Ancient proteins resolve the evolutionary history of Darwin's South
American Ungulates
Frido Welker1,2, Matthew J. Collins1, Jessica A. Thomas1, Marc Wadsley1, Selina Brace3,
Enrico Cappellini4, Samuel T. Turvey5, Marcelo Reguero6, Javier N. Gelfo6, Alejandro
Kramarz7, Joachim Burger8, Jane Thomas-Oates1, David A. Ashford1, Peter Ashton1, Keri
Rowsell1, Duncan M. Porter9, Benedikt Kessler10, Roman Fisher10, Carsten Baessmann11,
Stephanie Kaspar11, Jesper Olsen4, Patrick Kiley12, James A. Elliott12, Christian Kelstrup4,
Victoria Mullin13, Michael Hofreiter1,14, Eske Willerslev4, Jean-Jacques Hublin2, Ludovic
Orlando4, Ian Barnes3 & Ross D. E. MacPhee15
1University
2Max
of York, York, YO10 5DD, United Kingdom.
Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
3Natural
History Museum, London SW7 5BD, United Kingdom.
4University
5Institute
of Zoology, Zoological Society of London, London NW1 4RY, United Kingdom.
6CONICET
7Museo
of Copenhagen, DK-1165 Copenhagen, Denmark.
- Museo de La Plata, B1900FWA La Plata, Argentina.
Argentino de Ciencias Naturales "Bernardino Rivadavia", C1405DJR Buenos Aires, Argentina.
8Johannes
9Virginia
Gutenberg-University, Anselm-Franz-von-Bentzel-Weg 7, D-55128 Mainz, Germany.
Polytechnic Institute & State University, Blacksburg, VA 24061 USA.
10University
11Bruker
of Oxford, Oxford, OX3 7FZ, United Kingdom.
Daltonik GmbH, Bremen, Germany.
12Department
13Trinity
of Materials Science and Metallurgy, University of Cambridge, Cambridge, CB3 0FS
College Dublin, Dublin 2 , Ireland.
14Institute
for Biochemistry and Biology, Karl-Liebknecht-Str. 24-25, 14476 Potsdam OT Golm, Germany
15American
Museum of Natural History, New York, NY, United States of America, 10024.
No large group of recently-extinct placental mammals remains as evolutionarily
cryptic as the ~280 genera grouped as “South American native ungulates”
(SANUs). To Darwin1,2, who first collected their remains, they included perhaps
the ‘strangest animal[s] ever discovered’. Today, much like 180 years ago, it is no
clearer whether they had one origin or several, arose before or after the
Cretaceous/Palaeogene (K/Pg) transition 66.2 Ma (million years ago) 3, or are
more likely to belong with the elephants and sirenians of superorder Afrotheria
than with
the euungulates (cattle, horses, and
allies)
of
superorder
Laurasiatheria4–6. Morphology-based analyses have proven unconvincing because
convergences are pervasive among unrelated ungulate-like placentals. Ancient
DNA approaches have also been unsuccessful, probably due to rapid DNA
degradation in semitropical and temperate deposits. Here we apply proteomic
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analysis to screen bone samples of the late Quaternary SANU taxa Toxodon
(Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative
protein sequences. For each SANU we obtained ≈90% direct sequence coverage of
collagen (I) α1 and α2 chains, representing approximately 900 of 1140 amino
acid residues for each subunit. A phylogeny was estimated from an alignment of
these fossil sequences with collagen (I) gene transcripts from available
mammalian genomes or mass spectrometrically (MS/MS) derived sequence data
obtained for this study. The resulting consensus tree agrees well with recent
higher-level mammalian phylogenies7–9. Toxodon and Macrauchenia form a
monophyletic group whose sister taxon is not Afrotheria or any of its constituent
clades as recently claimed5,6, but instead crown Perissodactyla (horses, tapirs,
and rhinos). These results are consistent with the origin of at least some SANUs 4,6
from “condylarths,” a paraphyletic assembly of archaic placentals. With ongoing
improvements in instrumentation and analytical procedures, proteomics may
produce a revolution in systematics like that achieved by genomics, but with the
possibility of reaching much further back in time.
SANUs are conventionally organised into five orders (Litopterna, Notoungulata, Astrapotheria,
Xenungulata and Pyrotheria) that are sometimes grouped together as a separate placental
superorder (Meridiungulata)10. They appear very early in the Palaeogene record and evolved
thereafter along many divergent lines, as their abundant fossil record attests. Most lineages had
become extinct by the end of the Miocene, although a few species of litopterns and
notoungulates persisted into the Late Pleistocene. Despite continuing interest in their
evolutionary history (e.g.,5,11–14), phylogenetic relationships of the major SANU clades to one
another and to other placentals remain poorly understood (see Supplementary Information).
Although some recent investigations (e.g.,4–6) have suggested that basal South American
members of Litopterna conclusively group with certain Holarctic condylarths, and are thus best
placed in Euungulata (Laurasiatheria), several other studies claim to have identified potential
synapomorphies linking various SANU taxa with Afrotheria5,6,15,16. This latter view is broadly
consistent with such indicators as prolonged late Mesozoic faunal exchange between
Gondwanan landmasses17 and the possibility that Xenarthra (the other major endemic South
American placental clade) is also related to Afrotheria7–9,18. However, most of the character
evidence on which the SANU-Afrotheria sister-group hypothesis is based is in dispute19,20. In
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principle, a more definitive test of phylogenetic affinities could come from genomic data, but to
date the application of ancient DNA techniques has been limited and DNA survival is predicted
to be poor (Fig. 1a) (see Supplementary Information).
Type I collagen (COL1), a structural protein comprised of two separate chains, COL1α1 and
COL1α2 (coded by genes on separate chromosomes), is known to provide useful systematic
information (“barcoding”)21, and can be recovered over significantly longer time spans than
DNA22. Most of the 48 samples of Toxodon sp. and Macrauchenia sp. we analyzed for sequence
information came from localities in Buenos Aires province (see Supplementary Information and
Fig. 1b), especially from areas that experience subtropical to maritime-temperate climates23.
Peptide mass fingerprinting (ZooMS) (see Supplementary Information) of COL1 extracts 24
revealed variable levels of collagen preservation in the sample set (see Supplementary
Information and Extended Data Table 1). After screening, two samples each of Toxodon and
Macrauchenia displaying excellent COL1 preservation (See Extended Data Figure 1) were
selected for LC-MS/MS sequencing using a variety of LC-MS/MS platforms, and direct
radiocarbon dating (see Supplementary Information and Extended Data Table 1).
Combining analyses from a total of eight MS/MS runs, we were able to assemble near-complete
COL1 sequences for Macrauchenia (89.4%) and Toxodon (91.0%), similar to levels of sequence
coverage for modern samples. Comparative analyses with fossil and modern samples suggest
that our SANU COL1 sequences are authentic: COL1 amino acid sequence variation is located in
similar positions along both COL1 chains compared to collagen sequences derived from
genomic sources (Extended Data Figure 3) and deamidation ratios conform to expectations for
Pleistocene samples (Extended Data Figure 2), a criticism of previous pre-Holocene collagen
studies25. Independent manual de novo sequencing of product ion spectra for selected
phylogenetically relevant peptides was in full agreement with sequence assignments from
database searches. Furthermore, 86.70% and 94.41% of the assembled species consensus
sequences for Macrauchenia and Toxodon, respectively, was covered by a minimum of two
independent product ion spectra, with individual positions being covered by an average of 77.1
(for Macrauchenia) and 103.9 (for Toxodon) product ion spectra (see Extended Data Table 2).
Molecular evidence for the phylogenetic placement of the extinct SANUs Macrauchenia and
Toxodon was previously unavailable. To examine the phylogenetic position of these taxa, an
alignment of 76 mammalian COL1 sequences and one outgroup (Gallus) was constructed from
available mammalian genomic COL1 sequences in Genbank, as well as several MS/MS-derived
protein sequences obtained for this study. A Bayesian phylogenetic tree was estimated from the
data, with separate models of substitution applied to two partitions (COL1α1 and COL1α2). The
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resulting consensus tree (Fig. 2) is based solely on protein sequence data, but its topology
corresponds closely to branching relationships in Placentalia recovered in recent molecular
studies7–9. Furthermore, nodes poorly supported in this study (e.g. those within Laurasiatheria)
involve the same series of phylogenetic relationships that have proven difficult to resolve in
other studies5,7–9. To examine how alternative topologies could affect the position of our target
taxa we ran additional Bayesian analyses, using constraints mirroring differing mammal
phylogenies (Extended Data Figure 4, Supplemental Information).
In all phylogenetic analyses performed with our data (including the use of unconstrained
parsimony and probabilistic tree reconstruction methods), Macrauchenia and Toxodon formed a
strongly supported monophyletic pair that grouped exclusively with Perissodactyla (as
represented by extant Equus, Tapirus, and Ceratotherium). Neither showed any association with
the clades conventionally contained in Afrotheria (see Supplementary Information). In future,
and with more evidence, it may be appropriate to include these SANUs within an augmented
definition of Perissodactyla. At present we prefer to recognize Litopterna and Notoungulata as
part of a branch-based rankless taxon Panperissodactyla, uniting all taxa more closely related to
crown Perissodactyla than to any other extant taxon of placentals (see Supplementary
Information).
Despite poor resolution at the base of Laurasiatheria, the fact that Macrauchenia and Toxodon
were not recovered at a basal position within Euungulata would imply that the initial split
between Perissodactyla and Artiodactyla occurred earlier than the origin of the SANU clades.
Since fossil evidence indicates that both litopterns and notoungulates were already present in
South America by the Early Palaeocene26,27, this would suggest that the divergence events
leading to the modern orders must have occurred at, if not before, the K/Pg boundary (see
Supplementary Information, Extended Data Figure 5).
These observations do not constitute a full molecular test of SANU monophyly, as there is no
proteomic evidence available for members of the remaining orders (Astrapotheria,
Xenungulata, Pyrotheria). As far as it is now known, Xenungulata and Pyrotheria became extinct
in the late Palaeogene, but some members of Astrapotheria (sometimes considered the sister
group of Notoungulata28) persisted until the Middle Miocene (16.0-11.6 Ma29). This is well
beyond the extrapolated estimate of <4.0 Ma for good collagen survival in an optimal (cool)
burial environment22, although the empirical limits on collagen survival under differing
environmental conditions are poorly understood at present (see Supplementary Information).
The results presented here establish that, in principle, the ≈2,100 residues (i.e. one fifth of the
amino acid residues analyzed by Meredith, et al.9) comprising bone COL1 in placental mammals
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are sufficiently variable to provide reliable systematic information. Of course, a phylogeny
based on two genes may be sensitive to factors affecting phylogenetic resolution such as gene
lineage sorting, missing taxa, aberrant molecular rates, and selection acting on protein coding
sequences. Despite this, the topology derived from the collagen sequences in this study is in
broad agreement with other mammalian trees, and supports monophyletic placement of two
late Quaternary SANUs with a high degree of confidence. Reliable systematic information is an
essential foundation for many other enquiries in evolutionary biology, including patterns of
early Cenozoic mammalian divergence, radiation, extinction and palaeobiogeography. With
further development, molecular sequencing of degradation-resistant proteins such as bone
COL1 is sure to open new vistas in the study of vertebrate evolution.
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Acknowledgements
We thank the Museo Argentino de Ciencias Naturales "Bernardino
Rivadavia", Buenos Aires (MACN), the Museo de La Plata (MLP), and the Natural History
Museum of Denmark, Copenhagen (ZMK), for allowing us to sample fossil specimens in their
collections for this project. The American Museum of Natural History and the Copenhagen Zoo
provided samples of extant mammals suitable for collagen extraction. Mogens Andersen and
Kristian Gregersen of ZMK provided information on specimens in their care. Partly supported
by SYNTAX award "Barcode of Death", ERC Advanced Award CodeX, ERC Consolidator Award
GeneFlow, SYNTHESYS FP7 grant agreement 226506, EPSRC MIB, NE/G012237/1 & NSF OPP
1142052. JTO and DA are members of the York Centre of Excellence in Mass Spectrometry,
created thanks to a major capital investment through Science City York, supported by Yorkshire
Forward with funds from the Northern Way Initiative.
Author Contributions R.D.E.M., I.B. and M.J.C. conceived the project and coordinated the
writing of the paper with F.W. and J.A.T., with all authors participating. J.N.G., A.K., M.R., E.C. and
R.D.E.M collected fossil and extant mammal samples for protein extraction. M.W., S.B., I.B., J.A.T.,
J.B. and M.H. conducted DNA analyses. F.W., M.W., P.A., S.K., C.B., C.K., D.A., J.T-O., R.F., B.K., P.K.,
J.A.E., E.C., L.O., and M.J.C. performed protein analyses and interpretation of results. J.A.T., I.B.,
F.W., and M.W. conducted the phylogenetic analyses and constructed trees. S.T.T., J.N.G., M.R.,
D.M.P. and R.D.E.M. provided the historical, systematic, and palaeontological framework for this
study. J-J.H., E.W., and J.S. provided technical information. Final editing and manuscript
preparation was coordinated by M.J.C., R.D.E.M., and I.B.
Author Information. Raw MS/MS and PEAKS search files have been deposited to the
ProteomeXchange with identified PXD001411. Generated COL1 species consensus sequences
will be available in the UniProt Knowledgebase under the accession numbers C0HJN3-C0HJP8.
The authors declare no competing financial interests. Readers are welcome to comment on the
online version of the paper. Correspondence and requests for additional information on
methods and specimens should be addressed to F.W. (frido.welker@palaeo.eu), M.J.C.
(matthew.collins@york.ac.uk), I.B. (i.barnes@nhm.ac.uk) or R.D.E.M. (macphee@amnh.org)
FIGURES
Figure 1 | a, Predicted survival of an 80 bp DNA fragment after 10ka modelled using the
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rate given in30. b, Location of finds by Darwin1,2 and of samples used in this study. c,
Glutamine deamidation ratios for bone samples from the sequenced Pleistocene SANUs are
high compared to coeval horse (MACN Pv 5719) as well as modern hippo and tapir, providing
support for the authenticity of the ancient sequences (see Supplementary Information).
Figure 2 | Relationship of Toxodon (Notoungulata) and Macrauchenia (Litopterna) to
other placental mammals. 50% majority rule Bayesian consensus tree of COL1 protein
sequence data, with chicken (Gallus) as outgroup. Scale bar indicates branch length, expressed
as the expected number of substitutions per site. Major clades (orders and superorders) are
colour coded; species names in bold indicate collagen sequences derived from MS/MS rather
than genomic data, fossil taxa depicted in silhouette. Inset: in all tree-reconstructions
conducted (see Supplementary Information), Toxodon and Macrauchenia (dark gray) group
monophyletically at the base of crown Perissodactyla (light green) with 100% posterior
probability, forming Panperissodactyla.
Figure 1
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Figure 2
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Extended Data Figure Legends
Extended Data Figure 1. | Examples of MALDI-TOF-MS and MS/MS product ion spectra. a,
MALDI-TOF-MS ZooMS spectra for Toxodon (upper) and Macrauchenia (lower) were used to
screen for samples for the best collagen preservation. b. PEAKS alignment of matching
product ion spectra for Macrauchenia MLP 96-V-10-19 (specimen sample # MLP2012.12)
highlighting peptides aligning to the sequence GPNGEAGSAGPTGPPGLR. c & d, Annotated
PEAKS report of product ion spectra for the same peptide sequence detailed in b for Toxodon
(c) and Macrauchenia (d) detailing differences between both genera (gsT and gsA, highlighted)
and shared substitutions compared to Equus (gpA for Equus, gpT for Toxodon and
Macrauchenia). Note in panel b that both deamidation (N ⇢ D) and variable hydroxylation (P ⇢
h) were detected in different peptides covering this region of the sequence.
Extended Data Figure 2. | Comparison of levels of deamidation for samples in this study
with van Doorn et al.22 (diamonds). The Macrauchenia sample was
14C
dead, consistent with
observed levels of deamidation, which are lower than either Toxodon dated to 12 ka or Equus
sp. (Tapalqué) (not dated). Dotted lines indicate error ranges on Gln estimation for samples
which are not dated or undatable. The measurement approach used in this study - frequency of
deamidation in positions represented in at least 7 MS/MS spectra - is different from the
approach used in 22, so the absolute values may not be directly comparable.
Extended Data Figure 3. | Collagen type I substitution variability for placental mammals
(genomic and proteomic data) compared to the dasyurid marsupial Sarcophilus harrisii
(Tasmanian devil) as outgroup. Substitution variability scores range between 0 and 1 and
incorporate sequence coverage for a given number of species over a 15 amino acid moving
average (95% standard deviation in lighter tone). a. Along-chain variation in genomic
sequence variability (upper red) is similar to proteomic sequence variability (lower blue) for
both COL1α1 and COL1α2 chains. b. Molecular surface rendering (VMD98) of the collagen
unit cell taken from coordinates given in PDB 3HR2. Colours represent genomic (left) and
proteomic (right) sequence variability throughout the structure.
Extended Data Figure 4. | Bayesian constraint tree based on phylogeny published by
O’Leary et al. (2013, fig. 1). See Methods and SI 3.2 for further details and discussion.
Extended Data Figure 5. | Maximum clade credibility phylogeny from BEAST molecular
dating analysis. Branch lengths are measured in Ma (millions of years; scale axis indicates 100
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Ma intervals). Node labels show 95% highest probability densities for molecular dates (in Ma).
Fossil constraints provided in Table S3. Vertical dashed line indicates K/Pg boundary.
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Methods
ZooMS Screening
After zooarchaeology by MS (ZooMS) screening of selected Macrauchenia (n=26) and Toxodon
(n=22), four bone specimens were selected for MS/MS analysis. Using a combination of
enzymes we were able to obtain sequence coverage of around 90% for COL1 for both genera.
Subsamples of ~200 mg were taken from each bone or skin sample for COL1 extraction. Bone
samples were demineralised in 0.6 M HCL for 8 days at 4°C. The acid was removed and the
samples were washed three times with ultrapure water then heated at 70°C in 0.6 M HCl for 48
hours to gelatinise the COL1. Samples were then ultrafiltered using 30 kDa filters and washed
through with ultrapure water. 0.5 mL from each sample retentate was taken to dryness
overnight in a vacuum centrifuge. 100 µL of 50 mM ammonium bicarbonate solution (pH 8) was
added to each sample. The samples were then digested with trypsin (0.5 µg/uL, for sixteen
hours at 37°C). After enzyme digestion, samples were acidified with 2 µL of 5% (volume %)
trifluoroacetic acid (TFA). Samples were then concentrated using C18 ZipTips: the ZipTips were
prepared using a conditioning solution of 50% acetonitrile, 49.9% water, 0.1% TFA; the tips
were then washed with a washing solution of 0.1% TFA; the sample was then transferred over
the column 10 times; the tips were then washed again using 0.1% TFA solution; finally the
sample was eluted using the conditioning solution. For ZooMS analysis, 1 µL of each sample was
spotted in triplicate onto a ground steel plate with 1 µL of CHCA matrix solution (1% in 50%
ACN/0.1% TFA (v/v/v)). MS analysis was carried out on a Bruker ultraflex MALDI-TOF/TOF
mass spectrometer over the m/z range 800 - 4000 (Extended Data Figure S1). Screening
revealed large differences in COL1 spectral quality between samples. Of 46 SANU samples, only
five (3/20 Toxodon, 2/25 Macrauchenia) yielded good ZooMS spectra. One of the three Toxodon
samples (ZMK 22/1889) produced few strong MS/MS spectra and only four samples (two each
of Macrauchenia and Toxodon) were used in the main study.
MS/MS Sequence Analysis
Selected collagen extracts from pooled trypsin (0.4 µg/uL 16 h, 37°C) and elastase digests (0.8
µg/uL, 16 h, 37°C) of two specimens of each SANU sample were analysed on both Thermo
Scientific Orbitrap and Bruker maXis HD LC-MS/MS platforms. Additionally, Orbitrap and maXis
HD instruments were also used for sequencing collagen from modern aardvark (Orycteropus
afer), silky anteater (Cyclopes didactylus), hippopotamus (Hippopotamus amphibius) and South
American tapir (Tapirus terrestris), as well as Pleistocene Mylodon darwinii and Equus sp.
samples from South America.
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Hybrid Quadrupole-Orbitrap
Sample separation was performed on an Ultimate 3000 RSLCnano LC system (Thermo
Scientific). Peptides were first trapped on a Pepmap µ-pre-column (0.5 cm x 300 µm; Thermo
Scientific) and separated on an EASY Spray PepMap UHPLC column (50 cm x 75 µm, 2 µm
particles, 40°C; Thermo Scientific) with a 60 min multi-step acetonitrile gradient ranging from 2
– 35% mobile phase B (mobile phase A: 0.1% formic acid/ 5% DMSO in water; mobile phase B:
0.1% formic acid/ 5% DMSO in acetonitrile) at a flow rate of 250 nL/min. Mass spectra were
acquired on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer at a resolution of
70000 @ m/z 200 using an ion target of 3E6 and maximal injection time of 100 ms between m/z
380 and 1800. Product ion spectra of up to 15 precursor masses at a signal threshold of 4.7E4
counts and a dynamic exclusion for 27 seconds were acquired at a resolution of 17500 using an
ion target of 1E5 and a maximal injection time of 128 ms. Precursor masses were isolated with
an isolation window of 1.6 Da and fragmented with 28% normalized collision energy.
Bruker maXis HD
Sample separation was performed on an Ultimate 3000 RSLCnano LC system (Thermo
Scientific). Peptides were first trapped on a Pepmap pre-column (2 cm x 100 µm; Thermo
Scientific) and separated on a PepMap UHPLC column (50 cm x 75 µm, 2 µm particles; Thermo
Scientific) with a 120 min multi-step acetonitrile gradient ranging from 5 – 35% mobile phase B
(mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile) at
a flow rate of 400 nL/min. A CaptiveSpray nanoBooster source (Bruker Daltonik GmbH), with
acetonitrile as a dopant, was used to interface the LC system to the maXis HD UHR-Q-TOF
system (Bruker Daltonik GmbH). Source parameters were set to 3 L/min dry gas and 150°C dry
heater; nitrogen ‘flow’ setting for the nanoBooster was set to 0.2 bar. Mass spectra were
acquired in the m/z range 150 – 2000 at a spectral acquisition rate of 2 Hz. Precursors were
fragmented with a fixed cycle time of 4 s using a dynamic method adapting spectra rates
between 2 and 10 Hz based on precursor intensities. Dynamic exclusion was set to 0.4 min
combined with reconsideration of an excluded precursor for fragmentation if its intensity rises
by a factor of 3.
Collagen type I sequence assembly
Product ion data from the maXis HD and Orbitrap platforms were analysed in three stages.
Initially MASCOT (Matrix Science) was used to search against the UniColl database to generate a
list of ranked peptides for each spectrum. Sequences derived from this exercise were added to a
local database of genomic and published collagen sequences and common laboratory
14 of 63
contaminants, and the original data was then re-analysed by PEAKS (v. 6, Bioinformatics
Solutions Inc.) using this new database (for example PEAKS output see Extended Data Figure
S1b-d).
As an independent check a limited number of the product ion spectra of peptides (previously
assigned by PEAKS) were also manually de novo interpreted (JT-O) without prior knowledge of
the assignment, in all cases with full agreement between the two approaches.
Generation of and searching against Unicoll, a non-redundant synthetic collagen
database
Publicly available COL1α1 and COL1α2 sequences were concatenated and aligned using Mafft31
with subsequent manual alignment of misaligned sites using Bioedit31 and Geneious v 4.632. A
custom python script was used to digest the COL1 with trypsin in silico. For each tryptic
fragment, all variable amino acid positions across the aligned sequences were recorded. A new
sequence was created for every permutation of these variable sites. These sequences were
concatenated and stored in FASTA format with a header indicating the position in the original
alignment. The result was a database with each entry a concatenation of sequences representing
every permutation of observed mutations for that particular tryptic fragment. One tryptic
fragment of the sequence (COL1α2 positions 870-905) was too variable to include without
exceeding available memory. Only the original observed variants were included for this part of
the sequence. Using this strategy it was possible to generate the equivalent of more than 10200
alternative collagen ‘sequences’ (c.f. 1082 is the upper estimate of the number of atoms in the
universe).
MS/MS datafiles were merged and submitted to Mascot with: enzyme set to Trypsin/P; variable
modifications for deamidated (NQ), Lys->Hyl(K), oxidation (M) and Pro->Hyp(P); peptide mass
tolerance +/-10 ppm; and fragment mass tolerance +/-0.07 Da. The structure of sequence
entries in Unicoll meant that it cannot accommodate missed cleavages. Select summaries
containing matched peptides with a Mascot score greater than 30 were exported into Excel for
each analysis. Peptides were identified by picking the highest scoring hits for each tryptic
fragment, if the score exceeded 40, while for matches with scores between 30 and 40, the
spectra were inspected manually to choose the best hit amongst the possibilities given by the
search engine.
Searching data using PEAKS
Product ion spectra were searched using the PEAKS software33 against a database comprising
genomic COL1 sequences plus fossil consensus sequences, composed of UniColl peptides hits,
15 of 63
missing and low coverage regions filled with conserved mammalian COL1 sequences (see 2.3
Phylogenetic Reconstruction Methods). Additionally, common laboratory contaminants were
included in database searches. Full PEAKS searches (Peptide de novo, PEAKS DB, PEAKS PTM
and SPIDER) were performed with peptide mass tolerance +/-10 ppm and fragment mass
tolerance +/-0.07 Da, in addition to respective platform and enzyme details. Searches were
performed allowing for deamidated(NQ), Lys->Hyl(K), oxidation(M) and Pro->Hyp(P). False
discovery rate (FDR) was put at 0.5% and peptide scores were only accepted with -10lgp scores
≥ 30 and ALC (%) ≥ 50. Where there was ambiguity in interpretation of the spectra peptides
were selected based upon knowledge of sequence constraints, PTMs and fragmentation
patterns.
Reference sequence authentication
In order to check the quality of our MS/MS COL1 sequences, we sampled a modern and fossil
sample for which we had independent genomic data, specifically (i) a modern aardvark sample
(Orycteropus afer) and (ii) a fossil equid bone from a geological formation rich in SANU fossils
with their respective genome sequences. The fossil sample had similar collagen yields and
ZooMS profile to the SANU samples used for MS/MS sequencing (Pleistocene horse, Tapalqué,
South America, Fig 1b) (section 2.1.6.1.). Our modern aardvark MS/MS sequence was identical
to that of the protein product inferred from the released genomic sequence. For the Pleistocene
Equus sp. sequence, two amino acid substitutions were detected (T>L, COL1α1, and H>D,
COL1α2), similar to the maximum number of differences observed in a recent study comparing
Equus genomes to the Equus ferus caballus reference genome34.
De novo sequence authentication
The absence of corresponding genomic data prevents similar comparisons with MS/MS-derived
sequence for the SANU species. Instead we assessed amino acid substitution locations along
both COL1α1 and COL1α2 chains in both our (and previously published35) fossil COL1
sequences with genomic data, using the COL1α1 and COL1α2 sequence of Tasmanian devil
(Sarcophilus harrisii) as an outgroup to eutherian mammals. C- and N-terminal telopeptides
were removed as they were rarely observed from fossil samples. COL1 position numbers are
given as a continuous count with COL1α1 and COL1α2 concatenated, with COL1α1 ranging from
position 1 to position 1014 and COL1α2 ranging from 1015 to 2028.
We find that the location of amino acid variation along the COL1α1 and COL1α2 chains is similar
among the different COL1 sequences obtained from genomic sources (Extended Data Fig. 3). We
identify several regions, mainly located in COL1α1, that appear to lack sequence variation
16 of 63
among the four major mammalian superorders. This can be a result of the functional
importance of some of these regions during COL1 fibril formation, α1 and α2 chain binding and
COL1 hydroxylation 36–38. Additionally, we observe a substitution rate in COL1α2 roughly twice
that observed in COL1α1.
Comparing COL1 sequences derived from MS/MS data in this and an earlier study35, with
genomic data for laurasiatheres reveals good correspondence in the location of substitutions
along the COL1α1 and COL1α2 chains between our results and genomic data (see Extended
Data Figure S3). Buckley’s MS/MS35 data for laurasiatheres are derived from a single species
(Manis tetradactyla). Sequence variation from those data compared with genomic data is
similar, although we note that several regions displaying high rates of amino acid substitution
are missing from the Manis consensus sequence provided (notably around positions 726-756,
991-1089, 1306-1364, 1423-1443 and 1899-1977).
Buckley35 provides two xenarthran and five afrotherian COL1 sequences obtained using mass
spectrometric sequencing. Regions with high substitution complexity are missing from the
consensus sequences provided, for Afrotheria (1024-1089, 1588-1599, 1740-1754) and
Xenarthra (1024-1089, 1207-1234, 1348-1364, 1588-1638, 1771-1806, 1921-1947). The
absence of such regions prohibits the inclusion of these sequences in our phylogenetic treebuilding, as the majority of informative positions are missing from the sequences provided.
For substitution locations, our data suggest structural and/or functional organization of these,
and their frequency, in specific regions of both chains. Additionally, the substitution rate for
COL1α2 is roughly double that of COL1α1.
We criticised claims of authentic collagen sequences retrieved from a Tyrannosaurus rex
sample39 based in part on the low levels of reported deamidation40, and more recently have
demonstrated an increase in glutamine deamidation in archaeological rather than modern
collagen, which correlated with thermal age (Extended Data Figure S2 and
41);
similar levels
have been reported for Pleistocene mammoths and equids34.
Deamidation ratios observed for glutamine here are consistent with ancient collagen of
equivalent thermal age (Extended Data Figure S1). The lowest levels of Gln to Glu deamidation
are observed in modern samples from hippo (1.8% ± 3.2) and tapir (5.7% ± 10.9) bone. The
highest levels of Gln deamidation occur in the radiocarbon dead Macrauchenia samples (Glu =
82.8.4% ± 14.3). The Toxodon samples are less deamidated (Glu = 59.2% ± 24.5), which is
consistent with a late Pleistocene date (12 ka). However, by contrast, the Pleistocene equid is
much better preserved (Glu = 18.9% ± 18.4) despite the fact that it cannot be much younger
17 of 63
than Toxodon (Figure 1c).
DNA extraction and sequencing
Approximately 250 mg of the three samples with the highest number of peaks in the mass
spectra from each species (see 2.1 Protein Extraction and Sequence Assembly Methods) were
used for DNA extraction. DNA extraction was performed as in the method described by42. PCR
primers were designed to target Perissodactyla and Laurasiatheria specific regions of the cytb,
COX1, 16S and 12S genes using mtDNA sequences downloaded from NCBI (Table S1). Primer
design was done using the program Primer3. PCR was performed for 60 cycles and samples
were visualized on 2.5% agarose gel. Products were successfully amplified from several samples
while PCR controls showed no amplification products. BLAST searches of the sequences
obtained revealed no homology to any previously derived sequence for several of the products,
while sequences from two Macrauchenia samples showed high similarity (98 and 99%,
respectively) to domestic pig sequences, a common contaminant in ancient DNA analyses
43.
A
Pleistocene horse bone from the same depositional context as some of the SANU specimens
yielded a sequence 98% identical to modern horse (Equus caballus), suggesting that the failure
to PCR amplify putative SANU DNA sequences was not due to technical problems, but a lack of
endogenous DNA in the samples investigated.
DNA Next Generation Sequencing (NGS) Approach
After failing to amplify endogenous DNA through Sanger sequencing of targeted PCR products,
we applied a non-targeted, NGS shotgun approach in a further attempt to identify whether
endogenous DNA could be obtained. Based on the collagen sequencing results Macrauchenia
sample 12-1641 (metapodial) was selected as the most likely candidate for NGS analyses. DNA
extractions of Macrauchenia sample 12-1641 followed protocols described in Brace et al.44 and
were carried out in the dedicated ancient DNA laboratory at Royal Holloway, University of
London. The library was constructed in a dedicated ancient DNA laboratory: Johannes
Gutenberg University, Mainz, using a modified version of the protocol in45. Modifications: the
initial DNA fragmentation step was not required; all clean up steps used MinElute PCR
purification kits. Blunt-end repair step: Buffer Tango and ATP were replaced with 0.1 mg/mL
BSA and 1 x T4DNA ligase buffer. The proceeding clean up step was replaced by an inactivation
step, heating to 750C for 10 minutes. Adapter ligation step: 0.5 mM ATP replaced the T4 DNA
Ligase buffer. The index PCR step followed a further protocol
46
using AmpliTaq Gold DNA
polymerase and the addition of 0.4 mg/mL BSA. The index PCR was set for 20 cycles with three
18 of 63
PCR reactions conducted per library. The indexed library was sequenced on an Illumina HiSeq
platform (Mainz, Germany) using a single lane, paired-end read, sequencing run.
Bioinformatics Methods and Conclusion
Paired-end reads were quality trimmed (q=10) with cut-adapt
47
and then sequences were
simultaneously adapter trimmed and the paired reads joined together with Seq-Prep (available
from https://github.com/jstjohn/SeqPrep). Reads shorter than 17bp were discarded. In the
absence of any close phylogenetic relative (required for the accurate genomic mapping of
reads), processed reads were de-novo assembled into contigs using clc_denovo_assembler
(available in CLC Assembly Cell v.4.2), with contigs shorter than 70bp discarded. Two
approaches were then used to investigate the data for mammalian genomic sequences (which
had proven successful for other aDNA NGS samples).
In order to examine whether there were any mammalian DNA sequences suitable for
phylogenetic analysis in our dataset, first, contigs were blasted using blastn to a local nucleotide
database, downloaded from NCBI. Custom perl scripts (available on request) were used to
assign taxonomic and gene information to BLAST hits. These results were searched for standard
orthologous mitochondrial and nuclear phylogenetic sequences. Each of the potential hits
blasting to mammalian sequences were inspected; however, all were assignable to bacterial
elements, and no blast hit could be attributed to mammalian genes.
Secondly, two separate BLAST databases were generated; one from the contigs and a second
from the processed reads, using the makeblastdb command in BLAST. These databases could
then be queried with mammalian (including perissodactyl) mitochondrial and nuclear
phylogenetic sequences of interest using blastn. Neither of these searches returned any
matching contigs. Thus, the NGS dataset yielded nothing of use for phylogenetic analysis, and
gave no indication that any Macrauchenia DNA had persisted in the sample.
Phylogenetic Reconstruction Methods
Prior to the advent of DNA based molecular phylogeny, variations in protein structure and
sequence had been used to explore evolutionary relationships 48,49. The comparative dataset for
this paper was built using consensus amino acid sequences for COL1α1 and COL1α2 generated
by MS/MS analysis for the target taxa Toxodon and Macrauchenia as well as representatives of
all extant major mammalian clades. Leucine (L) and isoleucine (I) were converted into
isoleucines as these are isobaric and low energy MS/MS sequencing is not capable of
discriminating between them. Partition Finder
50
was used to select the best-fit partitioning
scheme from the amino acid data. This was identified as two separate partitions, for Col1a1 and
19 of 63
Col1a2. Bayesian phylogenies were generated using MRBAYES v.3.2.1
51
with the amino acid
model estimated from the data (to allow model jumping between fixed-rate amino acid models,
the prior for the amino acid model was set as: prset aamodelpr=mixed). The proportion of
invariant sites, and the distribution of rates across sites (approximating to a gamma
distribution) were also estimated from the data. Two chains were run for 5 million generations
(sampled every 500), with convergence between chains assessed in TRACER v.1.6
52.
All
effective sample sizes (ESSs) of parameters were greater than 100. After burn-in was removed,
a majority rule consensus tree was constructed, using the sumt command in MrBayes, from the
trees sampled in the posterior distribution.
In order to test for the robustness of the results of the Bayesian analysis under other methods of
tree reconstruction, we also conducted maximum likelihood (ML) and maximum parsimony
(MP) analyses. We performed parsimony analyses running PAUP* v.4.0b1053, using the heuristic
search option with a random taxon addition sequence (1,000 repetitions) and TBR branch
swapping, and rooting the tree along the branch leading to Aves. A ML phylogeny was estimated
in RAxML v7.0.454. A Dayhoff model of protein sequence evolution with gamma-distributed
variation in rates across sites (corresponding to the PROTGAMMADAYHOFFF model in RAxML)
was applied to each partition. Twenty separate ML analyses were performed (using the “–f d”
command in RAxML), and the tree with the highest likelihood was chosen from this set.
Molecular Clock Analysis
Fossil calibrated phylogenies were constructed in BEAST v.1.7
55
with the Dayhoff amino acid
model (chosen under the MrBayes mixed model) together with the proportion of invariant sites
and the distribution of rates across sites (approximating a gamma distribution) applied to each
partition. Analyses were run under a strict clock (estimated from the data), with the Yule model
of speciation, for 10 million generations (sampled every 1000 generations). Clock and tree
parameters were linked across partitions. Prior distributions on the root and 33 other nodes
were applied based on an interpretation of the mammalian fossil record (see Table S3) 7,56. The
clock rate prior was set as an uninformative uniform distribution (upper=E100, lower=E-12).
All other priors were left as the default values in BEAUti 57. Full details of all prior distributions
for divergence times are presented in Table S3. As in the case of the MrBayes analysis,
convergence and effective sampling were assessed using Tracer 1.7. A Maximum Clade
Credibility (MCC) tree was constructed using TreeAnnotator (available with BEAST) from the
trees sampled in the posterior distribution.
20 of 63
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Heled, J., Kearse, M., Markowitz, S., et al. Geneious v4.7. Geneious (2010). at
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Ma, B. et al. PEAKS: powerful software for peptide de novo sequencing by tandem
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Supplementary Information - Table of Contents
S1 Materials
1.1 Specimens and Direct Sequencing
1.2 Localities
1.3 Dating
1.4 Estimating Thermal Age
S2 Methods Tables
S3 Systematic Commentary
3.1 Concepts of SANU Relationships, 1894-2013
3.2 50% Majority Rule Bayesian Consensus Tree (Main Text Fig. 2): Comparison to
Results of Other Recent Investigations
3.3 Testing Results: Additional Phylogenetic Trees
3.4 Darwin, Owen, and Initial Interpretations of SANUs
24 of 63
1. Materials
1.1 Specimens (Fossil and Directly Sequenced Modern)
The full list of SANU specimens sampled for collagen analysis is provided in Extended
Data Table S1 (total of 22 Toxodon and 26 Macrauchenia extractions). Before sampling,
all SANU fossils used in this study could be satisfactorily allocated to either Toxodon or
Macrauchenia on the basis of distinctive morphological features. To represent each
SANU, two high-quality specimens were selected and their collagen directly sequenced;
this information was then used to construct consensus sequences as explained in S2.3
Phylogenetic Reconstruction Methods. A sample from each of the high-quality
specimens was submitted for radiocarbon dating (Extended Data Table S1).
To improve the taxonomic resolution of our comparative collagen type I database, we
also directly sequenced samples representing several extant taxa: silky anteater
(Cyclopes didactylus, AMNH 99199); South American tapir (Tapirus terrestris, Zoological
Museum, Natural History Museum of Denmark), hippopotamus (Hippopotamus
amphibius, Zoological Museum, Natural History Museum of Denmark), and aardvark
(Orycteropus afer, AMNH 51910). In addition, to validate our approach we sequenced
and assembled COL1 sequences for two fossil specimens relevant to our study. These
were (i) a laurasiathere (Equus sp., MACN Pv 5719), from a locality in the Tapalqué area,
which also yielded many of our SANU specimens, and (ii) a xenarthran (Mylodon
darwinii MLP 94-VIII-10-32) from the famous end-Pleistocene locality of Cueva del
Milodon in southern Chile (see S2.1 Protein extraction and sequence assembly
methods).
1.2 Localities
The majority of the SANU specimens used in the analyses were collected in the late 19 th
or early 20th centuries; some lack adequate locality and stratigraphic data (Extended
Data Table S1). Most specimens probably represent the common Pleistocene species T.
platensis and M. patachonica, but as museum labels generally record genus only, we
reference our samples as Toxodon sp. and Macrauchenia sp. in the text.
Almost all of the specimens for which some locality information exists come from the
province of Buenos Aires, mostly from areas fairly close to the Atlantic coast. Localities
on labels generally refer only to rivers or towns, in or near which it may be assumed
collecting took place, although for some specimens, museum catalogs provided
additional geographical information. The distribution of all known localities and
estimates of their thermal age are provided in Extended Data Table 1.
1.3 Dating
On museum labels, specimens were generally allocated to “Formacion Pampeana” or
“Lujanense,” stratigraphic and land-mammal age designations that were used
interchangeably in the past (although they are not formally coeval) to refer to the late
25 of 63
Quaternary (i.e., 130 - 11.7 kyr) (for a discussion, including new estimates, see 1). To
test age inferences a limited program of radiocarbon dating was undertaken (Extended
Data Table S1). Both of the high-quality Toxodon samples (MLP 44-XII-29-5 and MACN
Pv 17710) returned relatively young age estimates that fell at or close to the
Pleistocene/Holocene transition and are among the youngest known for this taxon
(Anthony Barnosky, pers. commun., 9.22.2014). By contrast, the two high-quality
Macrauchenia samples (MLP 96-V-10-19 and MACN Pv 18952) did not return AMS
radiocarbon dates, despite yielding collagen adequate for sequencing by MS/MS. This
highlights the difference between radiocarbon analysis and protein sequencing with
regard to limits of detection: in MS/MS analysis, peptides can be released and
sequenced from samples unsuitable for radiocarbon dating because of contamination by
tannins and humic acids.
1.4 Thermal Age
Thermal age2 is a means of providing comparative age estimates which normalise for
differences in the thermal history of fossils, and reports them as ages assuming all had
been held at a constant 10 °C, ie. ageka@10°C; thermal ages for this paper were estimated
using the online tool www.thermal-age.eu. Seasonal fluctuations in monthly
temperature at the sites were estimated from the WorldClim dataset3. The extent to
which temperature decreased prior to the Holocene was estimated from the difference
in the 1° × 1° PIMP2 grid for the region at three time intervals: Modern (pre-industrial),
Holocene (6ka) and LGM4. These data were correlated against the equivalent time
intervals from estimated fluctuations in air temperature over the last 800ka5, and the
correlation used to transform the latter temperature series to reflect the temperature
change in this region.
Thermal age estimates are reported in Extended Data Table 1, and we used relative
rates of molecular degradation based upon the temperature dependence of DNA
depurination from 6 and the rate data estimated in bone from 7. We lack detailed site
information for most fossils (Supplemental Information 1.2) and consequently data on
sediment type, levels of hydration and burial depth over time, as well as storage history.
For all samples we therefore used a thermal diffusivity for sediments of 0.04844 (m2
day-1), a depth of 1.25 m, and ignored museum storage.
26 of 63
2. Methods Tables
Table S1. PCR primers to target Laurasiatheria and Perissodactyla specific mtDNA.
Perissodactyla
Gene Start
End
Size
Forward Primer
Reverse Primer
Tm1
COI
274
325
COI
1175
COI
51
ATAGCATTCCCCCGAATAAA
TGATGGTGGGAGGAGTCAGA
58.4
1230
55
ATTCGTCCACTGATTCCCCTTA
TGCTCAGGTTTGGTTGAGTG
61.9 59.9
1212
1453
241
CACTCAACCAAACCTGAGCA
CTGTAGACACTTCTCGTTTGG
59.9 53.5
CytB
7
117
110
AACATCCGGAAATCTCACC
TTCCTAGGAGGGAGCCGAAG
57.4 63.0
CytB
112
195
83
AGGAATCTGCCTAATCCTCCA
CGGATGAGAAGGCAGTTGT
60.0 58.8
CytB
7
195
188
AACATCCGGAAATCTCACC
CGGATGAGAAGGCAGTTGT
57.4 58.8
CytB
786
851
65
TGGAGCGTAGGATGGCGTA
60.3 62.7
16S
878
979
101
16S
977
1196
219
CACACGAGGGTTTTACTGTCTC
16S
223
362
139
TTAAGTTAAACACCCCGAAACCA
CAGCACTCCCCCTCATATTAAA
CTGCCTGCCCAGTGACATC
Tm2
61.7
TGGCCATTCATACAAGTCCCTA
62.9 59.3
TCACTCGGAGGTTGTTTTGTT
59.3 58.1
TTCACCTCTACCTATGAATCTTCTC
58.1 56.8
Laurasiatheria
Gene
Start
End Size
12S
249
350 101
AATTAAGCCATGAACGAAAGTT
16S
1039
1150 111
ATAAGACGAGAAGACCCTATGGAG
CytB
CytB
Forward Primer
Reverse Primer
Tm1 Tm2
TTAATTCGGGTTAATCGTATGAC
TCACCCCAACCTAAATTGTTA
52.7
53.0
57.9
55.1
453
517
64
AGCAATCCCATACATCGGAAC
CTTTGTCTACTGAGAATCCTCCTC
58.2
58.4
208
517 309
TGCCGAGACGTAAATTATGGT
CTTTGTCTACTGAGAATCCTCCTC
56.5
58.4
Table S2. Comparative dataset used for phylogenetic reconstruction, referencing
database sources (with accession numbers) or associated genome publication.
Order
Species
Database
Accession COL1ɑ1
Accession COL1ɑ2
Aves
Gallus gallus
UniProt
P02457
P02467
Afrosoricida
Echinops telfairi
Genbank
XM_004707059.1
XM_004702663.1
Afrosoricida
Chrysochloris asiatica
Genbank
XM_006832467.1
XP_006834349.1
27 of 63
Carnivora
Ailuropoda melanoleuca
Genbank
XM_002923729.1
NW_003217535.1
Carnivora
Canis lupus
Genbank
NM_001003090.1
XM_005628344.1
Carnivora
Felis catus
Genbank
XM_003996699.1
XM_003982764.1
Carnivora
Mustela putorius
UniProt
M3YVG8_MUSPF
M3XR96_MUSPF
Carnivora
Odebenus rosmarus
Genbank
XM_004395150.1
XM_004394078.1
Carnivora
Leptonychotes weddelli
Genbank
XP_006740794
XP_006730730
Carnivora
Panthera tigris
Genbank
XM_007086767.1
XM_007076695.1
Artiodactyla
Bos primigenius
UniProt
CO1A1_BOVIN
CO1A2_BOVIN
Artiodactyla
Bubalus bubalis
Genbank
XP_006041214.1
XP_006054012.1
Artiodactyla
Pantholops hodgsonii
Genbank
XP_005964709
XP_005985745
Artiodactyla
Hippopotamus amphibius
This study
This study
Artiodactyla
Orcinus orca
Genbank
XM_004282622.1
XM_004265587.1
Artiodactyla
Sus scrofa
ENSEMBL
ENSSSCG00000017581
ENSSSCG00000015326
Artiodactyla
Ovis aries
Artiodactyla
Tursiops truncatus
Genbank
XM_004313715.1
XM_004327093.1
Artiodactyla
Physeter catodon
Genbank
XM_007128545.1
XM_007105299.1
Artiodactyla
Camelus bactrianus
Genbank
XM_006174954.1
XM_006178536.1
Artiodactyla
Vicugna pacos
Genbank
XM_006218653.1
XM_006207629.1
Chiroptera
Myotis davidii
Genbank
XP_006774459.1
XP_006779282.1
Chiroptera
Myotis lucifugus
Genbank
XP_006105322.1
XP_006085025.1
Chiroptera
Myotis davidii
Genbank
XP_006774459.1
XP_006779282.1
Chiroptera
Pteropus alecto
Genbank
XM_006924793.1
XM_006911910.1
Chiroptera
Pteropus vampyrus
Genbank
GCA_000151845.1
GCA_000151845.1
Chiroptera
Eptesicus fuscus
Genbank
XP_008151928.1
XP_008147219.1
Cingulata
Dasypus novemcinctus
Genbank
XM_004484849.1
XM_004470707.1
Dasyuromorphia Sarcophilus harrisii
Genbank
XM_003768375.1
XM_003775331.1
Didelphimorphia Monodelphis domestica
ENSEMBL
ENSMODG00000011945
ENSMODG00000016732
Diprotodontia
International Sheep Genomics International Sheep Genomics
Consortium, 2010
Consortium, 2010 8
Macropus eugenii
Renfree et al. 20119
Renfree et al. 20119
Hyracoidea
Procavia capensis
ENSEMBL
ENSPCAG00000011915
ENSPCAG00000002641
Lipotyphla
Condylura cristata
Genbank
XM_004684338.1
XM_004676693.1
28 of 63
Lipotyphla
Erinaceus europaeus
Genbank
GCA_000296755.1
GCA_000296755.1
Lipotyphla
Sorex araneus
Genbank
XM_004608670.1
XM_004602187.1
Lagomorpha
Oryctolagus cuniculus
UniProt/Genbank
G1T4A5
NM_001195668.1
Lagomorpha
Ochotona princeps
Genbank
XP_004591119
XP_004582363
Macroscelidea
Elephantulus edwardii
Genbank
XP_006889362.1
XP_006891537.1
Monotremata
Ornithorhynchus anatinus
UniProt
F7ESN3_ORNAN
F6UFX0_ORNAN
Perissodactyla
Ceratotherium simum
Genbank
XM_004434679.1
XM_004431356.1
Perissodactyla
Equus asinus
Genbank
FJ594763.1
FJ594764.1
Perissodactyla
Equus caballus
Genbank
XM_005597481.1
XM_005609220.1
Perissodactyla
Tapirus terrestris
This study
This study
Perissodactyla
Equus sp. (Pleistocene)
This study
This study
Pilosa
Choloepus hoffmanni
(Absent)
ENSCHOG00000000328
Pilosa
Mylodon darwinii
This study
This study
Pilosa
Glossotherium sp.
This study
This study
Primates
Homo sapiens
Genbank
NM_000088.3
NM_000089.3
Primates
Macaca mulatta
Genbank
XM_001096194.2
NM_001266337.1
Primates
Microcebus murinus
ENSEMBL
ENSMICG00000007252
ENSMICG00000008483
Primates
Nomascus leucogenys
Genbank
XM_003281260.2
XM_003252507.2
Primates
Otolemur garnettii
Genbank
XM_003786490.1
XM_003782677.1
Primates
Pan paniscus
Prado-Martinez et al. 201310
Prado-Martinez et al. 201310
Primates
Pan troglodytes
Prado-Martinez et al. 2013
Prado-Martinez et al. 2013
Primates
Pongo abelii
Prado-Martinez et al. 2013
Prado-Martinez et al. 2013
Primates
Pongo pygmaeus
Prado-Martinez et al. 2013
Prado-Martinez et al. 2013
Primates
Saimiri boliviensis
Genbank
XM_003931264.1
XM_003921214.1
Proboscidea
Loxodonta africana
Genbank
XM_003414641.1
NW_003573425.1
Proboscidea
Mammuthus sp.
Buckley et al. 201111
Buckley et al. 2011 11
Proboscidea
Mastodon sp.
Buckley et al. 2011
Buckley et al. 2011
Rodentia
Cavia porcellus
Genbank
XM_003466865.2
XM_003474996.2
Rodentia
Chinchilla lanigera
Genbank
XM_005394176.1
XM_005388041.1
Rodentia
Dipodomys ordii
ENSEMBL
ENSDORG00000013502
ENSDORG00000003027
ENSEMBL
29 of 63
Rodentia
Cricetulus griseus
Genbank
XM_003503960.1
XP_003497018
Rodentia
Mesocricetus auratus
Genbank
XM_005075850.1
XM_005082899.1
Rodentia
Ictidomys tridecemlineatus
Genbank
XM_005321944.1
XM_005331821.1
Rodentia
Jaculus jaculus
Genbank
XM_004655568.1
XM_004656219.1
Rodentia
Mus musculus
Genbank
NM_007742.3
NM_007743.2
Rodentia
Rattus norvegicus
Genbank
NM_053304.1
NM_053356.1
Rodentia
Octodon degus
Genbank
XM_004633633.1
XM_004632623.1
Scandentia
Tupaia chinensis
Genbank/UniProt
XM_006166190.1
L9JEW6
Sirenia
Trichechus manatus
Genbank
XM_004377939.1
XM_004386146.1
Tubulidentata
Orycteropus afer
Genbank
XM_007941916.1
XM_007939805.1
Tubulidentata
Orycteropus afer (MS/MS)
This study
This study
Litopterna
Macrauchenia patachonica
This study
This study
Notoungulata
Toxodon platensis
This study
This study
Table S3. Divergence dates and fossil constraints for BEAST molecular dating
analysis.
Minimum: hard lower bound, cut off for log normal distribution 12; Maximum: soft
upper bound, 95% of log normal distribution; Log Normal Prior parameters for the
BEAST analysis; Fossil Information including current “earliest” anchor fossil, age, and
locality. Asterisk indicates constrained nodes were not recovered in free-rate Bayesian
analysis.
Divergence
ROOT:
Mammal–Bird
Synapsida)
Monotremata–Theria
Australosphenida)
Min.
Max. Log Normal Prior
Fossil/Other information
Ref.
(Sauropsida– 312.3
330.4
mean="5.75"
stdev="1.0"
offset="312.3"
Protoclepsydrops, from Joggins Formation of 13
Nova Scotia
(Theriimorpha– 162.9
191.1
mean="8.99"
stdev="1.0"
offset="162.9"
Amphilestes
&
Phascolotherium
Stonesfield Slate, Middle Jurassic
61.5
71.2
mean="3.086"
stdev="1.0"
offset="61.5"
Khasia, from Early Palaeocene Tiupampa 12
fauna of Bolivia
48.4
113.0
mean="20.55"
stdev="1.0"
offset="48.4"
Phosphatherium, Ypresian (Early Eocene) 12
proboscidean from Morocco
Opossum–Kangaroo/Devil
(Ameridelphia–Australidelphia)
Afrotheria: Afrosoricida–Paenungulata
from 12
30 of 63
Paenungulata
55.6
71.2
mean="4.965"
stdev="1.0"
offset="55.6"
Eritherium, minimum age 55.8 +/- 0.2 = 55.6 13
Mya
Elephantiformes– Elephantimorpha*
28.3
55.6
mean="8.69"
stdev="1.0"
offset="28.3"
Eritreum melakeghebrekristosi
26.8+/-1.5 Mya
Proboscidea: Mammuthus–Loxodonta*
6.8
23.03
mean="5.165"
stdev="1.0"
offset="6.8"
Loxodonta, from hominid locality in Chad, 13
radiometrically dated between 6.8 and 7.2
Mya
Xenarthra:
58.5
71.2
mean="4.041"
stdev="1.0"
offset="58.5"
Riostegotherium from Late Palaeocene dated 13
to 58.7 +/-0.2 = 58.5 Mya
61.5
131.5
mean="22.27"
stdev="1.0"
offset="61.5"
Extinct primate sister taxa such as 12
carpolestids and plesiadapids from Early
Palaeocene
61.5
131.5
mean="22.27"
stdev="1.0"
offset="61.5"
Extinct primate sister taxa such as 12
carpolestids and plesiadapids from Early
Palaeocene
55.6
65.8
mean="3.245"
stdev="1.0"
offset="55.6"
Altiatlasius, oldest euprimate known from 12
Late Palaeocene of Morocco
33.7
65.8
mean="10.215"
stdev="1.0"
offset="33.7"
Catopithecus, from Fayû m Quarry L-41, 12
Egypt, end of Priabonian
33.7
55.6
mean="6.97"
stdev="1.0"
offset="33.7"
Karanisia, from Priabonian of Egypt
Catarrhini: Homo–Macaca
23.5
34.0
mean="3.343"
stdev="1.0"
offset="23.5"
Proconsul, from Late Oligocene (Chattian) 12
Meswa Bridge in Kenya
Hominidea: Homo/Pan–Pongo
11.2
›13.7
mean="7.16"
stdev="1.0"
offset="11.2"
Sivapithecus, from Middle Miocene Chinji 12
Formation, Pakistan
Hominini: Homo–Pan
5.7
10.0
mean="1.37"
stdev="1.0"
offset="5.7"
Orrorin, from Late
Formation, Kenya
Glires
61.5
131.5
mean="22.27"
stdev="1.0"
offset="61.5"
Heomys, stem rodent from Early to Late 12
Palaeocene Doumu Formation, China
Lagomorpha: Oryctolagus–Ochotona
48.4
61.1
mean="4.043"
stdev="1.0"
offset="48.4"
Calcanei crown lagomorph from Early 13
Eocene (Ypresian) Cambray Formation, India
Rodentia
55.6
65.8
mean="3.245"
stdev="1.0"
offset="55.6"
Sciuravus, from Early Eocene (Wasatchian) 14
Wyoming, USA
Choloepus–Dasypus
Boreoeutheria:
Archontoglires–Laurasiatheria
Archontoglires:
Primates–Rodentia
Primates:
Strepsirrhini–other Primates
Anthropoidea: Homo–Saimiri*
(Old–New world monkey)
Strepsirrhini:*Otolemur–Microcebus
(Lemuriformes-Lorisiformes)
(Rodentia–Lagomorpha)
Miocene
dated
to 47
12
Lukeino 12
31 of 63
Mouse–clade (Hystricognathi): Cavia–Mus 52.5
58.9
mean="1.91"
stdev="1.0"
offset="52.5"
Birbalomys, from Early Eocene (Ypresian), 14
India.
Muridae: Mus–Rattus
10.4
14.0
mean="1.145"
stdev="1.0"
offset="10.4"
Karnimata, from Late Miocene (Tortonian), 14
Kenya
Myomorpha: Dipodomys–Mus*
40.2
56.0
mean="5.03"
stdev="1.0"
offset="40.2"
Ulkenulastomys, Early Eocene dipodid from 14
Obayla Svita, Zaysan Basin, Kazakhstan
Octodon–Chinchilla*
24.5
37.3
mean="4.075"
stdev="1.0"
offset="24.5"
Eoviscaccia, oldest chinchillid from Deseadan 13
(Late Oligocene)
Laurasiatheria
62.5
131.5
mean="21.95"
stdev="1.0"
offset="62.5"
Protictis,
(Texas)
Erinaceomorpha–Soricomorpha
61.1
84.2
mean="7.351"
stdev="1.0"
offset="61.1"
Adunator, oldest erinaceomorph known from 13
Early Eocene (Torrejonian)
Chiroptera: Mega–Microbats
48.6
65.8
mean="5.475"
stdev="1.0"
offset="48.6"
Icaronycteris,
from
Late
Palaeocene 14
(Clarkforkian Mammal Age, substage 3)
Perissodactyla
55.5
61.1
mean="1.783"
stdev="1.0"
offset="55.5"
Hyracotherium, beginning of the Eocene
Toxodon–Macrauchenia
64.0
84.2
mean="6.905"
stdev="1.0"
offset="62.5"
Molinodus suarezi and Tiuclaenus minutus. 15
Max.= base of the Campanian (83.5 +/- 0.7 =
84.2 Mya)
Bovidae: Bos–Ovis
18.0
28.55
mean="3.357"
stdev="1.0"
offset="18.0"
Eotragus, Early Miocene, Western Europe 12
and Pakistan
Cetartiodactyla
52.5
65.8
mean="4.265"
stdev="1.0"
offset="52.4"
Himalayacetus, from shallow benthic zone 12
SB8, 52.5 Mya (Ypresian)
Whippomorpha*
52.5
61.1
mean="2.738"
stdev="1.0"
offset="52.5"
Himalayacetus, from shallow benthic zone 13
SB8, 52.5 Mya (Ypresian)
Cetacea
33.8
48.8
mean="4.775"
stdev="1.0"
offset="33.8"
Llanocetus, from La Meseta Formation, 13
Seymour Island, Antarctica, latest Eocene
Carnivora
37.1
56.0
mean="6.015"
stdev="1.0"
offset="37.1"
Hesperocyon gregarious,
(Duchesnean) stem canid
Mustelidae–Pinnipedia
33.8
48.8
mean="4.775"
stdev="1.0"
offset="33.8"
Mustelavus priscus, stem musteloid, first 13
occurrence in latest Eocene
Early
Palaeocene
carnivoran 12
Middle
13
Eocene 13
32 of 63
3. Systematic Commentary
3.1 Concepts of SANU Relationships, 1894-2013
The problem of where to put South American native ungulates in the classification of
placental mammals may have started with Darwin and Owen, but it continues to the
present. The many apparent morphological resemblances between SANUs and modern
mammal groups living elsewhere indicated to the Argentinian paleontologist Florentino
Ameghino 16 that not only must they be related to SANUs, but that most of their basal
lineages originated in South America as well. Authors subsequent to Ameghino have
dismissed the majority of these resemblances--and therewith Ameghino’s
interpretation of their significance--as mere convergences (although some of his
inferences may deserve a new look, such as his placing Macrauchenia and most of
Litopterna within Perissodactyla on the basis of dental and pedal similarities). This
effort at systematic housekeeping does not solve, however, the problem of what, if
anything, SANUs really are 17–23. There are many reasons for this, among which are the
following: (1) some nominal orders (Pyrotheria, Xenungulata) are known only from
early, highly derived forms; (2) even the better known groups (e.g., Notoungulata) are
still largely defined by dental attributes; and (3) the initial radiation(s) of SANU taxa
evidently occurred very early in the age of placental mammals in South America, the
fossil evidence for which is notably meagre.
For this section we have compiled, in table form, all of the major published systematic
interpretations regarding how SANUs are related to one other and to other members of
Placentalia. Table S4 focuses on recent efforts and emphasizes character data and
phylogenetic conclusions, while Table S5 presents the majority of published taxonomic
concepts in list form. Resolving relationships for lineages that might have last shared a
common ancestor in the earliest Cenozoic or latest Mesozoic is clearly a difficult
problem. For ease of reference, however, we recognize a clade, Panperissodactyla define
it as the clade uniting all taxa more closely related to crown Perissodactyla than to any
other extant placental taxon. For ease of reference, however, a rankless clade
Panperissodactyla may be defined as including all taxa more closely related to extant
Perissodactyla than to any other extant placental taxon (thus including Notoungulata
and Litopterna on the basis of this study and the phylogenetic branching pattern in fig.
2).
33 of 63
Table S4: Recent hypotheses concerning higher-level phylogenetic affinities of
SANUs within Eutheria*
Phylogenetic
characters
Astrapotheria,
Litopterna,
Notoungulata,
Pyrotheria,
Xenungulata;
also
Didolodontidae
(South American
“condylarths”)
North
American 153 postcranial
“condylarths”
characters
(Arctocyonidae,
Hyopsodontidae,
Meniscotheriidae,
Periptychidae,
Phenacodontidae)
de Muizon & Litopterna
Cifelli 61
(Protolipternida
e:
Asmithwoodwar
dia, Miguelsoria,
Protolipterna);
also
Didolodontidae
(e.g., Didolodus)
and Kollpaniinae
(Tiupampan
“condylarths”,
e.g., Molinodus,
Pucanodus,
Tiuclaenus)
“Zhelestids”;
11 Dental
and
other
basal tarsal characters
eutherians
(e.g.,
Protungulatum);
North
American
Mioclaenidae
Gheerbrant
et al. 60
Litopterna,
Notoungulata;
also
Didolodontidae
and Kollpaniinae
Holarctic
Dental
condylarths
characters
(Arctocyonidae,
Mioclaenidae,
Phenacodontidae); 1
Palaeogene African
?condylarth
(Abdounodus)
Gelfo 61
Litopterna
(Protolipternida
e);
also
Didolodontidae
and Kollpaniinae
Astrapotheria
(Astrapotherium
),
Litopterna
(Diadiaphorus,
Theosodon),
Notoungulata
(Thomashuxleya
)
Horovitz
57
Conclusions
1. Didolodontidae more closely related to Yes
Hyopsodontidae and Meniscotheriidae than
to SANU orders
Close affinity to panPerissodactyla?
Comparative
sample
Close affinity to Afrotheria?
Formal cladistics
Bergqvist 55
South
American
ungulate
sample
analysis?
Reference
No
No
No
No
Probable convergence rather than close No
shared ancestry between Palaeogene African
ungulates and SANUs
No
No
Basal
eutherians Dental
(e.g.,
characters
Protungulatum);
North
American
Mioclaenidae
Panameriungulata are a natural group, but Yes
does not include Protolipternidae
No
No
55 ingroup taxa, 240 postcranial
including
skeletal
representatives of characters
most
living
eutherian
orders,
and several groups
of
Holarctic
condylarths;
11
outgroup taxa
1. SANUs are polyphyletic, in two groups Yes
(Litopterna + Notoungulata vs. Astrapotheria)
No
No
2. Meridiungulata and Notoungulata are not
natural groups
3. Astrapotheria and Xenungulata are closely
related
1. North American Mioclaenidae and South Yes
American Kollpaniinae, Didolodontidae and
Litopterna together may constitute a
monophyletic clade, Panameriungulata
2. “Meridiungulata” containing the 5 orders of
SANUs is probably polyphyletic, but
Notoungulata may eventually prove to be a
member of
Panameriungulata
2. SANU groups are nested in two separate
Holarctic condylarth clades basal to eutherian
crown group comprising carnivores, rodents,
lagomorphs,
macroscelideans,
hyraxes,
proboscideans,
artiodactyls
and
perissodactyls
34 of 63
Shockey & Pyrotheria
Anaya Daza (Pyrotherium)
Arsinoitherium
Tarsal
characters
Pyrotheria share derived tarsal characters No
with Embrithopoda (Afrotheria), possibly
reflecting close shared ancestry
Yes
No
24
Gelfo 25
Litopterna
(Protolipternida
e);
also
Didolodontidae
and Kollpaniinae
Basal
eutherians Dental
(e.g.,
characters
Protungulatum);
North
American
Mioclaenidae
and
Hyopsodontidae
Panameriungulata are a natural group, but do Yes
not include Protolipternidae
No
No
Williamson
& Carr 26
Kollpaniinae
(e.g., Molinodus,
Pucanodus,
Tiuclaenus)
3 “zhelestids”; 27 52 craniodental No close relationship between Mioclaenidae Yes
Holarctic
and
skeletal and either basal South American ungulates or
Mioclaenidae;
1 characters
Palaeogene African ungulates
Palaeogene African
?condylarth
(Abdounodus)
No
No
Comparison
with
perceived
unambiguous
synapomorphies of
Afrotheria
No
Yes
No
Comparison
with Delayed dental No evidence for Afrotheria-like delayed No
perceived
eruption
dental eruption in Notoungulata
unambiguous
synapomorphy
of
Afrotheria
No
No
Bond et al. 67 Astrapotheria,
Litopterna,
Notoungulata,
Pyrotheria,
Xenungulata
Comparison
with
perceived
unambiguous
synapomorphies of
Afrotheria
No
No
Kramarz
al. 27
Comparison
with Delayed dental No evidence for Afrotheria-like delayed No
perceived
eruption
dental eruption in Astrapotheria, Pyrotheria
unambiguous
or Xenungulata
synapomorphy
of
Afrotheria
No
No
Lorente et al. Astrapotheria,
28
Litopterna,
Notoungulata,
Pyrotheria,
Xenungulata
Comparison
with Presence
perceived
astragalar
unambiguous
cotylar fossa
synapomorphy
of
Afrotheria
No
No
Billet & de Notoungulata
Muizon 29
(unidentified
Late Palaeocene
taxon)
Range of eutherians, Petrosal
including
characters
Hyracoidea
(Procavia)
O’Leary et al. Litopterna
30
(Protlipterna),
Notoungulata
(Thomashuxleya
), Xenungulata
(Carodnia); also
Didolodontidae
82 fossil and extant 4541 phenomic 1. SANUs are polyphyletic, in two groups Yes
mammal
taxa, characters
(Litopterna vs. Notoungulata + Xenungulata)
including
71
eutherian taxa
Agnolin
& Bibliographic
Chimento 65 review
of
Astrapotheria,
Notoungulata,
Pyrotheria and
Xenungulata
Billet
Martin 66
& Notoungulata
(Adinotherium,
Nesodon,
Trachytherus)
et Astrapotheria,
Pyrotheria,
Xenungulata
3 dental/skeletal
characters
(delayed cheektooth
replacement;
>19
thoracolumbar
vertebrae; welldefined
astragalar
cotylar fossa)
3 dental/skeletal
characters
(delayed dental
eruption relative
to jaw growth;
>19
thoracolumbar
vertebrae;
presence
of
astragalar
cotylar fossa)
1. SANUs are polyphyletic
2. Astrapotheria and Notoungulata probably
closely related to clades within Afrotheria
3. Pyrotheria and Xenungulata less clearly
related to Afrotheria
4.
Litopterna
probably
Mioclaenidae, not Afrotheria
related
to
1. Supposed synapomorphies with Afrotheria No
have been incorrectly identified in most
SANUs
2. When present, these characters are highly
variable and/or limited to more derived
lineages
of Cotylar fossa is convergently developed in No
several
non-afrothere
eutherian
and
metatherian groups; does not represent
synapomorphy linking SANUs and Afrotheria
Hyracoidea share several probable derived No
petrosal characters with SANUs
Yes? No
Yes
2. Litopterna and Didolodus are nested within
35 of 63
No
(Didolodus)
Pan-Euungulata, forming part of a condylarth
clade that is sister to Perissodactyla +
Cetartiodactyla
3. Notoungulata + Xenungulata are nested
within Afrotheria
Kramarz
Bond 31
& Astrapotheria,
Pyrotheria,
Xenungulata
Comparison
with Delayed dental No evidence for Afrotheria-like delayed No
perceived
eruption
dental eruption in Astrapotheria, Pyrotheria
unambiguous
or Xenungulata
synapomorphy
of
Afrotheria
No
*Authors’ concepts vary considerably concerning what is or is not a “South American
condylarth” vs. “basal litoptern”. Allocations in this table are as presented in the cited
papers.
36 of 63
No
Table S5. Hypotheses of SANU Relationships, Allocations By Clade
SANU Taxon
Considered as
Suborder Toxodontia
Order Ungulata
Author
Lydekker (1894) 32
Pachyrucidae
Typotheriidae
Toxodontidae
Suborder Astrapotheria
Homalodontotheriidae
Astrapotheriidae
Suborder Litopterna
Proterotheriidae
Macraucheniidae
Archaeopithecidae
Prosimiae
Ameghino (1906) 16
Notopithecidae
Henricosborniidae
Hyopsodontidae
Clenialetidae
Eudiastatidae
Acoelodidae
Hyracoidea
Archaeohyracidae
Eutrachytheriidae
Typotheria
Hegetotheriidae
Protypotheriidae
37 of 63
Typotheriidae
Nesodontidae
Toxodontia
Xotodontidae
Haplodontidae
Toxodontidae
Colpodontidae
Hippoidea
Notohippidae
Pantostylopidae
Condylarthra
Phenacodontida
Catathleidae
Pantolambdidae
Arctocyonidae
Hyracotheriidae
Perissodactyla
Palaeotheriidae
Proterotheriidae
Macraucheniidae
Adiantidae
Carolozitteliidae
Proboscidea
Pyrotheriidae
Trigonostylopidae
Amblypoda
Albertogaudryidae
Astrapotheriidae
Lophiodontidae
Isotemnidae
Ancylopoda
Homalotheriidae
Leontiniidae
Notostylopidae
Tillodonta
38 of 63
39 of 63
Familia
incertae
sedis
Didolodus, Notoprotogonia, Order Condylarthra
Lambdaconus, Proectocion
Suborden Homalodotheria
Order Toxodontia
Cohort Ungulata
Osborn (1910) 33
Cohort Ungulata
Scott (1913) 34
Superorder
Notoungulata
Notostylopidae
Homalodotheriidae
Suborder Astrapotheria
Inc. sed. Albertogaudryidae
Inc. sed. Isotemnidae
Astrapotheriidae
Suborden Toxodontia
Inc. sed. Archaeohyracidae
Toxodontidae
Suborder Typotheria
Interatheriidae
Hegetotheriidae
Typotheriidae
Proterotheriidae
Order Litopterna
Macraucheniidae
Pyrotheriidae
Order Pyrotheria
Suborder Toxodonta
Order Toxodontia
Toxodontidae
Notohippidae
40 of 63
Leontiniidae
Suborder Typotheria
Typotheriidae
Interatheriidae
Hegetotheriidae
Notopithecidae
Archaeopithecidae
Archaeohyracidae
Suborder Entelonychia
Notostylopidae
Isotemnidae
Homalodontotheriidae
Suborder Pyrotheria
Pyrotheriidae
Astrapotheriidae
Order Astrapotheria
Trigonostylopidae
Macraucheniidae
Order Litopterna
Proterotheriidae
Didolodidae
41 of 63
Bunolitopternidae
Suborder Litopterna
Order Ungulata
Mammalia
Schlosser (1923) 35
Mammalia
Simpson (1934) 36
Macraucheniidae
Proterotheriidae
Adianthidae
Pyrotheria
Suborder Amblypoda
Notopithecidae
Suborder Typotheria
Order
Notoungulata
Interatheriidae
Hegetotheriidae
Typotheriidae
Archaeopithecidae
Archaeohyracidae
Notohippidae
Suborder Toxodontia
Nesodontidae
Toxodontidae
Arctostylopidae
Suborder
Entelonychia
Notostylopidae
Isotemnidae
Leontiniidae
Homalodontotheriidae
Trigonostylopidae
Suborder
Astrapotherioidea
Albertogaudryidae
Astrapotheriidae
Didolodontidae
Order Condylarthra
42 of 63
Macraucheniidae
Order Litopterna
Proterotheriidae
Arctostylopidae
Suborder
Notioprogonia
Order
Notoungulata
Henricosborniidae
Notostylopidae
Isotemnidae
Suborder
Entelonychia
Homalodotheriidae
Notohippidae
SuborderToxodontia
Toxodontidae
?Leontiniidae
Notopithecidae
Suborder Typotheria
Interatheriidae
Typotheriidae
?Archaeohyracidae
? Acoelodidae
Astrapotheriidae
Suborden
Astrapotherioidea
Trigonostylopidae
Suborder
Trigonostylopoidea
Pyrotheriidae
Order Pyrotheria
Order
Astrapotheria
43 of 63
Didolodontidae
Order Condylarthra
Proterotheriidae
Order Litopterna
Superorder
Protoungulata
Simpson (1945) 17
Macraucheniidae
Arctostylopidae
Suborder
Notioprogonia
Order
Notoungulata
Henricosborniidae
Notostylopidae
Oldfieldthomasiidae
Suborder Toxodontia
Archaeopithecidae
Archaeohyracidae
Isotemnidae
Homalodotheriidae
Leontiniidae
Notohippidae
Toxodontidae
Interatheriidae
Suborder Typotheria
Mesotheriidae
Hegetotheriidae
Suborder
Hegetotheria
Trigonostylopidae
Suborder
Trigonostylopoidea
Astrapotheriidae
Suborder
Astrapotherioidea
Pyrotheriidae
Order Pyrotheria
Superorder
Paenungulata
Litopterna
Mirorder
Meridiungulata
Grandorder
Ungulata
Order
Astrapotheria
McKenna (1975) 37
Notoungulata
44 of 63
Astrapotheria
Trigonostylopoidea
Xenungulata
Pyrotheria
Astrapotheria
Tachytelic lineage
Arctocyonid
ancestral stock
Soria (1989) 38
Notoungulata
Xenungulata
Pyrotheria
?Notopterna
Didolodontidae
Bradytelic lineage
Litopterna
Lucas (1993) 39
Dinocerata
Uintatheriomorpha
Pyrotheria (+Xenungulata)
45 of 63
Didolodontidae
Grandorder
Ungulata
Order Condylarthra
McKenna
(1997) 21
and
Bell
Mioclaenidae
Mirorder
Meridiungulata
Perutheriidae
Protolipternidae
Order Litopterna
Macraucheniidae
Notonychopidae
Adianthidae
Proterotheriidae
Henricosbornidae
Suorder
Notioprogonia
Order
Notoungulata
Notostylopidae
Isotemnidae
Suborder Toxodontia
Leontiniidae
Notohippidae
Toxodontidae
Homalodotheriidae
Archaeopithecidae
Suborder Typotheria
Oldfieldthomasiidae
Interatheriidae
Campanorcidae
Mesotheriidae
Archaeohyracidae
Suborder
Hegetotheria
Hegetotheriidae
Eoastrapostylopidae
Order Astrapotheria
Trigonostylopidae
46 of 63
Astrapotheriidae
Carodniidae
Order Xenungulata
Pyrotheriidae
Order Pyrotheria
3.2 50% Majority Rule Bayesian Consensus Tree (Main Text Fig. 2):
Comparison to Results of Other Recent Investigations
The tree depicted in fig. 2 in the main text was constructed by analysing all trees in the
posterior distribution of trees (excluding those generated during burn-in) (see S2.3
Phylogenetic Reconstruction Methods). Nodes with less than 50% support are shown as
polytomies. There are some anomalies in our results in comparison to recent large-scale
genomic, metagenomic, and morphological (or phenomic) analyses of placental
mammals and commentaries thereon 14,30,40–45 (e.g., positions of Scandentia,
Heterocephalus and Mammut; relative positions of Macropus, Monodelphis and
Sarcophilus within Marsupialia, and of felids, ursoids and pinnipeds within Carnivora).
These are probably mostly due to the limitations of collagen sequence data, although
some poorly resolved nodes in this study have also proven problematic in other recent
analyses (e.g., position of echolocating Chiroptera vis-à-vis Primates, relationships
within Paenungulata). Missing taxa, gene lineage sorting, as well as atypical rates of
molecular evolution or selection acting on the protein sequences may have additionally
affected phylogenetic resolution.
Basal diversification within the Placentalia is a historically unstable and debated node;
recent papers have favoured one or another of three different clades in the basal
position (Afrotheria vs. Xenarthra vs. Atlantogenata [Afrotheria + Xenarthra])14,45.
These clades have sometimes been recovered as an unresolved trichotomy41, although
some genomic analyses e.g. 14,30 have identified either Xenarthra (branches shaded olive)
or Atlantogenata rather than Afrotheria (dark brown) as the sister of other Placentalia,
in contrast to our portrayal (for recent discussion, see46. Extant Laurasiatheria (shades
of green and blue) and Euarchontoglires (red, pink, orange, light brown) form a wellsupported clade equivalent to Boreoeutheria. Xenarthra is internally organized as
expected, but there is a trichotomy at the base of weakly-supported Afrotheria (0.64).
Afroinsectiphilia43 was not recovered, and Orycteropus and Macroscelidea
(Elephantulus) form a clade that does not include either Chrysochloridae (Chrysochloris)
or Tenrecidae (Echinops), which group separately (for choice of ranks, see47).
Lipotyphla (dark green) stands as sister to other laurasiatheres grouped in another
weakly-supported trichotomy (0.56) consisting of Carnivora, Chiroptera, and
Euungulata (Perissodactyla + Artiodactyla).
Euungulata as currently defined is supported by several different data sets13,30,48–50.
Here it receives only marginal support (0.64), and the failure to recover Whippomorpha
(Cetacea + Hippopotamidae) within Artiodactyla, even though other relationships are
plausibly resolved, is problematic. By contrast, within crown Perissodactyla,
Hippomorpha (horses, asses, zebras) and Ceratomorpha (rhinos, tapirs) were recovered
with fully expected contents and relationships. The collagen sequence of the test fossil,
Equus sp. MACN Pv 5719 from the latest Pleistocene locality of Tapalqué, proved to be
nearly identical to that of modern Equus caballus. This was expected, and gives
47 of 63
additional credibility to sequence information gained from fossils of similar age and
taphonomic history. Further, our protein result is consistent with ancient DNA results
showing that (with the exception of Onohippidion) the Pleistocene horses of South
America had not diverged greatly from their North American forebears at the time of
their extinction51.
As noted in the main text, the strongly supported monophyletic clade containing
Toxodon and Macrauchenia is placed immediately next to crown Perissodactyla in all
trees investigated (see inset), and thus our results provide a significant test of certain
hypotheses presented by 30 (see also Extended Data Fig S4). With regard to the position
of Litopterna, our results are consistent with 30 in that both studies place the latter
order within Euungulata. They were able to refine the placement of Litopterna further
with phenomic evidence that tended to support the view 52,53 that this SANU order
(represented in their analysis by the basal South American taxon Protolipterna
ellipsodontoides, and also by the South American condylarth family Didolodontidae as
represented by Didolodus multicuspis) diverged from one of the clades within
paraphyletic North American Condylarthra and may therefore be described as part of
stem Euungulata. We do not have proteomic evidence for any members of Condylarthra
and therefore cannot stipulate which of the known taxa might be objectively considered
(in our preferred terminology) members of Panperissodactyla. However, Palaeogene
meniscotheres, phenacodonts, and mioclaenids52, among others26, are obvious
possibilities (Tables S4 & 5). It is also appropriate to note here that it has been claimed
that at least one condylarth group, the louisinine apheliscids, might be situated close to
the ancestry of at least one afrothere ordinal taxon, Macroscelidea (sengis or elephant
shrews)54,55. This novel allocation effectively illustrates why Condylarthra is, as has long
been suspected, a paraphyletic dustbin replete with taxa of generally primitive aspect
which may, after appropriate phylogenetic investigation, turn out to have dramatically
different affinities.
Of special significance is the fact that our Toxodon results do not support the inference
that Notoungulata belongs in or near Afrotheria, as some recent authors30,56 have
contended (Tables S4 & 5). Instead, Toxodon consistently groups with Macrauchenia
and thence with Perissodactyla, indicating that Litopterna and Notoungulata did not
diverge within different superorders of placentals but instead evolved exclusively
within Laurasiatheria. This being the case, the Palaeocene henricosborniid notoungulate
Simpsonotus praecursor can no longer be regarded as the oldest taxon within the
afrothere clade Pan-Paenungulata as defined by O’Leary et al.30, Table S2. Because we
have no collagen sequence data for the remaining SANU orders (Astrapotheria,
Pyrotheria, Xenungulata) we cannot comment on their placement, other than to
mention that if they eventually also prove to group with crown Perissodactyla, then
panperissodactyls would become one of the largest units within Laurasiatheria.
The recent paper by Beck and Lee57 is primarily concerned with exploring how different
clock models (over)estimate divergence dates in eutherian cladogenesis. However, it is
important to cite in the present context because it incorporates in a single study
representatives of four of the five nominal SANU orders--Astrapotheria
(Trigonostylops), Litopterna (Diadiaphorus), Pyrotheria (Pyrotherium), and
Notoungulata (Simpsonotus, Henricosbornia, and Thomashuxleya)--as well as several
condylarths (sensu lato) and a stem perissodactyl (Hyracotherium). In order to produce
trees conforming to well-supported concepts of eutherian relationships in the recent
48 of 63
literature, the authors had to run their 421-character matrix using various topological
constraints (unconstrained trees are reported in supplementary files). Thus
“full/monophyly” was enforced for their combination ‘Notoungulata’ (Simpsonotus,
Henricosbornia, Thomashuxleya, and Pyrotherium), meaning that these 4 taxa were
constrained to act as a closed monophyletic unit (no external taxa joining internal
nodes). Inclusion of Pyrotherium in ‘Notoungulata’ is undefended but presumably
reflects the study of Billet58. Less rigidly constrained combinations included
‘Paenungulata’, ‘Afrotheria’, and ‘Laurasiatheria’. Trigonostylops, Diadiaphorus, and
condylarths were unconstrained, but Hyracotherium was required to group with
‘Laurasiatheria’. In their two preferred trees, ‘Notoungulata’ was recovered as the sister
group of a clade containing Hyracotherium, Diadiaphorus, and Trigonostylops. This
agrees with the narrower determination made in the present paper, that notoungulates
(sensu stricto) and litopterns have no relationship to afrotheres, but are instead
uniquely related to stem perissodactyls. Placement within Laurasiatheria was
presumably forced by the constraint on Hyracotherium, but the result is certainly
plausible overall. Also noteworthy is the fact that unconstrained Periptychus,
Mioclaenus, and South American condylarths group in a separate clade that is sister to
the foregoing, in partial agreement with de Muizon and Cifelli52. Finally, their results
imply that astrapotheres and litopterns may be more closely related to stem
perissodactyls than they are to pyrotheres and notoungulates (sensu stricto), although
this may likewise be a consequence of the constraint regime and in any case this
inference goes beyond what the matrix was intended to do (Robin Beck, pers. commun.,
25/10/14).
Finally, Meredith et al.47 have criticized the “extremely short fuse” model advocated by
O’Leary et al.30, which specified that the conventional superorders of crown placentals
emerged within a few hundred thousand years of the K/Pg event. Morphology-based
phylogenies of Placentalia tend to place divergence times of all crown superorders at or
near the beginning of the Cenozoic 66 million years ago (Ma)59, but molecular
investigations usually predict much earlier origins, which obviously allows more time
for cladogenesis. A recent example is the paper by Zhou et al.50, which employed first
and second codon positions in multiple gene sets as a basis for estimating phylogenetic
relationships. They concluded that the major divergences among laurasiatheres
occurred in the Late Cretaceous, not the early Cenozoic as most
palaeontological/morphological studies have posited30,59. According to Zhou et al.50,
Euungulata diverged during the Campanian (83.6-72.1 Ma), either around the time of
the Campanian/Turonian transition ca. 83 Ma (range, 91-76) as indicated by their 97gene set, or during the later Campanian around 76 Ma (89-66 Ma) based on their 60gene set. Significantly, this provides a long lead time for cladogenesis within
euungulates: Perissodactyla is posited to have originated in the Middle Palaeocene ca.
58 Ma (61-55) in both gene models, which is only slightly or not at all earlier than the
latest Palaeocene/earliest Eocene origin (~56 Ma) favoured in many palaeontological
discussions. In addition to various controversial indications that placentals
phylogenetically related to euungulates were already present by the end of the
Mesozoic60, the emerging South American record, combined with the analyses
developed in this paper, strongly implies that by the start of the Palaeogene
cladogenesis within Panperissodactyla had already led to the separation of the crown
clade from some or all SANU lineages, as the next section examines with our molecular
dating analysis in BEAST.
49 of 63
3.3 Testing Results: Additional Phylogenetic Trees
The positions of several nodes in mammalian phylogeny have not been fully resolved by
recent analyses. For example, recent studies 13,14,61 have notably differed for such
elements as the placement of Afrotheria, Xenarthra, and Boreoeutheria; whether
Scandentia grouped with Glires or Primates; and the branching patterns of clades
within Laurasiatheria.
To examine whether differing topologies would have any effect on the position of
Toxodon and Macrauchenia, we performed additional analyses (Extended Data Figures
S4 & S5), constraining the nodes to those clades found in 13,14,61. In Extended Data Figure
S5 we depict the run made with the preferred tree presented by O’Leary et al. 30 (their
fig. 1), with the following nodes constrained: Eutheria, Afrotheria, Xenarthra,
Boreoeutheria, Euarchontoglires, Laurasiatheria, Euarchonta, Euungulata, Ferungulata,
Scrotifera, with the basal relationship at the root of the placental tree being defined as
(Xenarthra (Afrotheria, Boreoeutheria)). Results in this and all the other analyses (not
shown) were identical: Toxodon and Macrauchenia were found to group
monophyletically, with 100% posterior probability. Similarly, the panperissodactyl
grouping was found for all constrained analyses, with 100% posterior probability.
Furthermore, we found that an alternative topology, where Macrauchenia and Toxodon
are constrained to be part of a monophyletic group (SANU+Afrotheria), is significantly
less parsimonious than an unconstrained analysis (Templeton test, p>0.0001).
To examine the timings of divergence events, a molecular dating analysis was
performed in BEAST62. Extended Data Figure S4 shows the dated maximum clade
credibility tree from this analysis, which provides a perspective on the estimated
divergence times for SANUs in a larger context. Given the limited amount of genetic
information utilized to create this tree, extensive commentary is not warranted. For a
recent evaluation of issues and methods as they affect divergence dating in mammals in
particular, see63.
Our divergence estimates indicate that the last common ancestors of Notoungulata +
Litopterna and crown Perissodactyla separated during the latter half of the Cretaceous,
89-74 Ma (base of Coniacian to latter part of Campanian). As noted elsewhere, there is
no fossil evidence for any of these taxa or their putative ancestors during this interval.
By contrast, the estimate for the divergence of ceratomorph and hippomorph
perissodactyls (ca. 56-58 Ma, i.e., within the Late Palaeocene) is arguably consistent
with the long-standing palaeontological understanding that crown perissodactyls
emerged near the Palaeocene/Eocene transition, presumably from a condylarth
ancestry30,64.
The two SANU lineages represented in this study, taken as proxies for the orders
Litopterna and Notoungulata, are estimated to have diverged ca. 69-64 Ma. Although
this interval is not wide, it covers a period between the approximate base of the
Maastricthian (end-Cretaceous) and the middle of the Danian (Early Palaeocene), and
thus embraces the K/Pg boundary (66 Ma). The younger age estimate simply replicates
the minimum accepted palaeontological ages for both notoungulates and litopterns in
South America (see Data Tables S4 & 5). However, it should be emphasized that this
datum correlates well with increasing evidence for a broad turnover in both flora and
50 of 63
fauna in Patagonia at that time. The Early Palaeocene is the time when the arrival of
northern elements first becomes evident, but was perhaps initiated just prior to the
K/Pg boundary (see review by 65). Although brief intercontinental landbridge
connections, microcontinental collisions, and so forth have been hypothesized in the
past to explain why such transfers were concentrated at the very beginning of the
Cenozoic, acceptable evidence for a dryland route between the Americas at that time is
lacking66. Given increasing evidence for filtered dispersal of various mammalian groups
over major sea “barriers” in the later Cenozoic, we cannot assume that north-south
movements would have been likelier in the Palaeocene than the terminal Mesozoic. On
the other hand, if there was a Late Cretaceous interchange of land vertebrates (by
whatever means), as new discoveries certainly seem to indicate, it is indeed odd that it
apparently did not involve placental mammals67.
3.4 Darwin, Owen, and Initial Interpretations of SANU Relationships
Charles Darwin began his introduction to On the Origin of Species: “When on board
H.M.S. ‘Beagle,’ as naturalist, I was much struck with certain facts in the distribution of
the inhabitants of South America, and in the geological relations of the present to the
past inhabitants of that continent.”68. Two of these extinct past inhabitants were large
mammals described by Richard Owen69, the type specimens of which Darwin had
collected on the voyage. Toxodon platensis, “a part, very perfect, of the head”, he
purchased from a rancher in Uruguay “for a few shillings.” Darwin70. Darwin also found
jaw fragments and teeth of this species in Argentina. Macrauchenia patachonica was
collected by him in a channel of sandy soil in a sandstone cliff at Port St. Julian, Santa
Cruz province, on the coast of Southern Patagonia (see main text fig. 1b). It was his only
collection of the species.
Both Darwin and Owen were quite uncertain about the affinities of Toxodon. Darwin71
wrote that he was quite taken aback by
...the Toxodon, perhaps one of the strangest animals ever discovered: in size it
equalled an elephant or megatherium, but the structure of its teeth, as Mr. Owen
states, proves indisputably that it was intimately related to the Gnawers, the order
which, at the present day, includes most of the smallest quadrupeds: in many
details it is allied to the Pachydermata: judging from the position of its eyes, ears,
and nostrils, it was probably aquatic, like the Dugong and Manatee, to which it is
also allied. How wonderfully are the different Orders, at the present time so well
separated, blended together in different points of the structure of the Toxodon!
More importantly, Darwin soon realized that these gigantic animals might provide clues
to his understanding of species formation. His Red Notebook, begun on the voyage in
1836 and continued in 1837 after he reached England, contains his first known notes on
evolution, including: “if one species does change into another it must be per saltum - or
species may perish.” This follows notes on the relationships between the two living
species of rheas in Patagonia and the “extinct Guanaco” (Macrauchenia) and the living
guanaco 72. So, as early as 1837, Darwin realized that evolution of species might result
from geographical or geological factors.
51 of 63
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