Supplementary InformationMethods and Discussion
Morphological analysis
Samples
C. elaphus: Late Pleistocene fossil material from Britain and Germany (Lister, 1981, 1984, 1986), and modern material including representatives of a range of modern subspecies (Natural History Museum, London).
D. dama:Modern andLate Pleistocene fossil material from Britain (Lister, 1981, 1984, 1986; Natural History Museum, London).
D. mesopotamica: Modern material (Natural History Museum, London & Hebrew University, Jerusalem); Late Pleistocene material from Tabûn, Israel (Natural History Museum, London).
M. giganteus: Fossil material from the Irish late-glacial, and from Middle to Late Pleistocene interglacials of Britain and Germany (Lister, 1994).
Cervus eldi, C. canadensis, C.nippon, Axis axis and A.xis porcinus, living (Natural History Museum, London; American Museum of Natural History, New York).
Muntiacus: representatives of the range of living species and subspecies (Natural History Museum, London).
Details of specimens in text Fig. 2:
B: left, Cervus canadensis, modern, AMNH 123050; ……right, M. giganteus, Allerød, Ireland, NHM M18078. …..left, modern Cervus elaphus, Chapman Collection, no. 94; right, modern Dama dama, Chapman Collection no. 41.
C: left, modern Cervus elaphus, modern, Chapman Collection, no. 94NHM 1866.8.6.12; right, Megaloceros giganteus, Allerod, Ireland, National Museum of Ireland, DublinNHM 14171.
D: left, Cervus elaphus, Middle Pleistocene, West Runton, NHM M17508; right, Dama dama, Middle Pleistocene, Swanscombe Lower Gravel, Harrison Museum (photo reversed).
E: left, Cervus elaphus, Late Pleistocene, Torquay Museum (no number)Middle Pleistocene, Grays, NHM M22034; right, Dama sp., West Runton, HarrisM. giganteus, Late Pleistocene, Kent’s Cavern, NHM M14242. …………on Museum 30.21069.
Character coding
It is typical of cervid dental and skeletal morphology that characters show considerable intra-specific variability (Lister 1996, Pfeiffer 1999). To take account of this, every effort was made to score as many individuals of each species as possible on each character (Table S1). Variation was quantified as described by Lister (1996). Almost all characters were defined with two alternative character states, but in the scoring of individual specimens, dental and skeletal characters were scored on a five-point scale from showing the full expression of one state, through intermediate expressions, to the full expression of the alternative state. These were then coded as 100, 75, 50, 25 and 0% of one of the two states (chosen arbitrarily). Within each sample (i.e. one character scored on a sample of individuals of one species), a mean value was calculated. This gives some estimate of the degree of consistency of expression of the character states in the species.
For the purposes of analysis, Provided sample size was ≥4, a mean expression of ≥75% (¾3/4) for a character state was required for the sample to be coded with that character state for the purpose of analysis, and only if the sample size was ≥4. . For sample sizes of ≤3, the required figure was increased to 87.5% (7/8). Lower figures (e.g. 70% one state, 30% the other) were coded as polymorphic. This is a versionform of ‘confidence coding’ (Wiens, 1999).
Characters were discarded only if (i) (a) they were found to be too difficult to define or score consistently, or (ii) available sample sizes were too low; and/or (b) they showed polymorphism within the outgroup (Muntinacus spp.) and/or (c) they showed polymorphism in more than two ingroup species. . Characters polymorphic in no, one or two ingroup species were retained. Where only one specimen of a species was available for a given character, and its score was intermediate, it was coded as missing data for the p
urposes of analysis (Table S2).
In prelimimary cladistic analysis with PAUP4.0b10, characters coded as polymorphic were found to support one or another clade (based on the alternative character states) depending on whether the accelerated or delayed transformation algorithm was selected (Swofford, 2002). Since there was no objective reason for choosing between the modes of transformation, an alternative approach was taken. A character for which samples showed polymorphism, instead of being coded ‘0’, ‘0&1’, and ‘1’, was instead coded as a three-state ordered character ‘0’, ‘1’ and ‘2’ respectively (cf. Tables S1 & S2). This appropriately treats the polymorphic condition as though an intermediate stage in the replacement of one morphology by another, and corresponds to the ‘ordered scaling’ method described by Wiens (1999). The method is also particularly advantageous in the present project advantageous in that it gives equal weight to (a) a sample showing ‘intermediacy’ due to intermediate character expression in individual specimens; (b) a sample showing ‘intermediacy’ due to a mixture of specimens showing fully expressed, but different, character states; or (c) any combination of the two. All of these situations are commonly encountered, and cannot in practice be readily coded in such a way as to distinguish among them. In addition, the scoring of polymorphism as an intermediate in an ordered series allows PAUP to utlilise the information in tree-building, which it does not for characters entered as polymorphisms per se. Where only a single specimen was available and its morphology was intermediate between two character states, it was coded as ‘?’ for the purposes of analysis.
The re-coding of a two-state character into a three-state ordered character does, however, potentially double the weight of the character in cladistic analysis, as two steps are required to change from ‘0’ to ‘2’. For this reason, all such ordered characters were weighted by 1/(0.5.n-1), where n is the number of character states, using the ‘scaling’ option in PAUP (Swofford 2002).
In Table S1 the character types (ordered/unordered), character states, and their percentage expressions, are shown before these transformations., but the Table indicates (column L) which characters were subsequently transformed into multistate ordered characters and weighted x 0.5.Table S2 shows the transformed matrix ready for phylogenetic analysis, and indicates the weightings given to each character. In Table S2 also, character states are colour-coded to give an impression of the distribution of derived character states. Shades of yellow indicate degrees of derivation from the outgroup (assumed ancestral) condition in an ordered character. In the many cases where polymorphic two-state characters have been re-coded as ‘0, 1, 2’, pale yellow indicates the polymorphic condition (code 1), bright yellow the fixed derived state (code 2). For characters where the outgroup is itself polymorphic, taxa fixed for one of the alternative states are indicated in yellow, those fixed for the other state are indicated in blue.
All other characters (i.e. those not showing polymorphism in any species) were entered as unordered, with the exception of bulla size (character 10), which is an ordered series of 5 size categories.
Two originally single characters were ‘split’ for the purposes of the analysis, in order to incorporate a more complex coding situation. Character 5 is an ordered character defining the number of points at the top of the antler as ‘1’, ‘2’, or ‘3 or more’. Character 6 embodies the variety of different forms seen in antlers with 3 or more top points, and is unordered. For the purposes of this character, species showing only 1 or 2 top points are coded as ‘?’ (P. Forey, pers. comm.). In the second case, character 33 determines whether or not the distal humerus has a clear division between coronoid and radial fossae, while character 34 determines whether, if a division is present, the coronoid or radial fossa is larger. For the purposes of the latter character, species showing no division are coded as ‘?’.
Comparison with previous studies
In a preliminary study including a wider range of cervid taxa (Nock 2001, data not shown), some interesting convergences between the M. giganteus-D. dama clade and Rangifer. tarandus(reindeer) were evident, including palmated antlers and double-ridged axis (main text, Fig. 2 C). However, the position of the reindeer outside the Cervini is incontrovertible (Groves & Grubb 1987, Randi et al. 1998, Pitra et al. 2004).
In an important study, Pfeiffer (1999, 2002, 2005, in press2005) has undertaken a major analysis of cervid phylogeny based on morphological characters. This work broke new ground in the application of cladistic methodology to Quaternary mammal palaeontology, and in the extremely detailed observation and recording of characters. In several of her conclusions, Pfeiffer’s work and the present study are in agreement: the monophyly of the Megacerini (M. giganteus and earlier ‘giant deer’ species), and the lack of support for Axis as a natural genus including A. axis and A. porcinus.. However, in the key question of the relationship of DamaM. giganteus, the two studies differ dramatically, as Pfeiffer (1999, in press20025) places it in a clade with red deer, Cervus elaphus (red deer)- albeit with bootstrap values of 60% or less (Pfeiffer 2005) - , whereas our molecular and morphological data reject that topology in favour of a relationship with M. giganteusDama. Comparison of datasets and methodology suggests the following reasons for this difference:
1. Most of the characters here interpreted as synapomorphies of Dama and M. giganteus were not found in Pfeiffer’s study (Table S1).
2. Pfeiffer incorporated characters showing a considerable degree of intra-species variation or polymorphism,coding them as multi-state ‘grades’ (up to 6 ordered states) within the cladistic analysis; in our analysis, markedly variable characters were excluded. , or coded as multi-state with a maximum of three states.
3. We mostly attempted to restricted individual characters to dichotomies between two or three states of a single morphological entity. Pfeiffer more frequently coded characters as up to 6 ordered ‘grades’, or iIn some other characters,casesPfeiffer gave as (unordered) character states a considerable range of morphologies. An example of the effect of this is that whereas we find the presence of a posterior antler tine (the ‘back tine’, character 1, Table S1) to be shared between D. dama and M. giganteus against its total absence in our other taxa, Pfeiffer’s (in press20025) character 97 defines four varieties of posterior tine, with different species of Dama and M. giganteus scored as different character states which are not treated as homologous by the analysis.
34. We allowed PAUP4.0b10 to determine character polarity using an outgroup (Muntiacus spp.), whereas Pfeiffer determined polarities on the basis of the distribution and frequency of the character states among living and fossil cervids, the more widespread character state being treated as plesiomorphic.
Molecular Analysis
DNA sSampling
Bone samples of specimens identified by skeletal morphology as M. giganteus were obtained from two areas: Ireland (n=2; Liskelly Stream, Cork; Ballynamintra Cave, Waterford) and the eastern Urals/Western Siberia (n=6; Table S32).
For those extant taxa for which sequence data was not available, DNA was obtained either from frozen blood samples obtained from London Zoo (A. axis, D. dama), or conserved antler from the University Museum of Zoology, Cambridge (D. mesopotamica) (Table S3S4).
PCR amplification and sequencing
Primer sequences are given in Table S4 S5 and a summary of PCR reactions is given in Ttable S5S6. Post-mortem fragmentation of DNA obtained from the palaeontological specimens meant that PCR products could only be obtained when the target fragment was <200bp in size. Therefore a series of overlapping fragments were amplified to generate a contig of mitochondrial cytochrome b (cytb) sequence, and sequence for the ATPase 8 gene (ATP8) and the control region (CR).
Nuclear DNA
No nuclear copies of the mitochondrial gene sequences (numts) have been identified previously in cervids, but we found evidence of a cytb numt sequences across the Cervidae. Amplification and sequencing of the A. axis specimen with the primer combination Cerv_cytb_64F/288R generated a fragment of sequence that did not match with that following it. We designed primers (Table S4S5) that employed these sequence mismatches to preferentially amplify the alternative sequence and resolved 445 bp of sequence in D. mesopotamica, D. dama, A. axis and C. elaphus (accession numbers AM072746-AM0727469). The presence of insertions and deletions in this sequence, which would break the reading frame, and the high degree of, but not absolute, similarity between taxa argues that it is a numt element rather than a contaminant mitochondrial sequence. Multiple attempts to recover this sequence from the Megaloceros specimens failed, presumably due to the degradation of nuclear DNA, which would originally have been present at much lower copy numbers. We suggest that the low ratio of mitochondrial to nuclear DNA in bone and antler increases the chance of recovering numt sequences where they are present.
Molecular and mMorphological iIdentification
During preliminary sequencing, it became clear that there were two types of sequences being generated from the Urals/W. Siberian specimens. One type (from samples 49-1, 54 and 10351) had absolute homology with red deer (C. elaphus) sequences, while the other type (samples 50-1, 51-2) was a distinctive but unidentified sequence related to species within the Cervidae. Specimen 50-1 was a vertebra associated with a Megaloceros cranium from Redut, on the Maas river of western Siberia, with an AMS radiocarbon date of 7034 ± 34 BP (OxA 13015); specimen 51-2 was an ulna from a complete Megaloceros skeleton found at Kamyshlov Mire, western Siberia, dated to 7,065 ± 38 BP (Ox-A 13014). Samples 54 and 10351 were tooth and bone, respectively, from an isolated maxilla from Kul’metovsk Cave in the eastern Ural mountains, dated to 10260 ± 55 (OxA 10676). The size and morphology of this specimen makes identification to M. giganteus extremely likely, in which case the C. elaphus sequence is likely to be a contaminant.This specimen, and sSpecimen 49-1, a tooth from Grotto Sikiyaz-Tamak 7 in the Urals, dated to 10,355 ± 45 BP (Ox-A 12099), is are from an isolated series of milk molars whose identity as M. giganteus or C. elaphus cannot be verified morphologically (P. Kosintsev & A. Vorobiev, pers. comm.)
On the basis that specimen 51-2 allowed reliable and repeatable generation of novel sequence, and was absolutely identified, all other Ural/W. Siberian samples were dropped from subsequent analysis.
The two samples from Ireland both gave amplifiable DNA that was distinctive, but related to species within the Cervidae. Amplification from the Liskelly Stream, Cork, femur (uncalibrated AMS radiocarbon date 11839 ± 44 BP; KIA25455) was problematic due to presence of tannins and humic acids inhibiting the PCR, and this was highlighted by the proportion of extracts that exhibited inhibition (six out of ten). However, three non-continuous regions of the cytb (total of 254bp) were obtained (78bp using primers gid-L15245–gid-H15366; 109bp with gid-L15345–gid-H15497; and 67bp with gid-L15470–gid-H15605), and these were in agreement with the data from the Ballynamintra Cave, Waterford, phalanx (uncalibrated AMS radiocarbon date 11567 ± 42 BP; KIA25456).
Thus the two specimens that we considered to provide true Megaloceros sequences were the Ballynamintra Cave phalanx, with an uncalibrated AMS radiocarbon date of 11,567 ± 42 BP, and the Kamyshlov ulna, dated to 7,065 ± 38 BP. Due to the presence of only a single base difference between these two specimens, we used only the Urals specimen for phylogenetic analyses.
Figure S1 gives cloning data and figure Figure S2 shows pairwise distances between taxa in the analysis. Table S6 S7 gives molecular data from the radiocarbon analysis. Table S7 S8 shows base substitutions between the two Dama species. Table S8 S9 shows base substitutions between the two M. giganteus specimens and between them and Dama.
We note that sequences previously deposited on the EMBL database (AY347754-6, AY244492-5), and attributed to Megaloceros, show a high degree of homology with C. elaphus. We would suggest that identification of these specimens should be verified.
and Combined phylogenetic analyses using parsimony
For MP analyses of the combined molecular and morphological datasets, an heuristic search was conducted, weighting and ordering morphological data as before. With molecular characters treated as equivalentto a single unordered character change, i.e. equal weighting between one DNA nucleotide and one morphological character (Masters et al. 2005), a topology identical to the combined Bayesian, and DNA-only trees was returned (text Figure 1a). However, experimenting with the relative weightings of the two character types (Asher et al. 2004) shifted the topology. With molecular characters downweighted by a factor of ≤0.6, the Cervus canadensis - C. nippon pairing of the DNA-only trees changed to the C. elaphus – C. canadensis pairing of the morphology-only trees. The DNA-based topology of the basal ingroup taxa (C. eldi, Axis axis and Axis porcinus) was more robust, only transforming fully into the morphology-based topology when DNA characters were downweighted by a factor of ≤0.2.
References for Supplementary Information
Asher, R.J., Horovitz, I., Sánchez-Villagra, M.R. First Combined Cladistic Analysis of Marsupial Mammal Interrelationships. Molecular Phylogenetics and Evolution 33, 240-250 (2004).
Breda, M. 2005. The morphological distinction between the postcranial skeleton of Cervalces/Alces and Megaloceros giganteus and comparison between the two Alceini genera from the Upper
Pliocene–Holocene of Western Europe. Geobios (in press).
Götherström, A., Collins, M. J., Angerbjörn, A. & Lidén, K. Bone preservation and DNA amplification. Archaeometry44,395-404(2002).
Groves, C. & Grubb, P. Relationships of living deer. Iin: Biology and Management of the Cervidae (ed. Wemmer, C. M.) 21-59 (Smithsonian Institution, Washington, D.C., 1987).
Hasegawa, M., H. Kishino, and T. Yano. Dating the human-ape splitting by a molecular
clock of mitochondrial DNA. J. Mol. Evol. 22, 160- 174 (1985).
Lister, A.M. Evolutionary Studies on Pleistocene Deer. Unpublished PhD thesis, University of Cambridge, 320pp (1981).
Lister, A.M. Evolutionary and Ecological Origins of British Deer. Proc. R. Soc. Edinb. 82B, 205-229 (1984).
Lister, A.M. New results on deer from Swanscombe, and the stratigraphical significance of deer remains in the Middle and Upper Pleistocene of Europe. J. Archaeol. Sci. 13, 319-338 (1986).
Lister, A.M. The evolution of the giant deer, Megaloceros giganteus (Blumenbach). Zool. J. Linn. Soc. 112, 65-100 (1994).
Lister, A. M. The morphological distinction between bones and teeth of fallow deer (Dama dama) and red deer (Cervus elaphus). International Journal of Osteoarchaeology 6, 119-143 (1996).
Masters, J.C., Anthony, N.M., de Wit, M.J. & Mitchell, A. Reconstructing the evolutionary history of the Lorisidae using morphological, molecular, and geological data. American Journal of Physical Anthropology 127, 465-480 (2005).
Nock, D. What is the Giant Deer’s Position in relation to Other Living Cervid Species? Unpublished dissertation, Department of Biology, University College London, 106 pp. (1991).
Pfieffer, T. Die Stellung von Dama (Cervidae, Mammalia) im System pleisiometacarpaler Hirsche des Pleistozans. Cour. Forsch.-Inst. Senckenberg 211, 1-218 (1999).