bs_bs_banner Archaeometry 54, 5 (2012) 916–925 doi: 10.1111/j.1475-4754.2012.00666.x TRUTH IN THE BONES: RESOLVING THE IDENTITY OF THE FOUNDING ELITE THOROUGHBRED RACEHORSES* M. A. BOWER,1† M. G. CAMPANA,2 R. E. R. NISBET,3 R. WELLER,4 M. WHITTEN,5‡ C. J. EDWARDS,6§ F. STOCK,6 E. BARRETT,1 T. C. O’CONNELL,2 E. W. HILL,7 A. M. WILSON,8 C. J. HOWE,9 G. BARKER1 and M. BINNS10 1McDonald Institute for Archaeological Research, University of Cambridge, Downing Street, Cambridge CB2 3ER, UK 2Department of Archaeology, University of Cambridge, Downing Street, Cambridge CB2 3DZ, UK 3Sansom Institute for Health Research, University of South Australia, GPO Box 2471, Adelaide, SA 5001, Australia 4Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Lane, Hatfield, Herts AL9 7TA, UK 5Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK 6Smurfit Institute of Genetics, School of Genetics and Microbiology, Trinity College, Dublin 2, Ireland 7School of Agriculture, Food, Science & Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland 8Structure and Motion Laboratory, Royal Veterinary College, Hawkshead Lane, Hatfield, Herts AL9 7TA, UK 9Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK 10Equine Analysis Systems Inc., 668 Lansing Lane, Midway, Lexington, KY 40347, USA Our multidisciplinary study of historic Thoroughbred horses solves two long-running myster- ies in racing history. Eclipse, the greatest racehorse ever known, never lost a race. His skeleton is housed in the Royal Veterinary College, London; however, there is controversy over its authenticity. The 1880 Epsom Derby was won by Bend Or. In one of the great controversies of Thoroughbred racing, the owners of Bend Or were accused of swapping him with another horse, Tadcaster, whose maternal pedigree was more prestigious. Bend Or’s skeleton resides at the Natural History Museum, in London. Eclipse and Tadcaster were both extremely popular at stud, and the vast majority of racehorses today are descendents. We compared mitochondrial DNA haplotypes of living and historic Thoroughbred skeletons, including those of Eclipse and Bend Or. Additionally, we compared skeletal morphometrics of Eclipse’s skeleton with measurements taken at autopsy. Carbon and nitrogen stable isotopes of a range of skeletal elements were compared in order to establish that the Eclipse skeleton was that of a single horse. Our multidisciplinary data suggest that the putative skeleton is consistent with that of Eclipse. In contrast, mitochondrial DNA haplotype sharing indicated that the skeleton known as Bend Or is most probably that of Tadcaster. KEYWORDS: ANCIENT DNA, MITOCHONDRIAL DNA, HORSE EVOLUTION, THOROUGHBRED HORSES, MUSEOLOGY, MULTIDISCIPLINARY INTRODUCTION Eclipse was the greatest racehorse that ever lived; he never lost a race. Foaled during the solar eclipse of 1764, Eclipse sired over 350 foals and is an ancestor of 95% of all living racehorses. After his death in 1789, Vial de Saint Bel (founder of the Royal Veterinary College, London), *Received 23 September 2011; accepted 21 November 2011 †Corresponding author: email [email protected] ‡Current address: Max Planck Research Group on Comparative Population Linguistics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. §Current address: Research Laboratory for Archaeology & the History of Art, University of Oxford, Dyson Perrins Building, South Parks Road, Oxford OX1 3QY, UK. © University of Oxford, 2012 Resolving the identity of the founding elite Thoroughbred racehorses 917 performed an autopsy to determine why Eclipse was so fast (Vial de Saint Bel 1797). Although the skeleton (Fig. 1 (a)) is now kept at the Royal Veterinary College, rumours that it is not in fact the skeleton of Eclipse have circulated for many years (Clee 2009). At one time there were several skeletons that were claimed to be that of Eclipse, and at least seven purported hooves are currently in existence. One report states that Eclipse’s skeleton became part of the Royal Veterinary College’s teaching collection and was stored for some time in an attic, in a box with other disarticulated horse bones (Lanyon 1990). Bend Or was foaled at the Eaton stud in 1877 (Fig. 1 (b)). In 1880, ridden by the celebrated jockey Fred Archer, he won the Epsom Derby, the most prestigious of Great Britain’s Classic horse races, in a dramatic head-to-head finish (Fig. 1 (c)) (Times 1880a). The owners of Robert the Devil, the losing horse, raised a formal objection, claiming that Bend Or was actually a horse called Tadcaster (Times 1880b; Prior 1924), another chestnut colt, also owned by the Duke of Westminster. Bend Or (whose dam, or mother, was Rouge Rose) and Tadcaster (whose dam was Clemence) were both sired by the stallion Doncaster. A witness stated that a mix-up had occurred when the horses were transferred as yearlings from stud farm to trainer. The appeal failed. Because of his win, the horse believed to be Bend Or was popular at stud and sired many important winners. After the death of the horse, the skeleton was left to the Natural History Museum, London. The General stud book of England (GSB) was established in 1791 to record Thoroughbred horse pedigrees (Weatherby 1791), and to prevent confusion over horse identity and breeding. Figure 1 Eclipse and Bend Or. (a) The skeleton of Eclipse at the Royal Veterinary College, London. (b) Bend Or at stud. (c) The 1880 Epsom Derby finish, as Bend Or beats Robert the Devil ‘by a nose’ (Hulton Archive/Getty Images). (d) A painting of Eclipse by John Beer (by kind permission of the National Horseracing Museum, Newmarket). © University of Oxford, 2012, Archaeometry 54, 5 (2012) 916–925 918 M. A. Bower et al. In an alternative classification, developed by Bruce Lowe (1913) to correlate racing excellence with maternal lineages, horses have been assigned ‘Family Numbers’ based on their maternal lineage to one of the original female founders of the Thoroughbred breed. ‘Family Number’ (Lowe 1913) was defined according to the racing performance in English Classic races of the progeny of the maternal-line matriarchs (i.e., Family 1 has the greatest number of race winners tracing in maternal pedigree to a single female ancestor). As mitochondrial DNA (mtDNA) is maternally inherited, it can be used for tracing maternal bloodlines in Thoroughbreds (Hill et al. 2002; Harrison and Turrion-Gomez 2006). Thoroughbred horses from a single maternal lineage ought to share the same mitochondrial sequence. An analysis of present-day racehorses shows that this is not always the case. In a study of 281 Thoroughbred racehorses, maternal lineages were shown to contain two or more mitochondrial genotypes (Bower et al. submitted). The primary reasons for these discrepancies are historic errors in the General stud book, introduced early in the breed history, most of which can be traced to the foundation of mater- nal sub-lineages (Bower et al. submitted). Thus, maternal sub-lineage and genetic lineage cor- respond closely. In order to resolve the identities of these influential founders of the Thoroughbred racehorse, we undertook a multidisciplinary study of the skeletons of Eclipse and Bend Or. The results presented here represent the first time that ancient DNA, stable isotope, morphometric and pedigree analysis have been used together to solve a historic mystery. MATERIALS AND METHODS Analysis of DNA Ancient DNA was extracted and analysed following previously published protocols (McGahern et al. 2006). All appropriate ancient DNA authentication criteria were followed (Cooper and Poinar 2000), including the use of multiple controls (Spencer and Howe 2004) and replication of results in an independent institution. Amplification products were assessed for DNA damage and enzyme error by bacterial cloning (Bower et al. 2005). Sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/): HM581890–HM581905. Table 1 lists the historic Thoroughbreds analysed in this study. For the modern comparative horse samples, whole- genomic DNA was isolated from pulled hairs according to Allen et al. (1998) and from blood samples using a Nucleon DNA Extraction Kit (Amersham Biosciences), following the manufacturer’s instructions. Mitochondrial control region sequences from the historic and modern horse extracts were amplified by PCR as previously published (McGahern et al. 2006). Amplification of 743 bp of the equine mitochondrial D-loop was performed by PCR. Reactions were performed in 12.5 ml consisting of 1 ml DNA extract, 1 ¥ HiSpec additive (Bioline UK), 1 ¥ PCR buffer (Bioline UK), 2.41 mM MgCl2,25mM each of dNTP and 0.25 U Immolase hot start DNA Polymerase (Bioline UK). 0.4 mM of each primer was used. Forward Primer: 5′-ACCCTGGTCTTGTAAACCAG-3′, Reverse Primer: 5′-TGGTTGCTGATGCGGA-3′. Thermocycling conditions were as follows: initial activation at 94°C for 10 min was followed by 39 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 1 min. Sequencing reactions were carried out in both directions using the Big Dye® Terminator v3.1 cycle sequencing kit and internal sequencing primer 5′-GTTATGTGTGAGCATGGGC-3′. Sequencing products were analysed on an ABI 3100 Genetic Analyzer and base calling was performed using ABI Prism® AB DNA Sequencing Analysis Software v. 5.1.1. Sequences were deposited in GenBank (EU580148–EU580172). © University of Oxford, 2012, Archaeometry 54, 5 (2012) 916–925 Resolving the identity of the founding elite Thoroughbred racehorses