Truth in the Bones: Resolving the Identity of the Founding Elite Thoroughbred Racehorses*

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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).

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 maternal sub-lineages (Bower et al. submitted). Thus, maternal sub-lineage and genetic lineage correspond 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).

Table 1 The historic Thoroughbred horses sampled for this study

Historic Year of Year of Maternal lineage Element Sample Number of living Thoroughbred birth death (Lowe Family) analysed held by* maternal lineage relatives in comparative data set

Bend Or 1877 1903 Family 1 Mandible NHM 21 Corrie Roy 1878 c. 1900 Family 3 Metacarpus NHM 15 Donovan 1886 1905 Family 7 Humerus NHM 7 Eclipse 1764 1789 Family 12 Humerus, RVC 15 metacarpus, tibia, tooth Hermit 1864 1890 Family 5 Tooth BLA 6 Hyperion 1930 1960 Family 6 Tooth AHT 12 Ormonde 1883 1904 Family 2 Metacarpus NHM 18 Persimmon 1893 1908 Family 7 Mandible NHM 7 Polymelus 1902 1924 Family 3 Tooth UOC 15 St Frusquin 1893 1914 Family 22 Metacarpus NHM 12 St Simon 1881 1908 Family 11 Mandible NHM 13 Stockwell 1849 1871 Family 3 Metacarpus NHM 15 William the 1898 1917 Family 2 Metacarpus NHM 18 Third

*NHM, Natural History Museum, London; RVC, Royal Veterinary College, London; UOC, Zoological Museum, University of Cambridge; BLA, Blankley Stud, Lincolnshire; AHT, Animal Health Trust, Newmarket.

Sequences were aligned in Mega 4 (Kumar et al. 2004) using the Clustal-W algorithm (Higgins et al. 1994). A further 300 sequences from GenBank (AF481323–AF481305, EU580148–EU580172) were added to the alignment, for a total of 296 individual Thoroughbred horses. PhyML v. 2.4.4 for Linux (Guindon and Gascuel 2003) was used to estimate a maximum- likelihood tree, with an initial BIONJ tree (Gascuel 1997) using the HKY + gamma substitution model with four gamma categories. Transition/transversion ratios and the shape parameter for the gamma distribution were estimated from the data, both topology and edge lengths were opti- mized, and 1000 bootstrap replicates were run to estimate our uncertainty about the maximum- likelihood tree, given the methods used and the size of the data set (Felsenstein 1985).

Stable isotope Collagen was purified from bones using standard protocols (O’Connell and Hedges 1999). Bone powder was demineralized in 0.5 M HCl. The supernatant was discarded and the powder rinsed in water. The remaining collagen was gelatinized by heating in dilute HCl (pH 3.0) at 75°C for 48 h, and then lyophilized. Mass spectrometry was as follows: 1–4 mg was sampled, weighed precisely into tin capsules and analysed on a Costech automated elemental analyser coupled in continuous-flow mode to a Finnigan MAT253 isotope-ratio-monitoring mass spectrometer. Stable isotope concentrations were measured as the ratio of the heavier isotope to the lighter isotope relative to an internationally defined standard, Pee Dee Belemnite (VPDB) for carbon, and atmospheric nitrogen (AIR) for nitrogen. Isotopic results are reported as d values (d13C 15 13 13/12 13/12 and d N) in per mil (‰) values, where d CVPDB = [( Csample/ CVPDB)–1]¥ 1000‰ and 15 15/14 15/14 d NAIR = [( Nsample / NAIR)–1]¥ 1000‰. Based on replicate analyses of international and

© University of Oxford, 2012, Archaeometry 54, 5 (2012) 916–925 920 M. A. Bower et al. laboratory standards, measurement errors are less than 0.2‰ for d13C and d15N. The samples’ percentages of carbon and nitrogen by mass and the carbon/nitrogen ratios were also measured.

Morphometric analysis Measurements were taken using high-precision callipers. Skeletal measurements were normal- ized by scaling using the difference between the inch and Vial de Saint Bel’s inch (Vial de Saint Bel 1797; Butler 1814).

RESULTS AND DISCUSSION We obtained mitochondrial D-loop DNA from the bones of Eclipse and Bend Or. We proposed that if the putative skeleton of Eclipse has identical mtDNA to that of the real Eclipse’s dam’s female descendents (Family 12, n = 15), it is highly likely that the skeleton is indeed Eclipse. Likewise, if mtDNA from the skeleton claimed to be that of Bend Or matches the mitochondrial lineage of his dam Rouge Rose (Family 1, n = 21), then it is indeed that of Bend Or. Conversely, if the DNA matches the mitochondrial lineage of Clemence (Family 2, n = 18), the dam of Tadcaster, then the skeleton belongs to Tadcaster. Additionally, we obtained mitochondrial D-loop DNA from 10 further historic Thoroughbreds in order to test, with statistical robustness, whether historic Thoroughbreds could be accurately placed within their maternal pedigrees using mtDNA (Table 1). We compared these data with sequences obtained from 296 living Thorough- bred horses (Hill et al. 2002; Bower et al. submitted). A maximum-likelihood analysis was carried out in order to group living and historic Thoroughbreds and explore the relationships between maternal lineages. All of the historic horses, with the exception of Eclipse and Bend Or, gave rise to mtDNA sequences that conﬁrmed their recorded maternal lineage (Fig. 2). We undertook further analyses (morphometric, stable isotopic and pedigree analysis) to explore why this might be.

Eclipse Historical accounts record that the putative skeleton of Eclipse was stored in boxes with a number of other horse skeletons (Lanyon 1990). As extensive autopsy measurements of Eclipse are available (Vial de Saint Bel 1797), we made corresponding measurements from the proposed Eclipse skeleton. Comparison was only possible for measurements not influenced by mounting of the skeleton (i.e., the appendicular skeleton, including the scapula, humerus, radius, metacarpus, femur, tibia and metatarsus). Although digit lengths were also described at autopsy (Vial de Saint Bel 1797), the distal phalanges may be from another horse (Lanyon 1990) and thus were not included. Vial de Saint Bel recorded his autopsy measurements in a unit that he referred to as an inch. However, the inch was not standardized during the 18th and early 19th centuries (Butler 1814), and no definition is available of the length of Vial de Saint Bel’s inch. In addition, he rounded his measurements to the nearest inch and did not record whether the left or the right bone was measured. We measured the maximum length of each bone. Table 2 shows the autopsy measurements (as recorded) and the mean of the high-precision measurements obtained from the skeleton (left/right in inches). The average difference between Vial de Saint Bel’s measurements and the skeletal measurements was 13%, with a range of between 4 and 17%. Some discrepancy between skeletal and autopsy measurements is expected, since we do not know the precise anatomical landmarks on which Vial de Saint Bel based his measurements. Accurate identification of

Figure 2 Phylogenetic analysis. An unrooted maximum-likelihood tree of mitochondrial D-loop sequences from 12 historic Thoroughbred horses, including Eclipse and Bend Or and 296 living Thoroughbred horses. The HKY + gamma model of evolution was used, with 1000 bootstrap replicates.

Table 2 Eclipse skeletal measurements. Comparison of measurements taken by Vial de Saint Bel during autopsy (Vial de Saint Bel 1795) and the corresponding modern measurements of the putative skeleton of Eclipse. Note that the inch was not deﬁned as a precise measurement until the 19th century (Butler 1814)

Bone Vial de Saint Bel’s autopsy High-precision measurements Percentage measurements (inches) of skeleton (inches) difference 1 standard deviation

Scapula 18 15.71 13 1 3 Humerus 12 12.54 4 1 4 Radius 16 14.55 9 1 3 Metacarpus 12 10.02 17 1 4 Femur 15 17.48 17 1 4 Tibia 19 15.69 17 1 3 Metatarsus 14 11.95 15 1 4 Head Length of head 23.6 n/a Neck 1.5 ¥ length of head 1.4 ¥ length of head 7 Forehead 7/22 ¥ length of head 7.3/22 ¥ length of head 4

© University of Oxford, 2012, Archaeometry 54, 5 (2012) 916–925 922 M. A. Bower et al. anatomical landmarks introduces the biggest variation in conformational measurements of living horses (Weller et al. 2006b). However, taking into account error associated with conformation assessment and population variation in modern Thoroughbred racehorses (Weller et al. 2006a,b), and allowing for the variation of the length of Vial de Saint Bel’s inch, we conclude that the measurements of the proposed skeleton of Eclipse correspond closely to those of the horse that Vial de Saint Bel examined. We then analysed DNA isolated from four discrete anatomical features of the skeleton: one tooth (lower M3) and three bones—metacarpus, humerus and tibia. The mtDNA sequences from each sample were identical, suggesting that all originated from the skeleton of a single horse. To further confirm that skeletal elements pertained to a single individual, we measured the three bones’ carbon and nitrogen stable isotope ratios (O’Connell and Hedges 1999) (the tooth was excluded due to lack of material). The variation between the skeletal elements fell within the range expected from a single animal (Table 3) (Balasse et al. 1999). Previous SNaPshotTM profiling of all DNA isolates identified a chestnut horse, which is in agreement with the recorded phenotype (Campana et al. 2010). While these data indicate that the skeleton originated from a chestnut-coloured horse with the same proportions as Eclipse, mtDNA D-loop sequence data revealed that the skeleton belonged to maternal Family 20, and not to Family 12 as listed in the current (46th) edition of the General stud book. According to the current GSB, Eclipse’s dam was Spilletta. Spilletta’s dam, Mother Western, was a sister to the Whiteshirt mare, with their dam (Eclipse’s great-grandmother) being ‘Old Montagu mare’ (Fig. 3 (a)). An analysis of mtDNA from 15 living Thoroughbred horses designated as Family 12 revealed that all living horses descended from Mother Western (Eclipse’s maternal grandmother, n = 3) in the female line had an identical sequence to Eclipse and Family 20 (n = 9). Eclipse’s haplotype was also shared by Family 19 (n = 1) and Family 2 (n = 1), but since these were single individuals, they were excluded from the data analysis. Those descended from the Whiteshirt mare (n = 12) shared mtDNA sequence identity with each other and with Family 9 (n = 14) (Bower et al. submitted). We propose, therefore, that an error has been introduced to the GSB, at the time of the introduction of this pedigree in the fifth edition (1891) (as per Fig. 3 (a)). It has previously been suggested (Bobinski 1953) that two mares, one the progeny of the stallion Old Montagu and another owned by a Lord Montague (with a similar but different spelling), were confused and assumed to be the same horse. He concluded that Whiteshirt mare and Mother Western were not sisters. This is supported by our data. However, Bobinski (1953) places Lord Montague’s Mare in Family 68, while our results show that she should be in Family 20. We conclude from this that Lord Montague owned several mares, representing several maternal lineages. An 1840 pedigree of Eclipse (Whyte 1840) (Fig. 3 (b)) is at odds with both the

Table 3 Isotopic values for Eclipse based on puriﬁed collagen

Sample Amt% (C) d13C (‰) Amt% (N) d15N (‰) Atomic C/N ratio

Humerus 46.33 –22.74 14.91 5.98 3.63 Metacarpus 42.95 –22.66 14.10 6.07 3.55 Tibia 41.44 –22.20 14.59 5.74 3.31

The metacarpus and tibia measurements are the mean of three replicates, with a single humerus measurement due to lack of material. All values are within the expected range of variation for a single individual.

Figure 3 The ancestry of Eclipse. (a) According to the 1791 General stud book (Weatherby 1791). (b) According to Whyte (1840), whose record was published shortly after Eclipse’s death. (c) According to ancient DNA. Horses in red are mares and horses in blue are stallions (see online for a colour version of this ﬁgure).

GSB and Bobinski, and supports our ﬁndings. Thus, Family 12 should comprise two independent maternal lineages; one descending from the Montagu mare and the second descending from an unknown Family 20 mare from Lord Montague’s stable (Fig. 3 (c)). As only 3% of horses fall into the Family 20 group (9/296), it seems unlikely that a different, random horse of the same size and coat colour as Eclipse would also be in the Royal Veterinary College collection. We therefore suggest that the putative skeleton of Eclipse is the authentic skeleton, and that a revision of the GSB pedigree record for Eclipse is required.

Bend Or If the skeleton of the 1880 Derby winner, claimed to be Bend Or, is indeed Bend Or, the mtDNA sequence should be concordant with Family 1. If, however, the skeleton belongs to Tadcaster, then the mtDNA sequence should be concordant with Family 2. Families 1 and 2 are separated by eight nucleotide substitutions. The mtDNA sequences from the skeleton named Bend Or group clearly with those of Family 2 (n = 18) and not Family 1 (n = 21); therefore, the skeleton is that of Tadcaster. It would thus appear that the sacked groom was correct: two horses were switched during the transfer of horses from the Duke’s stud to his trainer, and Tadcaster won the 1880 Epsom Derby, not Bend Or. Therefore, all the descendents of Bend Or, through which the Darley Arabian line is transmitted to present-day Thoroughbreds, must now be assigned to Tadcaster.

Bend Or had close to 200 offspring in his lifetime and his descendents form a signiﬁcant part of the living Thoroughbred gene pool, including the famous Thoroughbreds Red Rum (1965), Shergar (1978) and Phar Lap (1926).

CONCLUSION Our multidisciplinary approach, including ancient DNA, stable isotope, morphometric and pedigree analysis, has enabled conﬁrmation of the authenticity of the skeleton of the celebrated racehorse Eclipse and has solved the mystery surrounding the winner of the 1880 Epsom Derby.

ACKNOWLEDGEMENTS The authors thank M. K. Jones, the Glyn Daniel Laboratory (University of Cambridge) and M. Spencer (University of Liverpool). The Natural History Museum, London, the Department of Zoology, University of Cambridge and the National Horseracing Museum, Newmarket, provided samples. The National Institute for Agricultural Botany and the Godwin Laboratory (Department of Geography, University of Cambridge) provided facilities. The Horserace Betting Levy Board, the McDonald Institute for Archaeological Research, the Isaac Newton Trust and the Leverhulme Trust funded this research. MGC is supported by an Overseas Research Studentship, the Cam- bridge Overseas Trust and Peterhouse College, Cambridge. AW holds a Royal Society Wolfson Research Merit Award. Reproduction of Figures 1 (b) and 1 (d) are by kind permission of the National Horseracing Museum, Newmarket.

AUTHOR CONTRIBUTIONS Conceived and designed the experiments: MAB, MGC, MB, AMW and GB. Performed the experiments: MGC, MW, RW, CJE and FS. Analysed the data: MAB, RERN, MGC, RW and EB. Wrote the paper: MAB, RERN, CJH and MGC. Provided data: EWH and EB. All authors read, edited and approved the manuscript. Sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/): HM581890– HM581905. The authors declare no competing ﬁnancial interests. Correspondence and requests for materials should be addressed to [email protected].

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