al. [10]. The specificity of the Eco47III restriction site to the pallidum subspecies was previously demonstrated by Centurion- Lara and coworkers [20]. We further performed a Blast search and found that 100% of similarity, including the recognition sequence for Eco47III (AGCGCT), was found only for T. pallidum subspecies pallidum, with an E-value of 1610254. Other T. pallidum subspecies or other species of Treponema, failed to present the Eco47III restriction site. PCR products from both the arp gene and the 59UTR of the 15-kDa lipoprotein gene were cloned into TOPO- TA (Invitrogen). Positive clones were sequenced in both strands using M13 forward and reverse primers. Due to its specificity to T. pallidum subspecies pallidum, in Langebio only the amplification of the 119-bp fragment of the 15- kDa lipoprotein gene was conducted. PCR products were cloned into pJET1.2/blunt (Fermentas) and positive clones were se- quenced in both strands using pJET1.2 forward and reverse sequencing primers. Supporting Information Figure S1 Acidic Repeat Protein (arp) gene fragment of Treponema pallidum subspecies pallidum, aligned with clone sequences from sample ELS551, obtained at the Universidade dos Ac¸ores (Portugal). A box shows the NaeI References 1. Luger A (1993) The origin of syphilis. Clinical and epidemiologic considerations on the Columbian theory. Sex Transm Dis 20: 110–117. 2. Hershkovitz I, Rothschild BM, Wish-Baratz S, Rothschild C (1995) Natural variations and differential diagnosis of skeletal change in bejel (endemic syphilis). In: Dutour O, Palfi G, Berato J, Brun JP, eds. L’Origin la syphilis en Europe- Avant ou apre`s 1493. Toulon, France: Centre Archeologique du Var. pp 81–87. 3. Hudson MM, Morton RS (1997) Fracastoro and syphilis: 500 years on. The Lancet 348: 1495–1496. 4. Blondiaux J, Alduc-Le Bagouse A (1994) A treponematosis dated from the Late Roman Empire in Normandie, France. In: The Origin of Syphilis in Europe: Before or After 1493? Proceedings of an International Colloquium. Toulon, Paris: Centre Arche´ologique du Var-Editions France. 5. Henneberg M, Henneberg R (1994) Treponematosis in an Ancient Greek colony of Metaponoto, southern Italy, 580-250 BC. In: The Origin of Syphilis in Europe: Before or After 1493? Proceedings of an International Colloquium. Toulon, Paris: Centre Arche´ologique du Var-Editions France. 6. Roberts C (1994) Treponematosis in Gloucester, England: a theoretical and practical approach to the pre-Columbian theory. In: The Origin of Syphilis in Europe: Before or After 1493? Proceedings of an International Colloquium. Toulon, Paris: Centre Arche´ologique duVar-Editions France. 7. Pa´lfi G, Dutour O, Borreani M, Brun JP, Berato J (1992) Pre-Columbian syphilis from the Late Antiquity in France. Int. J Osteoarchaeol 2: 245–261. 8. Stirland A (1991) Evidence for pre-Columbian treponematosis in medieval Europe (England). In: The Origin of Syphilis in Europe: Before or After 1493? Proceedings of an International Colloquium. Toulon, Paris: Centre Arche´- ologique du Var-Editions France. 9. Drancourt M, Raoult D (2005) Palaeomicrobiology: current issues and perspectives. Nat Rev Microbiol 3: 23–35. 10. Kolman CJ, Centurion-Lara A, Lukehart SA, Owsley DW, Tuross N (1999) Identification of Treponema pallidum subspecies pallidum in a 200-year-old skeletal specimen. J Infect Dis 180: 2060–2063. 11. Montiel R, Garc´ıa C, Ca n˜adas MP, Isidro A, Guijo JM, et al. (2003) DNA sequences of Mycobacterium leprae recovered from ancient bones. FEMS Microbiol Lett 226: 413–414. 12. Donoghue HD, Spigelman M, Greenblatt CL, Lev-Maor G, Bar-Gal GK, et al. (2004) Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA. Lancet Infect Dis 4: 584–592. 13. Malgosa A, Montiel R, D´ıaz N, Solo´rzano E, Smerling A, et al. (2005) Ancient DNA: A modern look at the infections of the past. In: Pandalai SG, ed. Recent Research Developments in Microbiology Vol. 9, Part II. Kerala: Research Signpost. pp 213–236. 14. Hershkovitz I, Donoghue HD, Minnikin DE, Besra GS, Lee OY, et al. (2008) Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean. PLoS One 3(10): e3426. 15. Bouwman AS, Brown TA (2005) The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis. J Archaeol Sci 32: 703–713. The Molecular Study of Ancient Syphilis restriction site used to analyze amplifications at the Universitat Auto` noma de Barcelona (Spain). (JPG) Figure S2 59UTR fragment of the 15-kDa lipoprotein gene of Treponema pallidum subspecies pallidum, aligned with clone sequences from samples ELS551 and ELS558, obtained at the University of the Azores (Portugal). A box shows the Eco47III restriction site, specific to this subspecies. (JPG) Figure S3 59UTR fragment of the 15-kDa lipoprotein gene of Treponema pallidum subspecies pallidum, aligned with clone sequences from samples ELS551, ELS558, and ELS944, obtained at Langebio, CINVES- TAV-IPN (Mexico). A box shows the Eco47III restriction site, specific to this subspecies. (JPG) Author Contributions Conceived and designed the experiments: RM AM. Performed the experiments: RM ES ND BAA-S MG-R MPC AI. Analyzed the data: RM. Contributed reagents/materials/analysis tools: RM NS AM. Wrote the paper: RM AM. 16. Barnes I, Thomas MG (2006) Evaluating bacterial pathogen DNA preservation in museum osteological collections. Proc Biol Sci 273: 645–653. 17. von Hunnius TE, Yang D, Eng B, Waye JS, Saunders SR (2007) Digging deeper into the limits of ancient DNA research on syphilis. J Archaeol Sci 34: 2091–2100. 18. Ho EL, Lukehart SA (2011) Syphilis: using modern approaches to understand an old disease. J Clin Invest 121: 4584–4592. 19. Centurion-Lara A, Molini BJ, Godornes C, Sun E, Hevner K, et al. (2006) Molecular differentiation of Treponema pallidum subspecies. J Clin Microbiol 44: 3377–3380. 20. Centurion-Lara A, Castro C, Castillo R, Shaffer JM, Van Voorhis WC, et al. (1998) The flanking region sequences of the 15-kDa lipoprotein gene differentiate pathogenic treponemes. J Infect Dis 177: 1036–1040. 21. Peng RR, Wang AL, Li J, Tucker JD, Yin YP, et al. (2011) Molecular typing of Treponema pallidum: A systematic review and meta-analysis. PLoS Negl Trop Dis 5: e1273. 22. Garc´ıa C, Ferna´ndez J (1999) The Hermitage of La Soledad through archaeology. In: Garc´ıa C, Ferna´ndez J, eds. La ermita de La Soledad a trave´s de la arqueolog´ıa. Huelva: Diputacio´n provincial de Huelva. pp 57–92. 23. Marangoni A, Moroni A, Tridapalli E, Capretti MG, Farneti G, et al. (2008) Antenatal syphilis serology in pregnant women and follow-up of their infants in northern Italy. Clin Microbiol Infect 14: 1065–1068. 24. Armangil D, Canpolat FE, Yig˘it S, Demir HA, Ceyhan M (2009) Early congenital syphilis with isolated bone involvement: a case report. Turk J Pediatr 51: 169–171. 25. Shen HJ, Sun MY, Liu AM (2009) [Clinical features of 121 infants below 3 months of age with congenital syphilis]. Zhonghua Er Ke Za Zhi 47: 871–873. [In Chinese]. 26. Jaffe HL (1972) Metabolic, degenerative, and inflammatory diseases of bones and joints. Philadelphia: Lea and Febiger. 1101 p. 27. Bauer WH (1944) Tooth buds and jaws in patients with congenital syphilis: Correlation between distribution of Treponema pallidum and tissue reaction. Am J Pathol 20: 297–319. 28. Je GC, Chang YL, Suk KL (1979) [Tooth Germ Changes in Congenital Syphilis]. The Seoul Journal of Medicine 20: 297–306. [In Korean]. 29. Bauer MF, Caravati CM, Jr. (1967) Osteolytic lesions in early syphilis. Br J Vener Dis 43: 175–177. 30. Dismukes WE, Delgado DG, Mallernee SV, Myers TC (1976) Destructive bone disease in early syphilis. JAMA 236: 2646–2648. 31. Hansen K, Hvid-Jacobsen K, Lindewald H, Sørensen PS, Weismann K (1984) Bone lesions in early syphilis detected by bone scintigraphy. Br J Vener Dis 60: 265–268. 32. Safont S, Isidro A, Malgosa A, Subira` ME (1996) Paleopatolog´ıa perinatal en los enterramientos de la Ermita de la Soledad (Huelva). In: Pe´rez-Pe´rez A, ed. Salud, enfermedad y Muerte en el Pasado. Barcelona: Fundacio´ Uriach 1838. pp 41–47. 33. Alduc-Le Bagousse A (1988) Estimation de l’aˆge des non-adultes: maturation dentaire et croissance osseuse. Donne´es comparatives pour deux ne´cropoles PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36371