successful or not, 615 
negative controls to identify potential contamination. The negative controls were carried 616 
along with all laboratory experiments and were also sequenced, the positive control till the 617 
first step of library preparation. 618 
Cambridge (PSN550) and Tartu (PSN923, PSN951, PSN441, and BEL024): root portions of 619 
teeth were removed with a sterile drill wheel or broken off and briefly brushed to remove 620 
surface dirt with full strength household bleach (6% w/v NaOCl) using a disposable 621 
toothbrush that was soaked in 6% (w/v) bleach prior to use. They were then soaked in 6% 622 
(w/v) bleach for 5 minutes. Samples were rinsed twice with 18.2 MΩcm H2O and soaked in 623 
70% (v/v) Ethanol for 2 minutes and allowed to dry. Samples were incubated for 72 hours at 624 
room temperature in a buffer of 2 ml/100 mg sample weight of 0.5M EDTA Buffer pH 8.0 625 
(Fluka) and 50 μl/100 mg sample weight of Proteinase K 10 mg/ml (Roche). Extracts were 626 
concentrated to 250 μl using Amplicon Ultra-15 concentrators with a 30 kDa filter (Millipore) 627 
and purified according to manufacturer’s instructions using buffers from the MineluteTM PCR 628 
27 
 
Purification Kit (Qiagen) with the following changes: 1) the use of High-Volume spin columns 629 
(Roche); 2) 10X PB buffer instead of 5X; and 3) samples incubated with EB buffer (Qiagen) 630 
at 37°C for 10 minutes prior to elution in 100 μl or 50 μl (BEL024) EB buffer. Only one 631 
extraction was performed per sample for screening and 30 μl or 50 μl (BEL024) used for 632 
libraries. 633 
3.2 Library Preparation 634 
3.2.1 Double-stranded DNA Libraries 635 
Tübingen and Zurich: The double-stranded DNA library preparation is a two step procedure. 636 
In the first step 20 µl of the extracted DNA were converted into double-stranded libraries 637 
[152]. In the second step, sample-specific barcodes were added to both ends of the DNA 638 
libraries [155]. The indexed sequencing libraries were then amplified again with Herculase II 639 
Fusion using the following conditions: 1X Herculase II buffer, 0.4 µM IS5 and 0.4 µM IS6 640 
primer [152] Herculase II Fusion DNA polymerase (Agilent Technologies), 0.25 mM dNTPs 641 
(100 mM; 25 mM each dNTP), and 0.5 - 4 µl barcoded library as a template in a total 642 
reaction volume of 100 µl. The amplification thermal profile was executed as described: 643 
initial denaturation for 2 min at 95°C, denaturation for 30 sec at 95 °C, 30 sec annealing at 644 
60 °C, 30 sec elongation at 72 °C for three to 20 cycles following by a final elongation step 645 
for 5 min at 72 °C. Afterwards, the amplified DNA was purified by a MinElute purification step 646 
and DNA was eluted in 20 µl TET. We measured the concentration of the amplified 647 
sequencing libraries either by using Bioanalyzer (Agilent Technologies) and a DNA1000 lab 648 
chip from Agilent Technologies or by Tape Station (Roche). 649 
Tartu: The shotgun library for sample BEL024 was produced in the following manner: Library 650 
preparation was conducted using a protocol modified from the manufacturer’s instructions 651 
included in the NEBNext® Library Preparation Kit for 454 (E6070S, New England Biolabs, 652 
Ipswich, MA) as detailed in [153]. Libraries were amplified using the following PCR set up: 653 
28 
 
30μl DNA library, 1X PCR buffer, 2.5 mM MgCl2, 1 mg/ml BSA, 0.2 μM inPE1.0, 0.2 mM 654 
dNTP each, 0.1 U/μl HGS Taq Diamond and 0.2 μM indexing primer. Cycling conditions 655 
were: 5’ at 94°C, followed by 18 cycles of 30 seconds each at 94°C, 60°C, and 68°C, with a 656 
final extension of 7 minutes at 72°C. Amplified products were purified using MinElute 657 
columns and eluted in 35 μl EB (Qiagen). Three verification steps were implemented to 658 
make sure library preparation was successful and to measure the concentration of 659 
dsDNA/sequencing libraries – fluorometric quantitation (Qubit, Thermo Fisher Scientific), 660 
parallel capillary electrophoresis (Fragment Analyser, Advanced Analytical) and qPCR. 661 
3.2.2 UDG-treated DNA Libraries 662 
The damage patterns of ancient DNA potentially cause sequencing artefacts. These 663 
artefacts are problematic for genome-wide analyses. To avoid those potential sequencing 664 
artefacts, double-stranded UDG-treated DNA libraries were produced for genome-wide 665 
analyses of the samples processed in Tübingen and Zurich. Therefore, 30 µl of the extracted 666 
DNA was initially treated with UDG [96]. The indexed double-stranded DNA library 667 
preparation and the amplification steps were performed according to the well-established 668 
protocols [152, 155]. 669 
The library of PSN550 was prepared at the MPI-SHH Jena (Germany) following [154] based 670 
on the original UDG protocol by [96]. Two libraries from 25 µl DNA extract each were 671 
prepared and pooled after indexing.  672 
The samples PSN923, PSN951, and PSN441, prepared at UTIG Tartu, follows the UDG 673 
treatment step according to [154] and continues with Adapter ligation as described for the 674 
non-UDG library of BEL024. 30 µl of extract were used as template DNA for the initial USER 675 
treatment reaction. 676 
 677 
 678 
29 
 
3.3 Enrichment strategies 679 
Most of the samples were enriched for the human mitochondrial genome, for three specific 680 
leprosy genes, and at least for the complete M. leprae genome. The enrichment for the 681 
human mitochondrial genome and the three specific leprosy genes were performed by 682 
applying an in-solution capture method [156, 157]. Libraries potentially suitable for a whole-683 
genome analysis were enriched for the complete leprosy genome either by an array capture 684 
technique [158] or by a myBaits in-solution capture procedure (Arbor Biosciences). 685 
3.3.1 Human mitochondrial capture 686 
Selfmade DNA baits [156] covering the complete human mitochondrial (mt) DNA sequence 687 
were used to enrich the DNA libraries for the human mt genome's libraries by an in-solution 688 
capture approach [157]. 689 
3.3.2 Mycobacterium leprae gene screening 690 
For screening the samples for M. leprae DNA, the libraries were enriched for three specific 691 
M. leprae genes [44, 45]. These genes are the gyrA gene, specific for all Mycobacteria, and 692 
the proS and RLEP genes, typically for M. leprae [44]. For the enrichment procedure [157], 693 
DNA baits were produced [156] covering the genes' located DNA segments on the 694 
Mycobacterium leprae genome. 695 
2.3.3 Mycobacterium leprae genome-wide enrichment 696 
For genome-wide enrichment, UDG treated DNA libraries were used. The enrichment was 697 
either performed by an array capture as previously successfully applied and described [44, 698 
45, 158] or by an in-solution capture approach [118] using myBaits Whole Genome 699 
Enrichment kit (Arbor Bioscences). Briefly, the array capture approach was performed by 700 
two rounds of hybridization of the DNA libraries onto the DNA arrays, containing millions of 701 
probes covering the entire M. leprae genome [44, 45, 47]. For the in-solution capture, RNA 702 
30 
 
baits were designed [159], following the manufacturer's protocol, myBaits manual v4 (Arbor 703 
Bioscences). For the final amplification, we used Herculase II Fusion polymerase according 704 
to the following implementation: 1X Herculase II buffer, 0.4 µM IS5, and 0.4 µM IS6 primer 705 
[152], Herculase II Fusion DNA polymerase (Agilent Technologies), 0.25 mM dNTPs (100 706 
mM; 25 mM each dNTP), and 5 µl enriched libraries as a template in a total reaction volume 707 
of 100 µl. The thermal amplification profile was executed as described: initial denaturation 708 
for 2 min at 95°C, denaturation for 30 sec at 95°C, 30 sec annealing at 60°C, 30 sec 709 
elongation at 72°C for 14 cycles following by a final elongation step for 5 min at 72°C. 710 
Finally, the amplified DNA was purified by a MinElute purification step and eluted in 20 µl 711 
TET. 712 
Samples PSN923, PSN951, PSN441 and BEL024 were processed at the University of Tartu 713 
(Estonia) with a different in-solution custom myBaits target enrichment kit from Arbor 714 
Biosciences (4X tiling, 70 bp, 19 bp spacing). UDG treated libraries were captured for all 715 
samples but BEL024, which was not UDG treated. Target enrichment was performed 716 
(individual reactions) following the manufacturer's instructions (myBaits manual v4) in one 717 
round of capture with the following exception: half-reactions of baits were used for all 718 
samples. We performed a second round of capture for sample PSN951. All samples were 719 
amplified using Kapa Hifi Hotstart ReadyMix (2X), and all reactions were purified using 720 
Qiagen MinElute columns with a two-step elution and a final elution volume of 30 µl. 721 
3.4 DNA sequencing 722 
Sequencing was performed either at the Max Planck Institute for Science of Human History 723 
in Jena, at the Functional Genomic Center Zurich in Zurich or at the Institute of Genomics 724 
Core Facility at the University of Tartu (UTIG). At the Max Planck Institute for Science of 725 
Human History, the sequencing was performed on an Illumina HiSeq4000 platform. Both 726 
paired-end and single-end sequencing procedures were applied. Single-end sequencing 727 
was executed using 1*75+8+8 cycles, paired-end using 2*50+8+8 cycles. Sequencing at the 728 
31 
 
Functional Genomic Center Zurich was performed applying a paired-end sequencing 729 
approach using either 2*75+8+8 cycles or 2*150+8+8 cycles. The libraries were sequenced 730 
on an Illumina NextSeq500 platform or Illumina HiSeq4000 or HiSeq2500 platforms. 731 
Samples sequenced at the UTIG were either single-end sequenced on an Illumina 732 
NextSeq500 executed applying 1*75+8+8 cycles for an initial screening. The UDG libraries 733 
were sequenced on the same sequencing platform. Paired-end sequencing of these libraries 734 
was executed using 2*150*8+8 cycles. All three sequencing centres used Illumina standard 735 
kits and protocols for sequencing. 736 
 737 
3.5 Genome-wide analysis - Read processing, mapping, and variant 738 
calling 739 
All libraries enriched for the entire M. leprae genome were screened using the EAGER 740 
pipeline version 1.92.55 [97]. In brief, the quality of the sequencing reads was inspected with 741 
FastQC version 0.11.5 [98], all libraries were adapter trimmed and read pairs were merged 742 
using AdapterRemoval version 2.2.1a [99] and subsequently aligned to the M. leprae 743 
reference genome (TN chromosome, NC_002677.1) using CircularMapper version 1.0 [97] 744 
with a minimum quality score of 20, a maximum edit distance of n = 0.01 and seeding 745 
disabled (recommended parameters that were tested best for aDNA [100, 101]). Relaxed 746 
mapping parameters were chosen to take post-mortem damage into account. Duplicates 747 
were removed using MarkDuplicates (https://broadinstitute.github.io/picard), and the 748 
mapping was evaluated with QualiMap version 2.2.1 [102]. The ancient origin of the reads 749 
was verified using DamageProfiler version 1.0 [103]. If a library tested positive (1-fold 750 
coverage > 60%), the sample was processed further. 751 
Therefore, all non-UDG treated, adapter-clipped libraries were trimmed by 2bp to remove 752 
bases damaged by postmortem damage. Subsequently, all libraries (UDG and non-UDG 753 
32 
 
treated) were merged by sample and mapped against the M. leprae reference genome as 754 
described above, only the maximum edit distance was set to n = 0.2. In addition, the 755 
UnifiedGenotyper from the Genome Analysis Toolkit (GATK) version 3.8.0 [104, 105] was 756 
used to generate a mapping assembly and SNP calling. 757 
3.5.1 Processing of published samples 758 
All published modern and ancient strains [44, 45, 47, 49, 106, 114–121] were mapped 759 
against the M. leprae reference genome as newly sequenced, trimmed samples described 760 
above. For the strains where no sequencing reads were available (TN, Br4923), sequencing 761 
reads were simulated using Genome2Reads [160] and mapped identically to the other 762 
samples. 763 
2.5.2 SNP typing 764 
The genotyping of all 19 newly reconstructed strains was performed using an established 765 
method [42]. Briefly, there are 84 informative markers (78 SNPs and six InDels in 766 
homopolymeric tracts) used for the classification in 16 SNP subtypes of M. leprae [42]: 1A-D, 767 
2E-H, 3I-M, and 4 N-P. For a more straightforward application, the SNP types (SNP type 1–768 
4) and the SNP subtypes (A-N) can be determined using a combination of three and 16 loci, 769 
respectively [42]. Deeper resolution in SNP subtyping was also recently published and the 770 
corresponding specific markers were also applied in our analysis [46]. 771 
3.5.3 SNP alignment and SNP Effect analysis 772 
The SNP alignment of all modern and ancient published strains [44, 45, 47, 49, 106, 114–773 
121] and the newly sequenced strains was conducted using a modified version of 774 
MultiVCFAnalyzer version 0.85.2 [123] (Issue: 775 
https://github.com/alexherbig/MultiVCFAnalyzer/issues/5; Pull request: 776 
https://github.com/alexherbig/MultiVCFAnalyzer/pull/6). The reference base was called if the 777 
33 
 
position was covered by a read at least one/three times and the quality score was at least 778 
30. The base was called a SNP if the quality score was at least 30 and 90% of the mapped 779 
reads contained this variant. A SNP was used when it was called in at least one sample. If it 780 
was not covered or heterozygous in other samples it was set to ‘N’ there. In addition, all 781 
positions were excluded that occur in known repeat regions and rRNA and the positions 782 
covered by the negative control sample SK12 [44]. M. lepromatosis was used as an 783 
outgroup. The pairwise distance of aligned sequences was calculated using snp-dists [124] 784 
by considering only the differences of A, C, G, and T (Additional file 3: Table S5). 785 
To investigate the effects of the unique SNPs in our samples that are shared among the 786 
newly reconstructed strains, the VCF files for the samples generated in this study were 787 
processed using the genomic variant annotations and functional effect prediction toolbox 788 
SnpEff version 4.3t [122] to annotate the variants and determine their functional effects. 789 
SnpEff was run using default parameters. 790 
3.5.4 Phylogeny 791 
The phylogenetic placement of the newly reconstructed strains was performed based on the 792 
SNP alignment. Only positions that are covered by at least 80% of the included genomes 793 
were considered (partial deletion). 794 
In addition, three different parameter sets were applied to obtain three SNP alignments 795 
differing by size and quality = 796 
(1) Using all strains with a 1-fold coverage of at least 60% of the genome to assess 797 
the placement of all newly reconstructed low-coverage genomes UF800, 798 
COR_XVIII, and UF8. This results in 197 strains and a SNP alignment of 4199 799 
positions. 800 
34 
 
(2) Using all strains with a 3-fold coverage of at least 60% of the genome. This 801 
results in 192 strains and a SNP alignment of 3549 positions. 802 
(3) Using all strains with a 3-fold coverage of at least 60% of the genome, and all 803 
hypermutated strains (85054, Amami, S15, Br14-3, Br2016-15, Zensho-4, 804 
Zensho-5, and Zensho-9) excluded. This results in 184 strains and a SNP 805 
alignment of 2851 positions. 806 
A maximum parsimony (MP) and maximum likelihood (ML) tree were calculated based on 807 
the SNP alignment (1) and (2). The MP analysis was performed using MEGAX [108] and 808 
500 bootstraps. The ML tree was determined with RAxML-NG version 1.0.0 [107] and 100 809 
bootstraps using the following command: 810 
raxml-ng --all --msa snpAlignmentIncluding.fasta --model GTR+G --tree pars{10} --811 
bs-trees 100 --threads 16 812 
All trees were visualized with FigTree version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) 813 
and rooted based on the placement of M. lepromatosis . 814 
3.5.5 Estimation of divergence time (BEAST analysis) 815 
The SNP alignment (3; see section 2.5.4) was used for phylogenetic timescale estimation 816 
and Bayesian phylogenetic inference. The analysis was performed using BEAST 2.6.3 [110] 817 
with Bayesian Model test, relaxed log-normal clock and Bayesian Skyline tree prior. The age 818 
of each sample was used for the molecular clock calibration. Ancient samples were assigned 819 
uniform priors across the most probable age range (95% calibrated 14C age estimate or 820 
archaeologically assigned) for the tip-date parameter. Two MCMC chains of 100 million 821 
steps were run with every 100th step logged and combined using LogCombiner (part of the 822 
BEAST package) with 10% burnin steps discarded. Chain convergence and mixing was 823 
35 
 
inspected in Tracer v1.7.1 [111]. ESS for all but two BMT parameters (gamma shape and 824 
proportion of invariable sites) exceeded 200. 825 
Maximum Clade Credibility tree was chosen using TreeAnnotator (part of the BEAST 826 
package) and visualised using FigTree v1.4.4 [109]. 827 
3.5.6 Temporal signal 828 
The temporal signal in the M. leprae data set was tested using the Date Randomization Test 829 
[112] to assess the applicability of the sample age information to calibrate the molecular 830 
clock. The BEAST inference was repeated 10 times using point sample ages (lower end of 831 
the age range was used for the ancient samples) randomly reassigned to samples in the 832 
data set (otherwise with settings identical to the main BEAST analysis described in the 833 
previous paragraph). The mean substitution rate estimate of 95% Credibility Interval for the 834 
main BEAST analysis did not overlap with any of the 10 estimated 95% Credibility Intervals 835 
in the randomized tip-date runs (Additional file 1: Fig. S6), which indicates sufficient temporal 836 
signal for reliable molecular clock calibration. In addition, the temporal signal was 837 
investigated using TempEst [113] resulting in R2=0.32 and a correlation coefficient of 0.56 838 
(Additional file 1: Fig. S7). 839 
3.6 Human mitochondrial DNA analyses and molecular sex 840 
determination 841 
For the analysis of the mitochondrial DNA, the captured data were analyzed by the EAGER 842 
pipeline [97]. Reads were mapped against the human mitochondrial genome (NC_012920.1) 843 
using CircularMapper [97] with the following settings: a BWA seed length (-l) of 1000 to 844 
effectively turn off seeding, BWA Max # Diff (-n) of 0.01 allowing fewer differences of reads to 845 
the reference sequence, and BWA quality filter of 30, to discard reads with a lower mapping 846 
quality than 30. Schmutzi Contamination Estimation [161] evaluated contamination rates of 847 
the sequenced DNA and jointly called the consensus sequence in FASTA format of the 848 
36 
 
analyzed samples by converting the bam. For consensus calling, bases with low quality are 849 
eliminated by applying a quality filter q=20. Only samples with final contamination below 5%, 850 
1st base damage at the 5’-end of the DNA above or equal to 9%, and coverage above 50 % 851 
are used for haplogroup determination using HaploFind [162] and HaploGrep2 [163]. 852 
For sex determination, the amount of nuclear DNA is mapped to the sex chromosome and 853 
autosomal sequences of the complete human genome hg19. For the mapping, the program 854 
BWA [164] is used with a BWA seed length (-l) of 32, BWA Max # Diff (-n) of 0.01, and a BWA 855 
quality filter (-q): 20. Sex determination is performed applying phyton script for sex 856 
identification [165], as well as following the methodology of the sex identification developed 857 
by Skoglund and colleagues [166]. 858 
Rx, the normalized ratio of the alignments to autosomes and sex chromosome X, is calculated. 859 
A 95% confidence interval is established. If the upper bound for Rx is lower than 0.60 the 860 
individuals’ sex is estimated as male and if the lower bound for Rx is higher than 0.80 861 
individuals are estimated as female. For samples with a value for Rx in between the sexes 862 
could not be assigned. 863 
We were able to determine the human mitochondrial haplogroup for six individuals and the 864 
genetic sex is estimated for three individuals. The results are represented in Additional file 1: 865 
Table S1. 866 
 867 
 868 
 869 
 870 
 871 
 872 
37 
 
Supplementary Figures 873 
 874 
 875 
Fig. S1: Radiocarbon dates of the M. leprae genomes. Ages are given in cal CE. 876 
 877 
38 
 
 878 
Fig. S2: Damage profiles of all newly reconstructed M. leprae strains, where shotgun data 879 
were available.880 
39 
 
 881 
Fig. S3: Uncollapsed (A) Maximum Parsimony and (B) Maximum Likelihood tree using all 882 
strains with at least 1-fold coverage at more than 60% of the positions (SNP alignment 1). 883 
Ancient strains are in bold, newly added strains highlighted in red. Genomes from animal 884 
reservoirs are italic and indicated with the corresponding animal. 885 
40 
 
 886 
 887 
Fig. S4: Uncollapsed (A) Maximum Parsimony and (B) Maximum Likelihood tree using all 888 
strains with a 3-fold coverage at more than > 60% of the positions (SNP alignment 2). 889 
Ancient strains are in bold, newly added strains highlighted in red. Genomes from animal 890 
reservoirs are italic and indicated with the corresponding animal. 891 
41 
 
 892 
Fig. S5: Uncollapsed Bayesian Maximum Clade Credibility time-aware tree for the leprosy 893 
genomes including only genomes with at least 3-fold coverage at least 60% of the genome 894 
sites and hypermutated strains excluded (SNP alignment 3). Nodes are labeled with median 895 
estimated age (years before 2016 CE, ie. before the youngest sample) and 95% Highest 896 
Posterior Density for the age estimate (violet bars) as well as posterior probability estimate. 897 
42 
 
 898 
Fig. S6: Date Randomization Test for the M. leprae dataset. BEAST analysis was performed 899 
for the original data set and ten replicates with randomly reassigned tip calibrations (ages of 900 
the samples). The lack of overlap between the timescale parameter estimates (here, the 901 
mean rate of nucleotide substitution) indicates a sufficient temporal signal for the molecular 902 
clock calibration and time-aware phylogenetic inference. 903 
43 
 
 904 
Fig. S7: Result TempEst [113] analysis for the M. leprae dataset. The plot visualizes the 905 
phylogenetic root-to-tip distance relative to sampling time in years before present with the 906 
year 2016 as present. 907 
 908 
 909 
 910 
 911 
 912 
 913 
 914 
 915 
 916 
 917 
  918 
44 
 
Supplementary Tables 919 
Table S1: Sample IDs, archaeological sample name archaeological dates, sample description, age at death, mitochondrial haplogroups, and 920 
archaeological and molecular sex of all analysed samples. 921 
Short ID Individual 
Sample 
Types 
Analyzed 
Lab ID Supplier Sample ID Country 
Archaeologi
cal Age 
14C 
age 
BP 
14C 
dates 
(in cal 
CE) 
Dating ID Age at death 
Archaelogi
cal Sex 
Molecul
ar Sex 
(after 
Mittnik 
et al., 
2016) 
Molecul
ar Sex 
(after 
Skoglu
nd et 
al., 
2010) 
mitochondr
ial 
Haplogrou
p 
Beja3 
Beja_3 
Upper 
premolar 
2 
TU521 
Beja 
individual no 
3 
Portugal medieval  n.a. n.a. n.a adult male? XX (female) 
XX 
(female) n.a. 
Beja_3 Maxilla TU522 
Beja_3 
Fibula 
shaft 
fragment 
TU949 
Beja_3 
Right rib 
shaft 
fragment 
TU950 
Beja4 
Beja_4 Lower incisor 2  TU519 
Beja 
individual no 
4 
Portugal medieval  n.a. n.a. n.a adult male XY (male) 
consiste
nt with 
XY but 
not XX 
n.a. 
Beja_4 Navicular right TU520 
Beja_4 
Tibia 
shaft 
fragment 
TU947 
Beja_4 
Unidentifi
ed foot 
fragment 
TU948 
Beja8 
Beja_8 Tibia TU523 
Beja 
individual no 
8 
Portugal medieval  n.a. n.a. n.a adult male XY (male) n.a n.a. Beja_8 
Left hand 
phalanx 
intermedi
ate 
TU951 
45 
 
Beja_8 
Unidentifi
ed foot 
fragment 
TU952 
Bergen Bergen Premolar Tooth TU594 
Nonneaeter 
Kloster, 
Bergen 
Norway; Era 
5 
Norway medieval 635-715 
1268-
1388 
MAMS-
31414 n.a. n.a. 
XY 
(male) 
XY 
(male) H2a1a 
Dryburn-
Bridge 
Dryburn_Bridge Tooth TU936 Dryburn Bridge 1 
UK, 
Scotland 
Early Bronze 
Age. 
Scotland 
n.a. n.a. n.a 6-7 years n.a. XY (male)  
XY 
(male) n.a. 
Dryburn_Bridge Tooth TU937 Dryburn Bridge 2 
Dryburn_Bridge Tooth TU938 Dryburn Bridge 3 
Dryburn_Bridge Septum  TU939 Dryburn Bridge 4 
IPT17 IPT_17 Bone fragment TU1082 
IPT’17; K-
8/9; 
VE2855;SK4
03; left fibula 
Portugal  17th c. – early 20th c. 
150-
210 
1642-
1911 
Beta-
514831 adult male n.a. n.a n.a. 
KirkHill 
Kirk Hill Temporal bone TU940 
Kirkhill St. 
Andrews 
UK, 
Scotland 
Early 
medieval 
915-
965 
1030-
1155 
SUERC-
91431 
25-35 
years female 
XX 
(female) 
XX 
(female) n.a. Kirk Hill 
Skull 
fragment TU941 
Kirk Hill Skull fragment TU942 
PAVd’09_I.3
4 
PAVd’09_I.34 Rib  TU524 PAVd’09_I.3
4 Portugal 
15th c. - 17th 
c. n.a. n.a. n.a adult male 
XX 
(female) 
consiste
nt with 
XX 
n.a. 
PAVd’09_I.34 Manual phalanx TU525 
PAVd’09_I.5 
PAVd’09_I.5 Maxilla TU526 
PAVd’09_I.5 Portugal 15
th c. - 17th 
c. 
      
adult female XX (female) 
XX 
(female) n.a. PAVd’09_I.5 Tibia  TU527 609-673 
1283-
1396 
MAMS-
31413 
Blockhuizen Leiden_Blockhuizen bone TU398 
Blokhuizen? 
Los boc 
doos 
leprosy? 
Netherlan
ds 
10th c. - 12th 
c.  n.a. n.a. n.a. adult  n.a. n.a. n.a n.a. 
COR_XVIII 
COR_XVIII 
Fibula 
shaft 
fragment 
TU1083 COR_XVIII 
(Barrejo, 
Cordiñanes 
de Valdeón, 
León) 
Spain 12
th c. -early 
13th c. 
n.a. n.a. n.a. 
adult male n.a. n.a n.a. 
COR_XVIII 
Tiny bone 
fragments 
of the 
ethmoid 
TU1084       
R7546-671 Russia_7546-671 Tooth TU11 7546-671 Russia n.a. 
139-
199 
1661-
1950 
MAMS-
31412   female 
The 
sample 
is 
consiste
nt with n.a. 
46 
 
consiste
nt 
XY but 
not XX 
with XY 
(male) 
but not 
XX 
(female) 
R7546-695 Russia_7546-695 Tooth TU12 7546-695 Russia n.a. n.a. n.a. n.a. n.a. n.a. 
XY 
(male) 
consiste
nt with 
XY but 
not XX 
F1e3 
R-Kich-
Malka 
Russia_Kich-
Malka Tooth TU10 Kich-Malka Russia 
end of 7th – 
first half of 
8th c. 
n.a. n.a. n.a. n.a. n.a. XX (female) 
XX 
(female) A12a 
SLS348B 
SantaLucia-
Segovia_348B Tooth TU1080 
Molar 1; 
Santa Lucia-
Segovia; 
348-B Spain   n.a. n.a. n.a. n.a. n.a. n.a. n.a n.a. 
SantaLucia-
Segovia_348B Tooth TU1081 
Molar 2; 
Santa Lucia-
Segovia; 
348-B 
S2385 
Santarem_2385 
Right 
upper 
PM2 
TU1009 VRP 2385 Santarem 
Portugal 
Late Roman/ 
Early 
medieval 
1730-
1790 172-383 
Beta-
524726 adult male 
The 
sample 
is 
consiste
nt 
consiste
nt with 
XY but 
not XX 
I3a 
Santarem_2385 Metacarpal TU517 
VRP 2385 
Santarem 
with XY 
(male) 
but not 
XX 
(female) 
Santarem_2385 
Upper 
premolar 
right 2  
TU518 VRP 2385 Santarem   
Santarem_2385 
Right, 
distal tibia 
bone 
fragment 
TU943 VRP 2385 Santarem   
Santarem_2385 Rib shaft fragment TU944 
VRP 2385 
Santarem   
Santarem_2385 
Left 
calcaneus 
bone 
fragment 
TU945 VRP 2385 Santarem   
Santarem_2385 Left intermedi TU946 
VRP 2385 
Santarem   
47 
 
ate 
cuneiform 
UF100 UF_100_40 
Proximal 
end of the 
second 
left rib 
TU1162 045/07 UF 100 UE 40 Spain 
12th c. -early 
13th c. n.a. n.a. n.a. 
16-17 
years male n.a. n.a n.a. 
UF101 
UF_101 
Second 
left 
maxillary 
premolar 
ZH1108 045/07 UF 101 UE 43 
Spain 12
th c. - early 
13th c. 
939-
983 
1027-
1157 
ETH-
111400 
25-30 
years male n.a. n.a n.a. 
UF_101 
Left 
portion of 
the 
maxillary 
bone 
ZH1119 045/07 UF 101 UE 43 
UF102 
UF_102 
Second 
right 
mandibul
ar 
premolar 
ZH1117 045/07 UF 102 UE46 
Spain 12
th c. - early 
13th c. n.a. n.a. n.a. 
17-20 
years male n.a. n.a n.a. 
UF_102 
Right 
metacarp
al 1 
ZH1118 045/07 UF 102 UE46 
UF103 
UF_103_49 Maxillary bone TU1163 
045/07 UF 
103 UE 49 
Spain 12
th c. -early 
13th c. n.a. n.a. n.a. 
27-35 
years male n.a. n.a n.a. UF_103_49 
Right 
mandibul
ar canine 
TU1164 045/07 UF 103 UE 49 
UF104 UF_104_56 
Proximal 
phalanx 1 
of the left 
hand 
TU1165 045/07 UF 104 UE 56 Spain 
12th c. -early 
13th c. n.a. n.a. n.a. n.a. female n.a. n.a n.a. 
UF11 
UF_11_69 
Left 
portion of 
the 
maxillary 
bone 
TU1172 045/07 UF 11 UE 69 
Spain 18th c. n.a. n.a. n.a Young adult female n.a. n.a n.a. 
UF_11_69 
Second 
right 
mandibul
ar molar 
TU1173 045/07 UF 11 UE 69 
UF18 UF_18_1123 
Proximal 
phalanx 4 
of the 
right foot 
TU1174 045/07 UF 18 UE 1123 Spain 18
th c. n.a. n.a. n.a n.a. n.a. n.a. n.a n.a. 
48 
 
UF21 
UF_21_1137 
First right 
mandibul
ar molar 
TU1170 045/07 UF 21 UE 1137 
Spain 16th c. 399-445 
1431-
1611 
ETH-
107778 
14-15 
years male n.a. n.a n.a. 
UF_21_1137 
Fragment 
of the 
ethmoid 
TU1171 045/07 UF 21 UE 1137 
UF25 
UF_25_1174 
Proximal 
phalanx 5 
of the 
right hand 
TU1166 045/07 UF 25 UE 1174 
Spain end of 13
th c. 
- 14th c. 
427-
473 
1423-
1466 
ETH-
107776 
17-20 
years n.a. n.a. n.a n.a. 
UF_25_1174 
Second 
right 
mandibul
ar molar 
TU1167 045/07 UF 25 UE 1174 
UF700 
UF_700 
Right 
metacarp
al 3 
ZH1113 045/07 UF 700 UE7006 
Spain 12
th c. - early 
13th c. 
909-
953 
1035-
1165 
ETH-
111399 
17-20 
years female n.a. n.a n.a. 
UF_700 
Left 
metacarp
al 2 
ZH1114 045/07 UF 700 UE7006 
UF701 
UF_701_7016 
Left 
portion of 
the 
maxillary 
bone 
TU1152 045/07 UF 701 UE 7016 
Spain 12
th c. -early 
13th c. n.a. n.a. n.a. n.a. female n.a. n.a n.a. UF_701_7016 
First right 
maxillary 
molar 
TU1153 045/07 UF 701 UE 7016 
UF_701_7016 
Head of 
the left 
humerus 
TU1154 045/07 UF 701 UE 7016 
UF702 
UF_702_7019 
Proximal 
phalanx 1 
of the 
right hand 
TU1155 045/07 UF 702 UE 7019 
Spain 12
th c. -early 
13th c. n.a. n.a. n.a. n.a. male n.a. n.a n.a. 
UF_702_7019 
Third left 
mandibul
ar molar 
TU1156 045/07 UF 702 UE 7019 
UF703 
UF_703_7027 
Left 
metacarp
al 3 
TU1157 045/07 UF 703 UE 7027 
Spain 12
th c. -early 
13th c. 
892-
938 
1040-
1208 
ETH-
107775 
25-35 
years female n.a. n.a n.a. 
UF_703_7027 
First right 
mandibul
ar 
premolar 
TU1158 045/07 UF 703 UE 7027 
49 
 
UF8 
UF_8_1054 
Right 
metatarsu
s 4 
TU1168 045/07 UF 8 UE 1054 
Spain 16th c. n.a. n.a. n.a n.a. female n.a. n.a n.a. 
UF_8_1054 
Second 
left 
mandibul
ar molar 
TU1169 045/07 UF 8 UE 1054 
UF800 
UF_800 
Right 
metacarp
al 1 
ZH1111 045/07 UF 800 UE 8008 Spain 12th c. - early 
13th c. n.a. n.a. n.a 
12-14 
years female n.a. n.a n.a. 
UF_800 
Right 
metacarp
al 3 
ZH1112 045/07 UF 800 UE 8008   
UF801 
UF_801 
Left 
metatarsu
s 5 
ZH1109 045/07 UF 801 UE 8011 
Spain 12
th c. - early 
13th c. n.a. n.a. n.a. 
16-20 
years n.a. n.a. n.a n.a. 
UF_801 
Proximal 
phalanx 4 
of the left 
hand 
ZH1110 045/07 UF 801 UE 8011 
UF802 
UF_802 
Proximal 
phalanx 2 
of the 
right hand 
ZH1115 045/07 UF 802 UE 8014 
Spain 12
th c. - early 
13th c. n.a. n.a. n.a. 
35-45 
years female n.a. n.a n.a. 
UF_802 
Left 
metatarsu
s 5 
ZH1116 045/07 UF 802 UE 8014 
UF803 
UF_803_8020 
Proximal 
phalanx 2 
of the 
right hand 
TU1159 045/07 UF 803 UE 8020 
Spain 12
th c. -early 
13th c. 
946-
992 
1023-
1157 
ETH-
107777 
25-30 
years female n.a. n.a n.a. UF_803_8020 
Third left 
mandibul
ar molar 
TU1160 045/07 UF 803 UE 8020 
UF_803_8020 
Medial 
phalanx 5 
of the left 
hand 
TU1161 045/07 UF 803 UE 8020 
BEL024 24  bone BEL024 / II24 24 Belarus 
10th c. - 12th  
c. 
898-
950 
135-
1203 
UBA-
44327 
25-30 
years male n.a. n.a n.a. 
PSN932 sk. 2012  bone CHRY023 PSN 932 UK, England 
10th c. - 12th  
c. 
915-
965 
1034-
1162 
UBA-
44325 n.a. n.a. n.a. n.a n.a. 
PSN951 sk. 2529 
Upper 
right 
canine 
tooth 
CHRY044 PSN 951 UK, England 
10th c. - 12th  
c. 
903-
953 
1034-
1162 
UBA-
44326 n.a. n.a. n.a. n.a n.a. 
50 
 
PSN550 sk42b 
Upper 
right 
canine 
tooth 
EDI006 PSN 550 UK, England 
5th c. - 7th c. 
(550-650 
CE) 
1423-
1475 575-650 
UBA-
44321 
25-36 
years female female female n.a. 
PSN441 JDS10 Upper left first molar JDS097 PSN 441 
UK, 
England 
12th c. -early 
16th c. 
691-
749 
1231–
1384 
SUERC-
71631 n.a. n.a. female female n.a. 
922 
51 
 
Table S2: Eager Report of the analysed samples (nonUDG - and UDG-treated merged and trimmed). 923 
 924 
Sample 
Name 
# reads 
after 
C&M 
Prior 
mapping 
# 
mapped 
reads 
Prior 
RMDup 
# of 
Duplicat
es 
Remove
d 
Mappe
d 
Reads 
After 
RMDu
p 
Endo
g. 
DNA 
(%) 
Clust
er 
Facto
r 
Mean 
Covera
ge 
std. 
dev. 
Covera
ge 
Covera
ge 
>= 1X 
in % 
Covera
ge 
>= 3X 
in % 
Covera
ge 
>= 5X 
in % 
# 
SNP
s 
DM
G 
1st 
Bas
e 3' 
DM
G 
2nd 
Bas
e 3' 
DM
G 
1st 
Bas
e 5' 
DM
G 
2nd 
Bas
e 5' 
Avg. 
frag. 
Lengt
h 
Med. 
frag. 
Lengt
h 
GC 
conte
nt 
In % 
BEL024 2907843
0 
823000
9 
656425
3 
16657
56 
28.3 4.94 43.86 11.85 97.71 97.51 97.41 111 0.01 0.01 0.02 0.02 86.06 74 56.87 
PSN550 1722602
0 
148292
2 
488509 99441
3 
8.61 1.49 23.71 7.59 97.64 97.43 97.27 98 0 0 0 0 77.92 76 57 
UF700 1828485
7 
503570
3 
415045
2 
88525
1 
27.54 5.69 19.45 9.2 97.53 96.91 95.39 105 0.04 0.03 0.05 0.04 71.79 68 55.43 
Bergen 1363804
52 
372181
75 
321346
82 
50834
93 
27.29 7.32 110.61 29.9 97.45 97.44 97.43 114 0.01 0 0 0 71.11 76 56.58 
PAVd’09_I.5 1952055
12 
686142
56 
638312
56 
47830
00 
35.15 14.35 96.82 23.75 97.45 97.44 97.42 115 0.02 0.01 0 0 66.15 74 56.72 
UF703 3243001
9 
526940
2 
399664
5 
12727
57 
16.25 4.14 26.94 18.03 97.44 96.19 93.73 92 0.01 0 0 0 69.17 67 54.42 
UF25 3332358
0 
504891
7 
348196
9 
15669
48 
15.15 3.22 33.09 24.92 97.4 95.73 92.92 167 0 0 0 0 69.01 66 54.02 
UF101 3199847
4 
395978
2 
297160
0 
98818
2 
12.38 4.01 21.28 13.77 97.39 95.69 92.41 99 0.04 0.03 0.05 0.04 70.37 68 54.05 
R7546-671 2143989
70 
213789
75 
204597
35 
91924
0 
9.97 23.26 16.51 11.79 97.16 94.96 90.16 103 0 0 0 0 58.71 59 53.14 
PSN441 1356370
4 
536992
4 
462808
5 
74183
9 
39.59 7.24 12.81 6.84 96.89 94.27 89.02 102 0 0 0 0 56.44 54 56.11 
52 
 
PSN923 2132154
0 
287051
3 
253042
2 
34009
1 
13.46 8.44 7.01 3.66 96.53 89.75 74.59 90 0 0 0 0 67.34 65 56.6 
PSN951 1562160
9 
145888
32 
135336
11 
10552
21 
93.39 13.83 18.09 11.67 96.31 92.35 87.07 90 0 0 0 0 56.04 53 55.31 
KirkHill 6792265
8 
227559
1 
191111
2 
36447
9 
3.35 6.24 6.86 5.64 94.85 81.01 62.36 74 0 0 0 0 61.51 58 53.63 
UF21 2961941
4 
166018
7 
145100
1 
20918
6 
5.61 7.94 4.11 3.26 92.14 67.7 39.31 51 0.01 0 0.01 0 64.21 60 55.3 
UF803 9033898
2 
122037
3 
945382 27499
1 
1.35 4.44 6.18 6.18 91.01 69.77 50.67 110 0 0 0 0 73.49 74 53.73 
UF800 1471387
94 
499762 351764 14799
8 
0.34 3.38 3.34 3.82 86.27 52.63 27.09 49 0.01 0.01 0.02 0.02 73.79 72 54.52 
UF11 2512361
0 
232378
3 
197603
8 
34774
5 
9.25 6.68 6.71 8.08 85.67 61.81 45.56 71 0.01 0 0.01 0 63.05 60 51.8 
UF8 2973500
5 
784616 715814 68802 2.64 11.4 1.46 2.89 67.52 18.99 3.43 75 0 0 0 0 69.16 68 55.19 
COR_XVIII 1195886
32 
219283
1 
203473
8 
15809
3 
1.83 13.87 2.49 4.67 67.28 32.39 17.55 154 0 0 0 0 51.51 49 52.06 
Beja_3 8157301
4 
711528 678407 33121 0.87 21.48 0.56 3.62 29.66 2.23 0.66 276 0.02 0.01 0.01 0.01 55.36 51 54.57 
Beja_4 1205645
75 
178639
7 
176117
1 
25226 1.48 70.82 0.38 4.57 5.87 1.72 1.23 742 0.03 0.02 0.02 0.01 49.2 45 59.35 
Beja_8 1215915
90 
715294 700941 14353 0.59 49.84 0.23 3.95 2.65 0.88 0.62 266 0.02 0.02 0.01 0.01 53.08 47 59.43 
UF801 4597875
3 
5334 2879 2455 0.01 2.17 0.04 1.04 1.99 0.13 0.09 41 0.02 0.01 0.02 0.02 58.68 51 55.62 
53 
 
Dryburn_Bri
dge 
2084891
0 
5519 3117 2402 0.03 2.3 0.03 1.11 1.07 0.1 0.08 52 0.03 0.02 0.03 0.02 47.22 49 56.36 
Santarem 4112529
5 
12310 9133 3177 0.03 3.88 0.05 1.65 1.02 0.15 0.12 51 0.08 0.04 0.04 0.03 52.03 49 56.75 
Karganaee 1972542
5 
2167 839 1328 0.01 1.63 0.02 0.98 0.26 0.1 0.07 17 0.03 0.02 0.02 0.01 56.84 55 57.36 
R7546-695 5514560
9 
10340 8961 1379 0.02 7.5 0.02 0.96 0.24 0.11 0.08 21 0.03 0.02 0.02 0.02 55 51 56.18 
UF102 4720599 2395 2105 290 0.05 8.26 0 0.2 0.24 0.03 0.01 4 0.01 0 0.04 0.02 53.93 46 55.67 
Russia 
Kich-Malka 
1084442
80 
6760 5666 1094 0.01 6.18 0.02 0.74 0.2 0.08 0.06 6 0.23 0.03 0.06 0.02 49.28 46 55.73 
Russia 
Sajanskaja 
3605519
0 
10270 8934 1336 0.03 7.69 0.02 0.93 0.2 0.1 0.07 20 0.03 0.02 0.02 0.01 53.55 50 56.17 
UF701 8430829 2501 1985 516 0.03 4.85 0.01 0.41 0.17 0.05 0.04 7 0.01 0.02 0.05 0.01 47.7 44 56.87 
Lagos34 1127945
3 
2316 1196 1120 0.02 2.07 0.02 0.9 0.16 0.07 0.05 34 0.42 0.03 0.02 0.01 54.36 51 55.3 
UF103 5584524 2652 1541 1111 0.05 2.39 0.02 0.85 0.16 0.07 0.06 48 0.02 0.02 0.02 0.02 52.79 50 55.71 
UF18 2256635 1524 1422 102 0.07 14.94 0 0.09 0.11 0.01 0 2 0 0 0 0.07 55.03 48 56.71 
UF702 3728426 869 343 526 0.02 1.65 0.01 0.37 0.11 0.05 0.04 18 0.03 0.04 0.07 0.04 42.63 42 56.09 
UF100 2115386 320 109 211 0.02 1.52 0 0.17 0.08 0.03 0.02 3 0.04 0 0.05 0 44.07 43 56.55 
54 
 
UF802 2670874 232 59 173 0.01 1.34 0 0.17 0.07 0.03 0.02 3 0.1 0 0.04 0.06 45.64 43 56.07 
Leiden 
Blockhuizen 
3041762 558 380 178 0.02 3.14 0 0.16 0.05 0.03 0.02 4 0.05 0.02 0.02 0 46.61 46 55.44 
UF104 2060458 208 101 107 0.01 1.94 0 0.12 0.05 0.01 0.01 1 0.04 0 0.04 0.04 43.19 41 56.42 
SantaLucia 
Segovia 
237189 34 5 29 0.01 1.17 0 0.03 0.02 0 0 0 0 0 0 0 44 43 56.19 
IPT_17 109208 19 10 9 0.02 2.11 0 0.01 0.01 0 0 0 0 0 0 0 48.56 46 57.67 
 925 
55 
 
Table S3: SNP subtyping [42, 45–47, 106] 926 
 M. leprae coordinates (strain TN as reference)    
  
164287
5 
293568
5 14676 
310277
8 
110423
2 7614 
152705
6 
231205
9 711197 Monot* 
Genoty
pe 
(deeper 
resoluti
on)** branch 
UF800 T A C C C C G C T 2F 2F 2F 
R7546-671 T A C C C C G C T 2F 2F 2F 
UF11 T C C C G T G C T 3I 3I-1 3 
UF21 T C C C G T G C T 3I 3I-1 3 
UF8 T C C C G T G C T 3I 3I-1 3 
PAVd’09_I.
5 T C C C G T G C T 3I 3I-1 3 
PSN923 T C C C G T G C T 3I 3I-1 3 
KirkHill T C C C G T G C T 3I 3I-1 3 
JDS5097 T C C C G T G C T 3I 3I-1 3 
UF101 T C C C G T G C T 3I 3I-1 3 
UF700 T C C C G T G C T 3I 3I-1 3 
PSN951 T C C C G T G C T 3I 3I-1 3 
Bergen T C C C G T G C T 3I 3I-1 3 
PSN550 T C C C G T G C T 3I 3I-1 3 
BEL024 T C C C G C G C T 3L 
New 
(3Q) 4 
COR_XVIII T C C C G C G G C 3K 3K-0 0 
UF803 T C C C G C G G C 3K 3K-0 0 
UF703 T C C C G C G G C 3K 3K-0 0 
U25 T C C C G C G G C 3K 3K-0 0 
* according to the 16 loci described by Monot et al. [42, 106]. 927 
** according to the loci described by Truman et al. [106]. 928 
 929 
 930 
56 
 
 931 
Table S4: Result table of the SNP effect analysis (Additional file 2) 932 
 933 
Table S5: SNP distance matrix based on alignment 2 (2.5.4 Phylogeny). (Additional file 3) 934 
 935 
 936 
Table S6: Unique SNPs within the newly reconstructed strains located within genes that are 937 
related to virulence factors (according: http://www.mgc.ac.cn/cgi-bin/VFs/compvfs.cgi).  938 
 939 
Gene 
name 
Strain Position of SNP 
(TN reference) 
Coverage Protein function associate with 
virulence factor  
leuD PSN951 2029811 29X Amino acid and purine 
metabolism / Leucine synthesis 
mce1A UF800 3093139 3X Mammalian cell entry (mce) 
operons 
ml0049 UF700 61431 13X Secretion system / ESX-1 
(T7SS) 
ml0135 BEL024 184256 59X Cell surface components / PDIM 
(phthiocerol dimycocerosate) 
and PGL (phenolic glycolipid) 
biosynthesis and transport 
ml1539 UF703 1856699 11X Secretion system / ESX-5 
(T7SS) 
ml2534 UF703 3016347 8X Secretion system / ESX-3 
(T7SS) 
 940 
 941 
 942