Vol 450 | 20/27 December 2007 | doi:10.1038/nature06343

ARTICLES
Whales originated from aquatic
artiodactyls in the Eocene epoch of India
J. G. M. Thewissen1, Lisa Noelle Cooper1,2, Mark T. Clementz3, Sunil Bajpai4 & B. N. Tiwari5
Although the first ten million years of whale evolution are documented by a remarkable series of fossil skeletons, the link tothe ancestor of cetaceans has been missing. It was known that whales are related to even-toed ungulates (artiodactyls), but
until now no artiodactyls were morphologically close to early whales. Here we show that the Eocene south Asian raoellid
artiodactyls are the sister group to whales. The raoellid Indohyus is similar to whales, and unlike other artiodactyls, in the
structure of its ears and premolars, in the density of its limb bones and in the stable-oxygen-isotope composition of its teeth.
We also show that a major dietary change occurred during the transition from artiodactyls to whales and that raoellids wereaquatic waders. This indicates that aquatic life in this lineage occurred before the origin of the order Cetacea.
Phylogenetic analyses of molecular data on extant animals strongly
support the notion that hippopotamids are the closest relatives ofcetaceans (whales, dolphins and porpoises)1–3. In spite of this, it isunlikely that the two groups are closely related when extant andextinct artiodactyls are analysed, for the simple reason that cetaceansoriginated about 50 million years (Myr) ago in south Asia, whereas
the family Hippopotamidae is only 15 Myr old, and the first hippopotamids to be recorded in Asia are only 6 Myr old4. However, analyses of fossil clades have not resolved the issue of cetacean relations.
Proposed sister groups ranged from the entire artiodactyl order5,6, tothe extinct early ungulates mesonychians7, to an anthracotheroid
clade8 (which included hippopotamids), to weakly supporting hippopotamids (to the exclusion of anthracotheres9,10).
The middle Eocene artiodactyl family Raoellidae11–14 is broadly
coeval with the earliest cetaceans, and both are endemic to south
Asia. Raoellids, as a composite consisting of several genera, have been
added to some phylogenetic analyses5,10, but no close relation towhales was found because raoellid fossils were essentially limited todental material11–14. We studied new dental, cranial and postcranialmaterial for Indohyus, a middle Eocene raoellid artiodactyl from
Kashmir, India (Fig. 1). All fossils of Indohyus were collected at a
middle Eocene bone bed extending for about 50 m at the locality
Sindkhatudi in the Kalakot region of Kashmir on the Indian side of
the Line of Control. Our analysis identifies raoellids as the sistergroup to cetaceans and bridges the morphological divide that separated early cetaceans from artiodacyls. This has profound implications for the character transformations near the origin of cetaceans
and the cladistic definition of Cetacea, and identifies the habitat inwhich whales originated. Taken together, our findings lead us topropose a new hypothesis for the origin of whales.
Cetaceans and raoellids are sister groups
To investigate the importance of raoellids in cetacean phylogeny, we
excluded raoellids from our initial phylogenetic analysis of artiodactyls plus cetaceans. Our data set differed from previous analyses10 by
the addition of several archaic anthracotheres, and some correctedscores for pakicetid cetaceans. This analysis found stronger support
for hippopotamid–cetacean sister-group relations than the previous
analysis10, consistent with molecular studies1–3. However, the base of

the artiodactyl cladogram is poorly resolved (see Supplementary
Information for details on phylogenetic runs). In a second cladistic
analysis (Fig. 2), we added the raoellids Khirtharia and Indohyus aswell as several archaic ungulate groups (condylarths) and found thatraoellids and cetaceans are sister groups and that they are the basal
node in the Cetacea/Artiodactyla clade, consistent with some previous analyses that used different character sets5,6. Our analysis is thefirst to show that raoellids are the sister group to cetaceans, resolving
the biogeographic conundrum and closing the temporal gap between
cetaceans and their sister. Relations between most artiodactyl families
higher in the tree are poorly resolved, and our data lack implications
for the relations between these families. Our analysis strongly argues
that raoellids and cetaceans are more closely related to each otherthan either is to hippopotamids.
Indohyus shares with cetaceans several synapomorphies that are
not present in other artiodactyls. Most significantly, Indohyus has athickened medial lip of its auditory bulla, the involucrum (Figs 1 and3), a feature previously thought to be present exclusively in cetaceans.
Involucrum size varies among cetaceans, but the relative thickness of
medial and lateral walls of the tympanic of Indohyus is clearly within
the range of that of cetaceans and is well outside the range of othercetartiodactyls (Fig. 3). Other significant derived similarities between
Indohyus and cetaceans include the anteroposterior arrangement ofincisors in the jaw, and the high crowns in the posterior premolars.
Characterizing Cetacea
Until now, the involucrum was the only character occurring in
all fossil and recent cetaceans but in no other mammals5,15,16.
Identification of the involucrum in Indohyus calls into question
what it is to be a cetacean: it requires either that the concept ofCetacea be expanded to include Indohyus or that the involucrum
cease to characterize cetaceans. We argue that the content
of Cetacea should remain stable and include Pakicetidae,
Ambulocetidae, Remingtonocetidae, Protocetidae, Basilosauridae,
Mysticeti and Odontoceti6,7,9,10,17. Thus, Cetacea remains a monophyletic group, whereas Artiodactyla remains a paraphyletic group(because Raoellidae are included but Cetacea are excluded).
An alternative classification would render both Cetacea andArtiodactyla monophyletic by including Raoellidae in Cetacea and1
Department of Anatomy, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272, USA. 2School of Biomedical Sciences, Kent State University, Kent, Ohio 44242,
USA. 3Department of Geology and Geophysics, University of Wyoming, Laramie, Wyoming 82071, USA. 4Department of Earth Sciences, Indian Institute of Technology, Roorkee,
Uttarakhand 247 667, India. 5Wadia Institute of Himalayan Geology, Dehra Dun, Uttarakhand 248 001, India.

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by limiting Artiodactyla to those clades one node above the raoellid/
cetacean node (Fig. 2). We do not prefer this classification because it
causes instability by significantly altering the traditional content ofboth Artiodactyla and Cetacea.
Characters identified as synapomorphies for Cetacea in some ofour most parsimonious trees include: long external auditory meatus,
double-rooted P3/, lack of P4/ protocone, M1-2/ metacones present
but small, and lack of M1-2/ hypocone. None of these features characterize all modern and extinct cetaceans; the dental characters, forinstance, cannot be scored in toothless mysticetes. In addition, all ofthese characters are found in some mammals unrelated to cetaceans.
We attach particular importance to two character complexes thatcharacterize basal cetaceans, constitute synapomorphic suites for theorder, and are of great functional importance. All fossil and recent
cetaceans differ from most other mammals in the reduction of crushing basins on their teeth: there are no trigonid and talonid basins in
the lower molars, and the trigon basin of the upper molars is verysmall (for example in pakicetids and ambulocetids) or absent.

aCrushing basins are large in raoellids (Fig. 1a, b) and other basal
ungulates. This implies that a major change in dental function
occurred at the origin of cetaceans, probably related to dietary change
at the origin18. Reduced crushing basins also occur in mesonychids,
archaic ungulates long thought to be closely related to cetaceans.
However, mesonychian molars have wear facets very unlike thoseof cetaceans7,18, whereas wear facets in raoellids are more similar towear facets in early cetaceans14.
The second character complex that identifies cetaceans is the shape of
the postorbital and temporal region of the skull. In early cetaceans, thisregion is long and narrow19. This affects the sense organs: the olfactory
peduncle is long and narrow and the orbits are set close together near
the roof of the skull. It also affects oral function, the nasopharyngeal
duct is narrow, and the out-lever of the masticatory muscles is long,increasing the closing speed of the jaws. We speculate that the importance of different sense organs was related to these changes, or thatchanging diet led to a change in food-processing organs.
Arctocyon
Hyopsodus
Phenacodus
Meniscotherium

Lateraltympanic
wall

Eoconodonb

Khirtharia
Indohyus
Pakicetus
Ambulocetus
Rodhocetus
ArtiocetuscTympanic bulladehDiacodexis
Gujaratia
Cebochoerus
Mixtotherium
Siamotherium
Amphirharagatherium
Gobiohyus
Homacodon
BunomeryxijMedullarycavityMicrobunodon
Anthracokeryx
ElomeryxfEntelodon
Perchoerus
Tayassu
Sus
Choeropsis
HippopotamuskgCortical
bone

l

1 cm

Merycoidodon
Agriochoerus
Protoceras
Heteromeryx
Leptoreodon
Hypertragulus
Leptomeryx
Amphimeryx
XiphodonEotylops
Poebrotherium
Cainotherium
Tragulus

1 cm

Figure 1 | Osteology of Indohyus and cross-sections of long bones of
Eocene cetartiodactyls. a, b, Oblique lateral view of skull RR 208 (a) andventral view of skull RR 207 (b). c–h, Posterior views of humerus (RR 149,c) and femur (RR 101, d), plantar views of metacarpal (RR 138, e) andproximal manual phalanx (RR 19, f), dorsal view of astragalus (RR 224, g),and posterior view of metatarsal (RR 139, h). i–l, Histological mid-shaft
sections for humerus of the pakicetid Ichthyolestes (H-GSP 96227, i),humerus of Indohyus (RR 157, j), femur of Indohyus (RR 42, k) and femur of
the artiodactyl Cainotherium (IVAU unnum, l). Both scale bars are 1 cm; thescale bar near d goes with a–h, and that near l goes with i–l.

Cetacea Raoellidae

Middle ear
cavityOther Artiodactyla

Involucrum

Crushingbasins

Hapalodectes
Dissacus
Ankalogon
Sinonyx
Pachyaena gigantea
Pachyaena ossifraga
Mesonyx
Synoplotherium
Harpagolestes

Mesonychidae

Andrewsarchus

Figure 2 | Phylogeny of artiodactyls, cetaceans and archaic ungulates. The
figure shows a consensus cladogram produced by heuristic searches with
PAUP (random addition sequence, 1,000 repetitions), using a publisheddata set10. See Supplementary Information for further details.
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Indohyus was aquatic
Behaviourally, the earliest whales (pakicetids) were aquatic
waders5,20–23. This led us to investigate whether Indohyus was aquatic
too. Cortical bone thickness in secondarily aquatic tetrapods is commonly increased at the expense of the medullary cavity, a patterncalled osteosclerosis24. Osteosclerosis occurs in early whales20,21,25,
manatees26,27, sea otters28, Hippopotamus29, beavers29, pinnipeds29
and Mesozoic marine reptiles26,30. Osteosclerosis provides ballastthat allows some aquatic taxa to be bottom walkers (hippopotamids)
and others to maintain neutral buoyancy in water (manatees)30.
Histological sections indicate that the limb bones of Indohyus arealso osteosclerotic (Fig. 1i–l), in a similar manner to those of pakicetid cetaceans. Our survey of cortical bone thickness in the limb
bones of terrestrial artiodactyls shows that this pattern is unusual
for that order: in mid-bone cross-sections of the femur, the medullary cavity makes up between 0.60 and 0.75 of the width of the bone,whereas in aquatic mammals the values are lower (Hippopotamus,
0.55; pakicetids and ambulocetids, 0.25–0.57). In Indohyus this ratio
is 0.42, suggesting that Indohyus was osteosclerotic and thus aquatic.
We interpret the limb osteosclerosis of Indohyus to be related tobottom walking and not to slow swimming, because the limbs are
gracile and not modified into paddles.
To investigate further the hypothesis that Indohyus was aquatic, westudied the stable isotopes of its enamel, a tissue relatively resistantto preburial and postburial alteration of isotopic composition31.
Enamel d18O values are influenced by the oxygen isotope composition of the food and water ingested by an animal as well as by certain
physiological processes (such as sweating, panting and respiration)32.
For aquatic species, the flux of environmental water by means of
direct ingestion and transcutaneous exchange overwhelms all otheroxygen sources33 and can cause the enamel d18O values of freshwatertaxa (for example Hippopotamus) to be 2–3% lower than those forterrestrial mammals33,34. Mean d18O values for four individuals of
Indohyus are at least 2% lower than those for our comparative sampleof Eocene terrestrial and semi-aquatic mammals from formations ofIndia and Pakistan of similar or slightly older age (Fig. 4)22,23.
Although not representative of the specific deposits from which
Indohyus was collected, oxygen isotope values for each ecological typefrom these sites (namely terrestrial, 24–28%; semi-aquatic, 23% orless) are surprisingly consistent regardless of age or location (Fig. 4).
This suggests that temporal and spatial variation in environmental
isotope values was relatively minor and was most probably insufficient to account for the extremely low d18O we have reported forIndohyus. We did not recover tooth material of other mammals at the

Indohyus site; until such material can be analysed, the most consistentinterpretation is that these low values are a result of the aquatic habitsof this species.
Supporting evidence for a semi-aquatic life of Indohyus comesfrom examination of its inter-individual variation in d18O values.
The overwhelming influx and mitigating influence of isotopicallyhomogeneous environmental water causes the variation in individual
d18O values for semi-aquatic and aquatic species (s.d. , 0.5%) to bemuch lower than that of terrestrial species (s.d. . 1.0%)33. This lineof evidence is especially relevant in our study because it does not
require an approximation of the mean environmental isotope values
for a site through analysis of the associated fauna. Variation in d18O
values for Indohyus (s.d. 5 0.4%) is extremely low and when compared with species of sufficient sample size (n . 3) it is similar to thatof the semi-aquatic archaeocete Pakicetus. Given that the influence ofphysiological and environmental factors on body water d18O values ismore strongly felt at smaller body sizes35, this low level of variation isparticularly compelling for Indohyus, with a body mass of less than
50 kg.
To explore the diet of Indohyus we studied carbon isotopes.
Enamel d13C values are defined by the carbon isotope compositionof an animal’s diet and can be used to identify the food webs andresources used by an animal36. The d13C values of primary producers
at the base of aquatic and terrestrial food webs overlap, but values for
freshwater phytoplankton are typically depleted in 13C relative tofreshwater macrophytes37, and both types of aquatic producer are
depleted in 13C relative to terrestrial C3 plants37–39. Consumers foraging within food webs fuelled by freshwater phytoplankton (forexample freshwater and brackish-water foraging Eocene whales) typically have lower d13C values than species foraging on aquatic macrophytes37 or on terrestrial resources33 (Fig. 4). Enamel d13C values for
Indohyus are higher than those for most early cetaceans and are mostsimilar to the d13C values in enamel for terrestrial mammals fromearly and middle Eocene deposits in India and Pakistan. Indohyus
could have been feeding on land or in water, but it was clearly eating
something different from archaeocetes such as Pakicetus andAmbulocetus. If the large crushing basins in the molars of Indohyus
were used for processing vegetation, these d13C values in enamel
could come from the ingestion of terrestrial plants or aquatic macrophytes. Alternatively, a more ominivorous diet would suggest that
Indohyus might have foraged on benthic, aquatic invertebrates in–6
Brackish-water
Anthracobunids andKhirtharia–820

Indohyus

15

–10
δ13C

Tympanic thickness (medial/lateral walls)

25

10

Terrestrial
mammals

Indohyus

–12
5

0
2.5

3.0

4.0
3.5
4.5
ln [Width occipital condyles (mm)]

5.0

5.5

Figure 3 | Plot of the ratio of the thickness of the medial tympanic wall tothat of the lateral tympanic wall against the natural logarithm of the width
across occipital condyles, showing that the ratio in Indohyus is similar tothat in cetaceans. In cetaceans (open squares), the medial tympanic wall is
inflated and called the involucrum, and the lateral tympanic wall is thinned
and called the tympanic plate. In artiodactyls (open triangles), the medial
and lateral tympanic walls are more similar in thickness, causing values on
the y axis to be closer to 1. See Supplementary Information for further
details.

Freshwater andbrackish-water
Archaeocetes

–14
20

22

24

26

28

30

δ18O

Figure 4 | Bivariate plot of d18O and d13C values for enamel samples of
early and middle Eocene mammals from India and Pakistan. Results areshown as means 6 s.d. for the sample population. Filled triangle, Indohyus;
open triangle, Khirtharia; open squares, terrestrial mammals; filled squares,
brackish-water anthracobunids; filled circles, freshwater/brackish-waterarchaeocetes. See Supplementary Information for details.

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For analysis of stable isotopes, we prepared powders by following publishedmethods (see Methods and Supplementary Tables 4 and 5). Multiple samples
were collected for each species to provide an estimate of population means forcarbon and oxygen isotope values33. About 5 mg of enamel powder was collectedfrom each specimen (tooth) for study of carbon and oxygen isotope values.
Most fossils of Indohyus were collected by the late Indian geologist A. Ranga
Rao, who discovered the locality about 25 years ago (acronym RR); additional
fossils were collected by S.B. and B.N.T. at the same locality (acronym IITR-SBKal-S).

10 cm

Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.

Figure 5 | Skeletal reconstruction of Indohyus. Hatched elements arereconstructed on the basis of related taxa.

freshwater systems. Although we cannot exclude the possibility ofaquatic foraging by Indohyus, d13C values in enamel do suggest thatthe diet of Indohyus differed significantly from that of Eocene whales.
A more refined interpretation of the dietary preferences of Indohyus
will require a study of tooth wear and tooth morphology.
Evolutionary hypothesis for whale origins
Indohyus was a small, stocky artiodactyl, roughly the size of the raccoon Procyon lotor (Fig. 5). It was not an adept swimmer; instead it
waded in shallow water, with its heavy bones providing ballast to keep
its feet anchored. Indohyus may have fed on land, although a specialized aquatic diet is also possible.
The modern artiodactyl morphologically most similar to Indohyus
is probably the African mousedeer Hyemoschus aquaticus.
Hyemoschus lives near streams and feeds on land, but flees into thewater when danger occurs40. Indohyus had more pronounced aquatic
specializations than Hyemoschus does, and it probably spent a considerably greater amount of time in the water either for protection orwhen feeding. As indicated by the evidence from stable isotopes,
Indohyus spent most of its time in the water and either came onshore
to feed on vegetation (as the modern Hippopotamus does) or foraged
on invertebrates or aquatic vegetation in the same way that the modern muskrat Ondatra does.
Raoellids are the sister group to cetaceans, and this implies thataquatic habitats originated before the Order Cetacea. The great
evolutionary change that occurred at the origin of cetaceans is thus
not the adoption of an aquatic lifestyle. Here we propose that dietarychange was the event that defined cetacean origins; this is consistent
with the cranial and dental synapomorphies identified. Molars ofIndohyus are markedly different from those of pakicetids, and it is
widely assumed that pakicetids ate aquatic prey18,23.
Our working hypothesis for the origin of whales is that raoellid
ancestors, although herbivores or omnivores on land, took to fresh
water in times of danger. Aquatic habits were increased in Indohyus(as suggested by osteosclerosis and oxygen isotopes), although it did
not necessarily have an aquatic diet (as suggested by carbon isotopes).
Cetaceans originated from an Indohyus-like ancestor and switched toa diet of aquatic prey. Significant changes in the morphology of theteeth, the oral skeleton and the sense organs made cetaceans different
from their ancestors and unique among mammals.
METHODS SUMMARY
We chose an existing character matrix10 as the basis for our phylogenetic analysis.
We corrected scores of some of the taxa, and made some changes in the taxa
included. Details on these taxa, the rationale for using them, and their scores are
given in Methods and in Supplementary Table 1.
Tympanic wall thickness was investigated to address the presence of theinvolucrum quantitatively. We calculated the ratio of medial tympanic wall
thickness divided by lateral tympanic wall thickness (see Methods andSupplementary Table 2). Bone histology was studied to investigate the presenceof osteosclerosis. It was quantified as the ratio of medullary cavity width
divided by bone width in the mediolateral plane (see Methods andSupplementary Table 3).

Received 26 June; accepted 3 October 2007.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank the late F. Obergfell for presenting us with thesediment blocks containing Indohyus fossils collected by A. Ranga Rao for
preparation and study; D. S. N. Raju and N. Raju for facilitating our research;
B. Armfield, R. Conley and A. Maas for fossil preparation; J. Dillard for preparing
Fig. 5; and J. Geisler and J. Theodor for providing additional information about theircladistic analyses. Laboratory research was funded by the National Science
Foundation (NSF) – Earth Sciences (grants to J.G.M.T. and M.T.C.). Collaborative
work was funded by the Indian Department of Science and Technology (to S.B.)
and the NSF – International Division (to J.G.M.T.) under the Indo-US Scientific
Cooperation Program. Laboratory analyses were supported by the Skeletal Biology
Research Focus Area of Northeastern Ohio Universities College of Medicine.
Author Contributions J.G.M.T. was responsible for anatomical and systematic
study, and scientific synthesis, L.N.C. for systematic and bone density study,
M.T.C. for the study of stable isotopes, and S.B. and B.N.T. for geological study andcollecting of Indohyus and comparative fossil samples.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. Correspondence and requests for materials should be
addressed to J.G.M.T. (thewisse@neoucom.edu).

1194©2007 Nature Publishing Groupdoi:10.1038/nature06343

METHODS
Systematic study. We chose an existing, published character matrix10 as the basis
for our phylogenetic analysis because this matrix is rich in characters and contains most relevant taxa. From this matrix we deleted those artiodactyls andcetaceans that are geologically young or modern and are well represented byfossil relatives (Odocoileus, Bos, Ovis, Remingtonocetus, Protocetus, Georgiacetus,
Basilosaurus, Balaenoptera, Physeter, Tursiops, Delphinapterus, Camelus andLama), as well as perissodactyls and non-ungulate taxa.
To this matrix, we added the anthracotheres Siamotherium, Anthracokeryx andMicrobunodon, because they are near the base of the anthracotheroid clade(Anthracotheriidae plus Hippopotamidae) and are sometimes thought to beclose to early whales4. Scores for these taxa, and the sources on which we based
the scores, are listed in Supplementary Table 1 (refs 4, 6, 41–46).
We chose Gujaratia pakistanensis (formerly Diacodexis pakistanensis)47 as outgroup for the analyses of cetaceans plus artiodactyls, and we chose Arctocyon andHyopsodus as outgroups for the (second) analysis that included all taxa (Fig. 2).
We corrected some of the scores for Pakicetidae10, because new fossils havebeen published for this family, in particular cranial material19 and postcranialmaterial21. Corrected scores for pakicetids are also listed in Supplementary Table
1 and were based on original material in the Howard-Geological Survey ofPakistan (H-GSP) collections, currently curated by J.G.M.T.
Raoellidae have been included in several previous phylogenetic analyses relating to early whales5,6. These authors based raoellid scores on Khirtharia andIndohyus. In the present analyses we have split scores for these animals, with
Khirtharia scores based mostly on published H-GSP material and one unpublished skull (H-GSP 1979; dentition published48, specimen now lost). Scores for
Indohyus are based on the material in the RR and IITR-SB collections; all raoellid
scores are listed, with the specimen number of the fossil on which the score wasbased, in Supplementary Table 1.
Study of tympanic walls. Tympanic wall thickness was investigated to address
the presence of the involucrum quantitatively. The involucrum is the thickened
medial wall of the tympanic bone (the ossified wall of the middle ear cavity). The
lateral tympanic wall of cetaceans is reduced in thickness (the tympanic plate).
To quantify these differences in tympanic walls, we calculated the ratio of medial
tympanic wall thickness to the lateral tympanic wall thickness. Lateral tympanic
wall thickness was measured with a micrometer (Dyer gauge) just inferior to
the tympanic ring, and medial tympanic wall thickness was measured directly
across from this site on the other (medial) side of the middle ear cavity (seeSupplementary Table 2).
Bone histology. Bone histology was studied to investigate the presence of osteosclerosis. Osteosclerosis is the thickening of the cortical bone. It was quantified as
the ratio of medullary cavity width divided by bone width in the mediolateralplane, because left and right cortical thickness plus medullary cavity thickness
equals bone width (see Supplementary Table 3). Measurements were taken on
the femur with callipers.
Fossil limb shaft fragments were embedded in Buehler low-viscosity epoxy
resin and sectioned with a diamond saw. Sections were mounted on frosted glass
slides by using epoxy resin. Mounted sections were then ground down andpolished to a thickness of about 75 mm by using a precision grinder with 600,
800 and 1,200 grit paper20 (Fig. 1i–l).
Study of stable isotopes. For the analysis of stable isotopes, three or more specimens of each species were analysed (when available; see Supplementary Tables 4
and 5) to provide a robust estimate of the population mean and s.d. for carbon
and oxygen isotope values33. About 5 mg of enamel powder was collected fromeach specimen, either by drilling directly from the tooth or by grinding enamelchips in an agate mortar and pestle. Before collection, contaminants were
removed by abrading the outer surface of the specimen.
Preparation of powders for analysis of stable isotopes followed publishedmethods31. Powders were first transferred to 1-ml microcentrifuge vials and thensoaked sequentially overnight in about 0.20 ml of a sodium hypochlorite solution (1–2 g dl21) and then in about 0.20 ml of calcium acetate buffered acetic acid(pH about 5.1). On addition of each reagent, samples were agitated for 1 min on
a Vortex Genie vortex mixer. After each soak, the supernatant was removed byaspiration and the residual powder was rinsed five times with deionized water.
Samples were then freeze-dried overnight and about 1.5 mg of powder from each
was weighed into individual test tubes for analysis on a Thermo-Finnigan gas
bench autosampler attached to a Thermo Finnigan DeltaPlus XP continuous-flow
isotope-ratio mass spectrometer at the University of Wyoming Stable Isotope
Facility.
All values for stable isotopes are reported in delta (d) notation, using theequation d(%) 5 1,000 3 (Rsample/Rstandard 2 1), where Rsample is the observed
isotope ratio of the sample (13C/12C or 18O/16O) and Rstandard is the accepted
ratio for an appropriate international standard (Vienna Pee Dee belemnite for
d13C; Vienna Standard Mean Ocean Water for d18O). Analytical precision istypically better than 0.1% for d13C values and 0.2% for d18O values (61s).
41. Ducrocq, S. The late Eocene Anthracotheriidae (Mammalia, Artiodactyla) fromThailand. Palaeontographica A 252, 93–140 (1999).
42. Ducrocq, S. Unusual dental morphologies in late Eocene anthracotheres(Artiodactyla, Mammalia) from Thailand: dental anomalies and extreme
variations. N. Jb. Geol. Paläont. MH 4, 199–212 (1999).
43. Suteethorn, V., Buffetaut, E., Helmcke-Ingavat, R., Jaeger, J. J. &Jongkanjanasoontorn, Y. Oldest known Tertiary mammals from South East Asia:
middle Eocene primate and anthracotheres from Thailand. N. Jb. Geol. Paläont. MH
9, 563–570 (1988).
44. Colbert, E. H. Fossil mammals from Burma. Am. Mus. Nat. Hist. Bull. 74, 419–424(1938).
45. Brunet, M. M. Découverte d’un crâne d’Anthracotheriidae, Microbunodon
minimum (Cuvier), á La Milloque (Lot-et-Garonne). C. R. Acad. Sci. Paris 267,
835–838 (1968).
46. Lihoreau, F., Blondel, C., Barry, J. & Brunet, M. A new species of the genus
Microbunodon (Anthracotheriidae, Artiodactyla) from the Miocene of Pakistan:genus revision, phylogenetic relationships and palaeobiogeography. Zool. Scr. 33,
97–115 (2004).
47. Bajpai, S. et al. Early Eocene land mammals from Vastan Lignite Mine, District
Surat, Gujarat, western India. J. Palaeontol. Soc. India 50, 101–113 (2005).
48. West, R. M. Middle Eocene large mammal assemblage with Tethyan affinities,
Ganda Kas region, Pakistan. J. Paleontol. 54, 508–533 (1980).

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