Contributions to Zoology, 77 (1) – 2008
Ancient DNA analysis indicates the first English lions originated from North Africa
Ross Barnett1, Nobuyuki Yamaguchi2, Beth Shapiro1, Richard Sabin3
Keywords: Panthera leo, Barbary lion, medieval, Royal Menagerie, Tower of London
The Royal Menagerie of England was established at the Tower of London in the 13th Century and served as a home of exotic animals until it was closed on behalf of the Duke of Wellington in 1835. Two well-preserved lion skulls recovered from the moat of the Tower of London were recently radiocarbon-dated to AD 1280-1385 and AD 1420-1480, making them the earliest confirmed lion remains in the British Isles since the extinction of the Pleistocene cave lion. Using ancient DNA techniques and cranio-morphometric analysis, we identify the source of these first English lions to lie in North Africa, where no natural lion population remains today.
The lion Panthera leo (Linnaeus, 1758) is a charismatic large cat that has been imported into Europe since early historic times. Lions were amongst the many exotic animals that were imported to Rome during the early Imperial Period for the gladiatorial games, although towards the end of the Roman Empire the increased scarcity of these animals in the wild forced combat shows to be largely replaced by exhibitions (Baratay and Hardouin-Fugier, 2002). Exotic wild animals do not appear to have been kept regularly in western Europe until the 13th century, when they were rediscovered through Western contact with the Byzantine and Muslim worlds (Baratay and Hardouin-Fugier, 2002). In England, the Royal Menagerie was established in 12th-13th centuries in Wood-stock near Oxford, and slightly later was relocated to the Tower of London, where the first residents were three leopards sent to Henry III by the Holy Roman Emperor Frederick II in 1235 (Hahn, 2003).
Although the Royal Menagerie and its animals are known from documentary records, few physical remains survive (O’Regan et al., 2005). Amongst the rare exceptions are two lion skulls that were recovered from the moat of the Tower of London during excavations in 1936-1937. These skulls were recently radiocarbon-dated to AD1280-1385 and AD1420-1480, making them the earliest confirmed lion remains in the British Isles since the extinction of the Pleistocene cave lion (P. l. spelaea) (O’Regan et al., 2005). The discovery of these first English lions attracted significant media attention (BBC, 2005). However, the geographical origin of these animals has not yet been investigated. Such knowledge would provide novel insights not only into the history of the Royal Menagerie, but also into patterns of animal trafficking during the Medieval period. Direct animal trade between Europe and sub-Saharan Africa was not well developed until the 18th century (Anonymus, 1876). Therefore, it may be reasonable to presume that the Tower lions were unlikely to have originated from sub-Saharan regions. Nevertheless, there is an undeniable possibility that sub-Saharan lions reached Europe as they could have reached shipping ports in North Africa and the Middle East through trans-Saharan trade routes that were well established by the early Medieval period (Yamaguchi, 2000b). Apart from a tiny population in northwest India, lions had been practically exterminated outside sub-Saharan Africa by the turn of the 20th century (Yamaguchi and Haddane, 2002; Patterson, 2004). In this context, if the foregoing first hypothesis turned out to be the case, the Tower lion skulls would possess significant value for the history of the lion, as well as the history of Medieval England.
Recent advances in ancient DNA (aDNA) techniques (e.g. Shapiro et al., 2004), in association with the available data concerning genetic profiles of the lion across its natural range (Dubach et al., 2005; Barnett et al., 2006a, 2006b) have made it possible to identify the origins of unprovenanced lion specimens, such as the Tower lions (Barnett et al., 2007).
In this paper we use aDNA techniques to extract and amplify mitochondrial DNA (mtDNA) from the two Tower lions, and compare the results with those of Barnett et al. (2006a, 2006b). We also conduct a cranio-morphometric analysis to investigate which lion population the Tower lions are morphologically similar to. Then, by combining both molecular and morphological results, we will try to determine the geographic origin of the first lions in England.
Materials and methods
Sampling and laboratory procedures
Small pieces of cortical bone (c. 5 × 5 × 2 mm) were sampled from the mandibles of the two Tower lion skulls (registration numbers NHM19220.127.116.11 and NHM1918.104.22.168) at the Natural History Museum, London, UK. Laboratory procedures were carried out as described in Barnett et al. (2006a) at the Henry Wellcome Ancient Biomolecules Centre (ABC), Oxford University, which is geographically isolated from modern molecular biology work and DNA amplification by polymerase chain reaction (PCR).
Data authenticity and analysis
Extraction of specimens NHM1922.214.171.124 and NHM 19126.96.36.199 took place in the ABC and was performed along with negative extraction controls. PCR amplification of a small hypervariable fragment of the mitochondrial control region was performed twice for each sample, each time incorporating negative amplification controls. The four resulting amplification products were then cloned using the TOPO TA system (Invitrogen, Carlsbad CA USA), sequenced on ABI377 automated sequencers (Perkin-Elmer, Wellesley MA USA), and aligned with previously published lion sequences (Barnett et al., 2006a, 2006b). A summary of the cloning results is presented in the Appendix. A total of 12 clones were sequenced from sample NHM 19188.8.131.52, and 10 from NHM1952. 10.20.16. Of these, only three sequences show evidence of postmortem DNA damage (E4, E6, and F5 in Table S1) and, in each instance, the damage occurs at nucleotide sites that are not known to be polymorphic in lions. A median-joining network was constructed from the resulting sequences using Network v184.108.40.206 (Bandelt et al., 1999).
To investigate the origins of the Tower lions independently of the molecular results, morphological investigation was undertaken using Asiatic and North African Barbary lion skulls kept in natural history collections in the UK and Europe. A skull was classified as subadult if cemento-enamel junctions of all canines were already visible above the alveoli of the cleaned skull and yet the basioccipital-basisphenoid suture, and/or frontal suture, was still open. If those sutures were closed, a skull was classified adult. Seventy five craniometric measurements were taken of the cranium and mandible, modified from Yamaguchi et al. (2004), using a metal caliper to the nearest 0.02 mm, except for those of 10 larger variables that were measured to the nearest 0.05 mm using a larger metal calliper (for details see Appendix). To test the measurement errors, five skulls were randomly selected and each measurement was taken three times on each skull. The coefficient of variation for each of the 75 variables was calculated, and the variables with average coefficient of variations of more than 2% were excluded from the analysis by accepting the arbitrary cut-off line for reliability and consistency in measurements used in Yamaguchi et al. (2004) (see Appendix). We have measured all Asiatic and North African Barbary lion skulls kept, and available for measurement, in major natural history collections in the UK and Europe. However, as not every skull was intact, some variables were excluded from the analysis for maximising both Asiatic and North African Barbary lion specimens to be included into the analysis whilst retaining as many variables as possible. We retained 57 variables with 23 individuals (Table 1).
Statistics for morphological analysis
All statistical analyses were carried out using the SPSS statistics package (version 13: SPSS Inc., Chicago, USA). A principal component analysis (PCA) was carried out to reduce the numbers of variables for the subsequent analyses, which were based on extracted principal components whose eigenvalues were larger than 1 (Tabachnick and Fidell, 2007). Then, a discriminant analyses (DA) was carried out to investigate if Asiatic and North African Barbary lion skulls could be distinguished, with the prior probabilities computed from the group sizes. A DA is designed to develop classification functions to classify each specimen best by following a priori groupings, so that it will usually result in a fairly good discrimination between the groups (Tabachnick and Fidell, 2007). Therefore, a cross-validation test was also carried out to check which group each case would be classified into if it was classified by the functions derived from all cases other than itself. Then, we tested if the Tower lions would be classified as either Asiatic or North African Barbary, or both.
Table 1. Lion skulls used for the morphological analysis. Museums are Natural History Museum London, Muséum National d’Histoire Naturelle Paris, Museum für Naturkunde der Humboldt-Universität, Berlin, Natural History Museum, University of Oxford, For-schungsinstitut und Naturmuseum Senckenberg, Frankfurt, Musée Zoologique, Strasbourg, and female is indicated by (f), male (m), adult (a), and subadult (sa). Individual ID numbers (e.g. P1, L1 or T1) are corresponding to those in Fig 2.
In addition to sexual size dimorphism that is common in the Felidae, it has been suggested that the skull morphological characteristics of captive lions differ from those of wild animals (Hollister, 1917). While a preliminary morphometric analysis suggested that both Tower lions were males based on the greatest length of skull and canine size (Gittleman and Van Valkenburgh, 1997), specimens did not have their sex recorded. Additionally, the specimens had spent at least some time in captivity. We therefore included into the analysis both male and female, and both captive and wild, individuals in our comparative data set (see Table 1). We deliberately did so to find out if a discriminant analysis (DA) would be able to separate the North African Barbary lion from the Asiatic lion regardless of sex and whether an animal was captive or wild.
Table 2. Classification results obtained by a discriminant analysis. The only one misclassified case in the cross-validation test was P6 in Table 1.
Approximately 130bp of the mtDNA control region (HVR1) was amplified from both samples. Although the Felidae are known to contain macrosatellites (numts) resulting from nuclear translocation of the mtDNA (Cracraft et al., 1998; Lopez et al., 1997), visual comparison of the sequences obtained from the Tower lions with previously published numts and lion mitochondrial sequences clearly indicated that the Tower lion sequences were indeed mitochondrial (for details see Barnett et al., 2006a, 2006b). Median-joining network analysis clarified that both consensus clone sequences were identical to the unique haplotype of the North African Barbary lion out of the 11 distinct haplotypes previously identified in the lion (Barnett et al., 2007). The sequences obtained from the Tower lions were also distinguishable from two haplotypes that characterised lions from India and Iran, which were the next most closely related haplotypes (Barnett et al., 2006a, 2006b).
A PCA based on the retained 57 variables resulted in seven principal components. Then, a DA based on those seven principal components extracted one canonical discriminant function, which was used in the analysis. The DA successfully separated Asiatic and North African Barbary lions, and the cross-validation test classified most (20 out of 21) specimens into the right groups (Table 2). The two Tower lions were both classified as the North African Barbary lion (Table 2 and Fig. 1).
Fig. 1. Discriminant function scores of the specimens analysed. Individual ID numbers are corresponding to those in Table 1. Note that the discriminant function was extracted to distinguish lions from Asia and North Africa, and not to separate sexes. Therefore, although both the Tower lions place themselves amongst female Barbary lions, this does not indicate that they are females.
Our results demonstrate that the two Tower lions share a unique mtDNA haplotype with the North African Barbary lion. Whilst written records have previously suggested that most lions brought into Medieval Europe originated from the region between North Africa and India (Anonymus, 1769; Baratay and Hardouin-Fugier, 2002), such records have not been able to distinguish lions which actually originated from North Africa and lions that had been brought from sub-Saharan Africa and then shipped to Europe from North African ports. Our results are the first genetic evidence to clearly confirm that the former was the case.
Previous work has shown that lions inhabiting the extensive stretch of land between northwest Africa and India are very closely related: characterised by a simple mtDNA haplotype structure, in particular in comparison to the ancestral population in sub-Saharan Africa, with the distance between the former and the latter being three to seven substitutions whilst only less than two within the former (Barnett et al., 2006a). Amongst the North African - Asian lion populations, our results (both genetic and morphological) suggest that the Tower lions belong to the North African Barbary lion in comparison to those originating from India and Iran.
Fig. 2. Map showing approximate sampling sites of the lion specimens analysed in Barnett et al. (2006a, 2006b), with mtDNA haplotypes (see Table 1). If the exact sampling location is not known, a dashed circle and dashed line are used. Two Tower lions possess the haplotype-11 that is unique to, and invariable within, the North African Barbary lion.
We expected that the skull morphometric analysis would not clearly separate North African Barbary lions from Asiatic lions if we included all the skulls measured into the analysis. This was because the well-known sexual dimorphism (e.g. Gittleman and Van Valkenburgh, 1997) and morphological difference between wild and captive individuals (Hollister, 1917) might make a clear-cut separation of the two geographic populations unlikely. Nevertheless, the discriminant analysis (DA) succeeded in doing so, suggesting that those two geographic populations would be distinguished from each other regardless of sex and whether an animal was wild or captive. We have not overlooked that the two Tower lions, which are likely to be males, appear amongst female Barbary lions in Figure 1. However, the DA was carried out to investigate if North African Barbary lions could be separated from Asiatic lions, and vice versa, and therefore, the analysis was not optimised for separating sexes.
Unfortunately, in spite of our extensive search across major natural history collections in Europe, Russia, Central Asia, and North America, no lion sample has been identified from the region between Libya and Iraq, from which lions totally disappeared by the mid 20th century (Nowell and Jackson, 1996; Patterson, 2004) although lions originated from that region were apparently imported into Europe during the 19th century (Edwards, 1996; Jardine, 1834). This makes it impossible to determine either mtDNA haplotype(s) or quantitative morphological characteristics that were present in this region, and potentially complicating the identification of the geographic origin of the Tower lions.
Historic records suggest that a single, contiguous population of lions existed from North Africa through the Middle East to India until the growth of civilisations along the Egyptian Nile and Sinai Peninsula (the narrow connection between Africa and Eurasia) some 4,000 years ago. The development of a human-dominated, narrow, land bridge effectively severed gene flow between North Africa and Near East Asia, which are also separated by more than 4,000 km of arid land (Nowell and Jackson, 1996; Yamaguchi, 2000a), and isolated lion populations to the west and east of this barrier. In the Near East, lions survived in southeastern Turkey until 1870, northern Syria (up until c. 1890s) and Iraq (the last record was in 1918) (Nowell and Jackson, 1996; Patterson, 2004). As we mentioned above, no genetic/quantitative morphological information is known for these eastern lions because no provenanced specimen is known to exist for investigation. However, their geographic proximity to lions in Iran (Nowell and Jackson, 1996), and their isolation from lion populations further west, may suggest that they are likely to belong to the Iranian haplotype (as well as Iranian/Indian morphological group), rather than those of the North African Barbary, and were not the source of the Tower lions.
Arid conditions in the eastern part of North Africa (Libya and Egypt) limited the size of the lion population in this region, even prior to the early 18th century, when lions finally disappeared from the eastern North African Mediterranean littoral zone (Nowell and Jackson, 1996; Yamaguchi and Haddane, 2002). Western North Africa (Morocco, Algeria and Tunisia), on the other hand, supported a relatively large lion population until fairy recently, and was one of the most important regions to supply lions to Europe between the 17th and first half of the 19th centuries (Anonymus, 1769; Yamaguchi and Haddane, 2002). Furthermore, western North Africa was the nearest region to Europe to sustain lion populations until the early 20th century, making it an obvious and practical source for medieval merchants. The foregoing argument may suggest that, while further evidence will be required to draw a firm conclusion, the first English lions originated in North Africa - probably North-west Africa.
We thank Paula Jenkins and Daphne Hills for providing access to samples. RB was supported by NERC and BS was supported by The Wellcome Trust. NY was supported by the European Union SYNTHESYS programme to visit Muséum National d’Histoire Naturelle, Paris, France, and Museum für Naturkunde der Humboldt-Universität, Berlin, Germany for skull measurements. We also thank Paula Jenkins, Daphne Hills, Louise Tomsett at Natural History Museum, London, Malgosia Nowak-Kemp at Natural History Museum, University of Oxford, Oxford, UK, Robert Asher, Peter Giere, Irene Thomas, Wolf-Die-ter Heinrich at Museum für Naturkunde der Humboldt-Univer-sität, Berlin, Katrin Krohmann, Thomas Martin at Forschungs-institut und Naturmuseum Senckenberg, Frankfurt, Germany, Jacques Cuisin, Francis Renoud, Daniel Robineau, Michel Tranier at Muséum National d’Histoire Naturelle, Paris, Marie-Dominique Wandhammer, Virginia Rakotondrahaja at Musée Zoologique, Strasbourg, France, for their kind support during NY’s visit to measure lion skulls. Two anonymous reviewers have contributed considerably with valuable comments, for which we are most thankful.
Received: 12 July 2007
Accepted: 6 December 2007
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List of craniometric parameters measured
All measurements are taken from the left side of the skull wherever possible. Numbers and letters in the square brackets are corresponding to those in the Figure. Measurement errors are indicated by coefficient of variation (%: Mean ± Standard Error: N = 5, 3 repeats each) in the round brackets.
Definitions for the points and measurements in the Figure
Definitions of general terms are as follows.
Vertical and Horizontal
These can be defined when a cranium or a mandible(s) is placed in the way shown in the Figure. For the mandible(s), simply place it on a horizontal surface. For the cranium, adjust the cranium to make the surface including the posterior ends of alveoli of both canines and both Pm4s horizontal.
Definitions for points
Table S1. A total of 12 clones were sequenced from sample NHM19220.127.116.11, and 10 from NHM1918.104.22.168, and compared to those of Barbary, Indian, and Iranian lions. Only three sequences show evidence of postmortem DNA damage (E4, E6, and F5) and in each instance the damage occurs at nucleotide sites that are not polymorphic in lions.
Definition for measurements