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Supporting Information Supporting Information Lacan et al. 10.1073/pnas.1100723108 SI Materials and Methods software (4). Two databases were compiled for both uniparental Ancient DNA Procedures. Drastic precautions were taken to avoid markers. The mtDNA database comprises 14,699 HVI hap- contaminations by modern DNA templates (1): pre-PCR and lotypes associated with their corresponding haplogroup. The post-PCR procedures were carried out in two separate labora- NRY database comprises 49 European populations representing tories located on two separate floors. Pre-PCR procedures were 10,488 Y-STR profiles. References used to compile these data- performed in a dedicated laboratory under laminar flux. Work- bases are available in Table S8. For maternal lineages, compar- benches, surfaces, and all equipment were systematically wiped isons were based on HVI haplotypes, and for paternal lineages, with bleach, rinsed with ultrapure water, and irradiated for at they were based on seven STR markers (DYS19, DYS389a, least 2 h with UV light before each manipulation. Laboratory DYS389b, DYS390, DYS391, DYS393, and DYS439) and on the access was limited to authorized personnel only who always wore seven male individuals for whom complete datasets were ob- gloves, overshoes, laboratory coats, and face masks. Pipettes, tained (195, 575, 584, 596, 615, 616, and 636). The pattern of plastic ware, and aerosol-resistant tips were sterile and used ex- genetic differentiation was visualized by multidimensional scaling clusively for ancient DNA work. DNA from people handling the plot (XLstat, version 7.5.2) and by plotting on a map all FST values anthropological material (members of the museum and labora- obtained in the comparison between the Treilles population and tory staff) was also analyzed to rule out recent contamination. each population in the database, using Surfer software (version DNA extracted from sheep or goat bone fragments also retrieved 8.0; Golden Software). in the ossuary were used as a negative control to detect potential The percentage of shared lineages between Treilles and each contamination that could have occurred during excavation. present-day population in the databases was graphically also Statistical Analyses. To study putative genetic relationships be- plotted on a map by using Surfer software (version 8.0; Golden tween individuals from the ossuary, kinship was determined from Software). autosomal STR profiles with ML-Relate software (2) and con- A haplotype network was generated for NRY haplogroup G2a* firmed with DNA•VIEW Software (3), with which the LR was from the Treilles data and all European data via the median- calculated assuming a prior probability of 0.5. joining algorithm of Network, version 4.5.1.6. To obtain the most Human specimens from necropoles cannot be of course con- parsimonious networks the reticulation permissivity was set to sidered as a population in a statistical sense. Furthermore an- zero. Datasets were preprocessed using the star contraction cient DNA data could not be obtained for all the specimens option in Network, version 4.5.1.6 (5). Because of the high level buried, and Y-haplotypes were not determined for all male of reticulation in the G2a* sample, Y-STR loci were subdivided individuals. However, to try to characterize affinities between the into two mutation rate classes based on observed STR allelic ancient Treilles specimens and current European populations, we variance and weighted as follows: 2 (low) for DYS391 and performed cross-population comparisons from HVI sequences DYS392 and 1 (high) for DYS389I, DYS389II, DYS19, DYS393, and partial Y-chromosomal haplotypes with the ARLEQUIN 3.1 and DYS390 (6). 1. Keyser C, et al. (2009) Ancient DNA provides new insights into the history of south 4. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): An integrated software Siberian Kurgan people. Hum Genet 126:395–410. package for population genetics data analysis. Evol Bioinform Online 1:47–50. 2. Kalinowski S, Wagner A, Taper M (2006) ML-Relate: a computer program for maxi- 5. Forster P, Torroni A, Renfrew C, Röhl A (2001) Phylogenetic star contraction applied to mum likelihood estimation of relatedness and relationship. Mol Ecol Notes 6: Asian and Papuan mtDNA evolution. Mol Biol Evol 18:1864–1881. 576–579. 6. Tishkoff SA, et al. (2007) History of click-speaking populations of Africa inferred from 3. Brenner CH (1997) Symbolic kinship program. Genetics 145:535–542. mtDNA and Y chromosome genetic variation. Mol Biol Evol 24:2180–2195. Fig. S1. Spatial distribution of the genetic matrilineal distances between Treilles samples and modern Western Eurasian populations. Lacan et al. www.pnas.org/cgi/content/short/1100723108 1of16 Fig. S2. Multidimensional scaling plot of genetic distances calculated for mtDNA data. The red square represents Treilles samples. Fig. S3. Spatial distribution of the genetic patrilineal distances between Treilles male samples and modern Western Eurasian populations. Lacan et al. www.pnas.org/cgi/content/short/1100723108 2of16 Fig. S4. Multidimensional scaling plot of genetic distances calculated for Y-chromosomal data. The red square represents Treilles samples. Lacan et al. www.pnas.org/cgi/content/short/1100723108 3of16 Fig. S5. Median joining network of Y-G2a haplotypes in current western European populations and in the Treilles male specimens (in red). Lacan et al. www.pnas.org/cgi/content/short/1100723108 4of16 Lacan et al. Table S1. Consensus STR autosomal profiles of the 24 Treilles human specimens Sample Quantity, name ng/μL D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 AMEL D5S818 FGA www.pnas.org/cgi/content/short/1100723108 − 137 6.20 × 10 3 (12)/13 (29)/30 (8/12) 12/12 16/17 6/6 12/12 11/12 17/(19) 12/15.2 17/17 (8/8) 12/16 X/Y 12/13 22/23 − 139 5.07 × 10 3 (11/13) 31.2/33.2 9/9 11/11 ——9/10 11/12 24/25 — (17/ — 14/15 X/Y 12/12 22/24 17) − 195 2.59 × 10 4 10/14 30.2/31.2 9/12 11/11 15/17 6/9,3 8/11 9/(13) 17/19 12/14 16/17 9/(11) (12)/14 X/Y 11/13 20/24 209 NA 12/14 30/30 (7)/10 (9/12) 18/18 6/(8) 9/(13) 9/9 (20/20) 13/14 (15)/ (11/ 13/19 X/Y 11/11 (19)/20 17 11) − 570 1.78 × 10 2 10/14 28/31 11/12 11/(12) 18/18 — 8/8 11/11 17/21 12/13 17/20 (8/11) (14)/17 X/Y 11/13 21/22 − 573 1.91 × 10 2 11/14 31.2/31.2 (10/12) 9/(11) 16/17 7/7 9/(10) (9/12) (17/17) 14/15 14/16 (11/ (16/19) X/X 12/12 21/23 11) − 575 1.23 × 10 2 11/16 27/28 10/12 12/12 15/18 6/7 8/11 9/12 17/21 12/13 17/17 8/11 14/16 X/Y 10/11 23/24 − 577 5.19 × 10 3 (10/10) (29/32.2) 10/12 10/12 (16/16) — 8/(12) (9)/11 (21/21) (14/14) ——(13)/14 (X/Y) (13/13) 24/25 − 579 1.97 × 10 2 14/15 29/32.2 12/(13) 10/(12) 18/18 6/9.3 8/12 8/12 20/21 14/14 (15)/ 8/11 12/15 X/Y 12/13 22/23 16 − 583 7.51 × 10 3 14/16 28/(30) 8/(12) 10/10 (15)/18 (9/9,3) 8/11 (14/14) 17/(20) 13/15 (15)/ — (14)/19 X/Y (10)/12 24/24 17 − 584 1.21 × 10 2 10/16 31.2/32.2 10/12 10/12 (15)/18 9/9 9/13 9/13 16/21 12/14 17/17 8/8 11/20 X/Y 11/(15) 20/24 − 587 6.75 × 10 3 10/(13) (24.2/ — (13/13) 16/16 — 8/8 ——13/14.2 (15/ (11/ — X/Y 12/12 — 24.2) 17) 11) − 588 3.38 × 10 3 11/14 24.2/30 11/(12) 10/10 (16)/18 9,3/ 10/12 (12)/13 23/23 14/16 14/18 — 13/17 X/Y 11/12 21/24 9,3 − 592 1.52 × 10 3 11/15 31/31.2 (10)/12 12/12 (17)/18 7/9.3 11/11 (12)/13 17/(23) 13/14 15/16 8/8 14/20 X/Y 11/12 24/24 − 593 1.63 × 10 2 ——12/12 (10/10) ——10/11 9/(11) ——(17/ — (12/17) (X/Y) — 25/26 17) − 596 1.18 × 10 2 13/13 29/33.2 9/10 10/11 16/17 6/9.3 8/11 9/12 16/24 13/13 14/16 8/8 15/16 X/Y 11/12 19/25 − 600 4.43 × 10 3 13/13 28/28 12/12 10/(11) 16/17 9/9.3 8/11 12/12 (17)/23 13/13 15/15 (8/8) 14/14 X/Y 12/12 23/25 604 NA 10/15 29.2/31.2 8/9 10/12 18/19 (6)/8 9/11 12/13 17/(25) 15/(16) 18/19 — 18/20 X/Y 11/12 (21)/26 611 Undet 14/14 (28/30) — (9/9) 14/15 9.3/ (8/11) ——(13)/14 16/17 (8/8) (12/17) X/Y (11/11) (19/19) 9.3 − 612 3.42 × 10 3 10/15 (28/28) — 10/10 (16)/18 (9/9.3) 8/8 11/11 17/20 13/15 15/15 (8/8) 12/19 X/Y 10/12 24/24 − 614 5.90 × 10 3 10/13 28/29 (8/10) 11/11 17/18 (7/7) (8/8) 11/12 (23/23) 12/12 14/14 — 18/19 X/X 10/13 21/21 − 615 1.85 × 10 2 11/12 28/28 (9/11) 12/12 15/15 9/(9.3) 8/11 8/13 20/20 15.2/ 14/14 11/11 12/18 X/Y 11/11 20/20 15.2 − 616 3.34 × 10 2 10/13 28/33.2 10/11 10/12 18/18 6/9.3 8/11 11/13 17/20 13/16 15/16 8/8 19/20 X/Y 11/12 22/24 − 636 2.01 × 10 2 10/10 (31.2)/ 8/9 12/13 15/18 (6)/8 9/11 12/14 17/24 15/16 17/19 8/8 15/18 X/Y 12/12 21/21 33.2 Research team 1 13/13 28/31 10/11 10/12 15/17 8/9 12/13 12/12 18/24 13/15 17/17 8/11 11/16 X/Y 11/12 21/ 23,2 2 13/14 28/29 8/10 11/11 15/18 7/9.3 11/11 11/12 24/25 13/15.2 14/17 8/10 12/15 X/Y 11/13 21/22 3 12/14 29/29 9/11 12/12 14/18 9/9.3 12/12 11/11 17/19 13/13 15/15 11/11 14/17 X/X 11/11 21/23 4 10/11 30/32.2 10/10 11/12 16/17 9/9.3 8/11 11/12 20/23 14/14 16/18 11/12 12/13 X/X 11/13 19/25 5 10/13 29/30 9/11 10/11 14/18 9/9.3 11/11 11/14 17/22 14/15.2 14/18 8/12 15/17 X/Y 12/12 20/23 Dashes denote that alleles could not be clearly amplified for the locus in question.
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