PALAIOS, 2008, v. 23, p. 574–585
Research Article
DOI: 10.2110/palo.2007.p07-054r
IDENTIFYING AQUATIC HABITS OF HERBIVOROUS MAMMALS THROUGH STABLE
ISOTOPE ANALYSIS
MARK T. CLEMENTZ,1* PATRICIA A. HOLROYD,2 and PAUL L. KOCH3
1
University of Wyoming, Department of Geology and Geophysics, 1000 E. University Avenue, Laramie, Wyoming 82071, USA; 2University of California,
Berkeley, Museum of Paleontology, 1101 Valley Life Sciences Building, Berkeley, California 94720, USA; 3University of California,
Santa Cruz Earth and Planetary Sciences Department, 1156 High St., Santa Cruz, California 95064, USA
e-mail: [email protected]
ABSTRACT
* Corresponding author.
2005), and the co-occurring embrithopod Arsinoitherium from the Jebel
Qatrani Formation, Egypt, interpreted as both a terrestrial grazer and
semiaquatic form (Thenius, 1969; Sen and Heintz, 1979; Court, 1993).
The second case reexamines published isotope values for the North American pantodont Coryphodon and associated early Eocene fauna from the
Bighorn Basin, Wyoming (Koch et al., 1995), to look for evidence of
aquatic adaptations to support previous interpretations based on postcranial and dental morphology. The third case applies our model to the
extinct rhinoceros Teleoceras, a genus whose aquatic adaptations have
been questioned (Prothero, 1992; MacFadden, 1998; Mead, 2000; Mihlbachler, 2003). Analysis of the ␦13C and ␦18O values of each of these
taxa and comparison with our hippo ecomorph model will enable us to
assess whether this model is appropriate for taxa outside of the hippopotamid clade and better document the recurrence of large, semiaquatic
mammals in terrestrial ecosystems.
Large-bodied, semiaquatic herbivores are an interesting ecological morphotype (i.e., ecomorph) best exemplified by two living artiodactyls—the
African river hippopotamus (Hippopotamus amphibius) and the pygmy hippopotamus (Choeropsis liberiensis; see Gatesy, 2002). Both hippopotamids
have several skeletal features associated with their semiaquatic habits—
graviportal limbs, reduced limb length, and raised orbits and nasal openings—
leading to the designation of this body type as the hippo ecomorph. Hippopotamids have a relatively short fossil record (middle Miocene–recent), but
latest phylogenetic analyses nest this group within the anthracotheres—an
extinct group of artiodactyls—which have a fossil record dating back to the
late middle Eocene (Boisserie, 2005; Boisserie et al., 2005a) and which are
suggested to be semiaquatic in their habits.
Several Cenozoic mammalian clades are thought to have independently
evolved semiaquatic lineages, based on morphological characters shared with
extant hippopotamids (e.g., Meehan and Martin, 2003). Aside from anthracotheres and hippopotamids in Africa and Eurasia, at least one species of
proboscidean may have been a semiaquatic herbivore. Moeritherium was an
early relative of elephants that had a long body with short limbs and elevated
orbits, and it inhabited river channels and estuaries in northern and western
Africa from late Eocene to early Oligocene (Osborn, 1909; Simons, 1964).
In North America, the hippo ecomorph may have been filled convergently
by taxa from three separate orders—Pantodonta, Perissodactyla, and
Artiodactyla—from the late Paleocene into the late Miocene. If so, the hippo
ecomorph was a recurring component of North American ecosystems for
over 55 million years (Meehan and Martin, 2003). Two of the most often
cited examples of this body type in the North American fossil record are the
pantodont Coryphodon from the early to middle Eocene (Lucas, 1998) and
the rhinoceratid Teleoceras from the middle Miocene to early Pliocene (15–
4.5 Ma; see Webb, 1983). Both were stout-bodied, short-legged herbivores
that were geographically widespread and abundant within continental ecosystems. In addition to morphological similarities with extant hippopotamids
(Simons, 1960; Rose, 1990, 2006; Prothero, 1998; Wall and Heinbaugh,
1999), specimens of both genera are found commonly in river channel deposits, both of which support interpretations of their aquatic habits (Lucas,
1998; Prothero, 1998).
Copyright 䊚 2008, SEPM (Society for Sedimentary Geology)
0883-1351/08/0023-0574/$3.00
Large-bodied, semiaquatic herbivorous mammals have been a recurring component of most continental ecosystems throughout the Cenozoic. Identification of these species in the fossil record has largely
been based on the morphological similarities with present-day hippopotamids, leading to the designation of this pairing of body type
and ecological niche as the hippo ecomorph. These morphological
characters, however, may not always be diagnostic of aquatic habits.
Here, enamel ␦13C and ␦18O values from living hippopotamuses were
examined to define an isotopic signature unique to the hippo ecomorph. Although ␦13C values do not support unique foraging habits
for this ecomorph, living and fossil hippopotamids typically have low
mean ␦18O values relative to associated ungulates that fit a linear
regression (␦18Ohippopotamids ⴝ 0.96 ⴞ 0.09· ␦18Ofauna ⫺ 1.67 ⴞ 2.97;
r2 ⴝ 0.886, p ⬍ 0.001). Modeling of oxygen fluxes in large mammals
suggests that high water-turnover rates or increased water loss
through feces and urine may explain this relationship. This relationship was then used to assess the aquatic adaptation of four purported
hippo ecomorphs from the fossil record: Coryphodon (early Eocene),
Moeritherium and Bothriogenys (early Oligocene), and Teleoceras
(middle–late Miocene). Only fossil specimens of Moeritherium, Bothriogenys, and large species of Coryphodon had ␦18O values expected
for hippo ecomorphs; ␦18O values for Teleoceras and a small species
of Coryphodon were not significantly different from those of the associated fauna. These results show that the mean ␦18O value of fossil
specimens is an effective tool for assessing the aquatic habits of extinct species.
INTRODUCTION
The purpose of this paper is to assess the validity of isotopic methods
for identification of aquatic and semiaquatic taxa in the fossil record and
to determine what conditions must be met before these methods may be
used. Although prior work has applied oxygen isotope analysis to hippopotamids (Bocherens et al., 1996), no study has adequately addressed
what factors generate characteristic oxygen isotope values in hippopotamids and, therefore, whether this approach is applicable to mammals outside of this clade.
Using published isotope data from living and fossil hippopotamids, we
construct a quantitative model for the hippo ecomorph that accounts for
environmental and physiological influences on oxygen isotope values.
This model is then applied to three test cases from the fossil record. The
first looks at the early Oligocene anthracothere Bothriogenys gorringei,
the early proboscidean Moeritherium, both of which have been hypothesized as semiaquatic mammals based on morphology and occurrence of
fossil remains in fluvial environments (Osborn, 1909; Simons, 1964; Coppens and Beden, 1978; Pickford, 1983, 1991; Ducrocq, 1997; Boisserie,
PALAIOS
AQUATIC MAMMAL ISOTOPES
How well these morphological criteria actually diagnose ecological
preferences is debated, and it is particularly difficult to determine whether
the few modern taxa can be used effectively as morphologic analogs for
organisms not closely related. Even the definition of aquatic or semiaquatic behavior in modern mammals is unclear. Most uses of the terms
are undefined. Where defined, the criteria are not necessarily ones that
can be readily applied to fossils. Fish and Stein (1991, p. 340) recognize
aquatic mammals as those ‘‘(1) that exhibit obvious external modifications of both the limbs and body which allow them to live successfully
in water, (2) that forage primarily in water, and (3) that use water as a
primary means to escape predation.’’ They characterized semiaquatic
mammals by their second and third criteria, but not the first. These criteria, however, are not directly observable in the fossil record, and there
are few clear-cut morphological characters that permit reliable inferences
to be made about the use of aquatic habitats by fossil organisms.
For these reasons, hypotheses about semiaquatic behavior should be
tested using alternative methods that are independent of morphology. The
stable isotope composition of fossil material has been shown to be an
excellent source of ecological information, making it a prime candidate
for independently testing ecomorphologic hypotheses. The oxygen isotope composition of fossil materials has been exploited as a proxy for
paleotemperature and salinity, but, more important, it has been used to
distinguish between terrestrial and aquatic species (Bocherens et al., 1996;
MacFadden, 1998; Clementz and Koch, 2001). Both the mean and the
variance in the oxygen isotope composition of individuals within fossil
populations have proven effective in identifying aquatic species and could
be used to do the same for hippolike species within ancient faunas.
Interestingly, despite their morphological adaptations to a semiaquatic
life and the fact that they spend a great deal of time in the water, modern
hippopotamuses obtain the bulk of their food outside of the water. Of the
two living species, the larger common hippo primarily grazes on terrestrial grasses at night (Field, 1970; Scotcher et al., 1978), whereas the
smaller pygmy hippopotamus is primarily a browser of semiaquatic vegetation, wild fruits, and low-growing ferns and herbs (Nowak and Paradiso, 1983). Carbon isotope (␦13C) values are already widely used in
paleodietary research to distinguish grazing and browsing behavior for
fossil hippopotamids (Bocherens et al., 1996; Zazzo et al., 2000; Boisserie
et al., 2005b) and may also be useful for assessing whether semiaquatic
species were foraging in terrestrial or aquatic food webs.
STABLE ISOTOPES AND THE MODERN HIPPO ECOMORPH
The ␦18O value of tooth enamel apatite [Ca5(PO4, CO3)3(OH, F)] is
dictated by the ␦18O value of an animal’s body water and the temperature
at which the mineral forms. As mammal body temperature is relatively
constant, variations in enamel ␦18O values are controlled by variations
in body water ␦18O values that, in turn, are controlled by the ␦18O value
of environmental oxygen sources as well as fluxes and fractionations
associated with physiological functions (Bryant and Froelich, 1995;
Kohn, 1996). Mean enamel ␦18O values typically correlate with surface
water ␦18O (␦18OSW) values (Luz and Kolodny, 1985; Delgado Huertas
et al., 1995), but within-population variance is high for published ␦18O
data from terrestrial species (Fig. 1; 1 SD ⱖ 1.0‰) as a result of temporal
and geographic variations in the ␦18O values of environmental oxygen
sources as well as individual differences in physiological response (Clementz and Koch, 2001). Published ␦18O data for aquatic species—
animals spending ⬎50% of their time in the water—show a similar correlation with ␦18OSW, but ␦18O values among individuals within a population typically vary much less (Yoshida and Miyazaki, 1991; Clementz
and Koch, 2001). The major oxygen flux for aquatic species comes from
the water that they inhabit (Hui, 1981; Andersen and Nielsen, 1983;
Kohn, 1996), which can result in low within-population variance (Fig. 1;
1 SD ⱕ 0.5‰) for species living in such isotopically homogeneous waters as seawater, large lakes, or rivers. Population-level variation in ␦18O
575
FIGURE 1—Bar graph depicting the mean levels of standard deviation (here denoted by s) for living populations (gray) and fossil accumulations (black) of aquatic
mammals, hippopotamids, and terrestrial mammals (Kohn et al., 1996; Leakey et al.,
1996; Zazzo, et al., 2000; Clementz and Koch, 2001; Cerling et al., 2003; FranzOdendaal et al., 2002; Harris and Cerling, 2002; MacFadden et al., 2004; Levin et
al., 2006). Error bars reflect 1 standard deviation from the mean, and numbers in
parentheses above each bar indicate the number of populations or accumulations
included in each. Statistically significant differences were detected among mean values (one-way analysis of ranks: H ⫽ 74.42, df ⫽ 5, p ⬍ 0.001); letters (A, B, C)
above each bar identify groups with statistically similar mean values (p ⬎ 0.05).
values for extant hippopotamids typically falls within the expected range
for aquatic and semiaquatic mammals with a few exceptions (Fig. 1).
Aquatic habits may not be the only cause for low ␦18O variation in
populations, however. At large body sizes (⬎4,500 kg), environmental
water is the dominant oxygen flux for mammals (⬃80% of total influx
for mammals; see Bryant and Froelich, 1995). For environments with
little seasonal or spatial variation in surface water ␦18O values, this may
reduce the variance for populations of extremely large mammals to that
of aquatic or semiaquatic species. Elephants are the only mammal species
to reach these extreme body sizes today (Silva and Downing, 1995). The
typical value for most elephant populations is on par with that of other
terrestrial species (mean SD ⫽ 1.1 ⫾ 0.3‰) and much higher than that
for aquatic species (Cerling et al., 2007), though there is a significant
range in reported variation in enamel ␦18O values (1 SD ⫽ 0.5‰–1.6‰).
This indicates that even though the effects of large body size may dampen
the enamel ␦18O variation for a population, these values should be distinguishable from those of aquatic and semiaquatic species.
Published ␦18O values for modern hippopotamids are consistently lower
than those for associated fauna (Fig. 2; see Bocherens et al., 1996; Kohn et
al., 1996; Leakey et al., 1996; Harris and Cerling, 2002; Levin et al., 2006).
A firm explanation for these low values has not been established, but they
are thought to relate to semiaquatic habits that lead to reduced evaporative
loss of 16O-enriched water from the body; nocturnal foraging that leads to
reduced evaporative water loss across the skin and consumption of plant
water that has not experienced 18O-enrichment due to evapotranspiration; or
the unique physiology of hippopotamids (Bocherens et al., 1996). This pattern
has also been reported for Pliocene- and Pleistocene-aged hippopotamids
(Bocherens et al., 1996; Zazzo et al., 2000; Franz-Odendaal et al., 2002),
suggesting it is a common characteristic of hippopotamids and may be useful
for defining the hippo ecomorph.
The ␦13C composition of the structural carbonate in tooth enamel apatite is directly related to that of an animal’s diet (Ambrose and Norr,
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CLEMENTZ ET AL.
FIGURE 2—Mean ␦18O values for one species of hippopotamid (Hippopotamus
amphibius) and associated fauna from seven localities in Africa (Kohn et al., 1996;
Leakey et al., 1996; Zazzo et al., 2000; Franz-Odendaal et al., 2002; Harris and
Cerling 2002; Cerling et al., 2003; Levin et al., 2006). Within the associated fauna,
species reported to be sensitive and insensitive to evaporative affects on plant water
(Levin et al., 2006) were separated from other taxa. Error bars represent ⫾1 SD
from the mean for each group. VSMOW ⫽ Vienna standard mean ocean water.
1993; Cerling and Harris, 1998). For herbivorous mammals, vegetation
is the primary carbon source. The ␦13C value of dietary plants is dependent on the photosynthetic pathway used by the plant (O’Leary, 1988;
Farquhar et al., 1989) and the ␦13C composition of the environmental
carbon source (Bunn and Boon, 1993; Boon and Bunn, 1994; Raven et
al., 2002; Cerling et al., 2004). For terrestrial vegetation, the metabolic
pathway used for carbon fixation during photosynthesis creates the largest
differences in ␦13C values for such plant types as C3 trees, shrubs, and
cool-climate grasses (mean ␦13C ⫽ ⫺27 ⫾ 3‰; C4 warm-climate grasses: mean ␦13C ⫽ ⫺13 ⫾ 2‰), but environmental stress (i.e., aridity,
salinity) and variation in the ␦13C composition of CO2 (closed canopy
forests: mean ␦13C ⫽ ⫺31.2 ⫾ 2.3‰;) can also create significant differences in plant ␦13C values (O’Leary, 1988; Farquhar et al., 1989;
Cerling et al., 2004).
In fresh-water environments, primary producers use the same photosynthetic pathways as those used by terrestrial plants, but their ␦13C compositions are also strongly controlled by the physical conditions associated with growth in an aquatic environment. Variation in the ␦13C composition, concentration, and source of dissolved inorganic carbon as well
as a significant reduction in the rate of diffusion of CO2 in water versus
air can cause the ␦13C values for fresh-water primary producers to significantly deviate from those of terrestrial plants (Osmond et al., 1981;
Raven et al., 2002). Mean ␦13C values for fresh-water floating, emergent,
and submergent C3 macrophytes (temperate: ⫺27.5 ⫾1.2‰; tropical:
⫺27.6‰) are similar to expected values for terrestrial C3 vegetation even
with the inherent variation imposed by the environment. Mean ␦13C values for fresh-water phytoplankton (temperate: ⫺28.6 ⫾ 1.3‰; tropical:
⫺33.3‰), however, are much lower (Hamilton et al., 1992; Forsberg et
al., 1993; Cloern et al., 2002) and suggest that enamel ␦13C values—
offset from plant ␦13C values by ⬃14.1‰ for large herbivorous mammals (Cerling and Harris, 1998)—can discriminate among consumers foraging on different primary producers in terrestrial and fresh-water ecosystems. Even with the absence of significant quantities of C4 grasses
from terrestrial ecosystems until the middle Miocene (Cerling et al., 1997;
Fox and Koch, 2003), differences in ␦13C values for terrestrial and aquatic C3 plants are still sufficient to distinguish among consumers foraging
in open, arid terrestrial habitats (higher ␦13C values), humid terrestrial
habitats or aquatic food webs fueled by fresh-water macrophytes (low
␦13C values), and deep forest or aquatic food webs fueled by fresh-water
phytoplankton (very low ␦13C values; see Clementz and Koch, 2001;
Darimont et al., 2007).
Semiaquatic mammals may be recognized by two stable isotope signals
that also characterize living hippopotamids. First, mean ␦18O values for
hippopotamids are significantly lower than those of the associated fauna.
The ability to apply this feature to taxa other than hippopotamids is examined in this study. Second, population-level variance in ␦18O values
for hippopotamids is generally lower than that of the associated fauna
when local surface waters are isotopically homogeneous. This characteristic has already been identified for marine mammal species (Clementz
and Koch, 2001) and should be useful when interpreting the aquatic habits of extinct species. In conjunction with the information provided by
␦18O values, enamel ␦13C values can be used to determine whether semiaquatic, herbivorous mammals were feeding on land in open habitats or
closed forests and in fresh-water food webs fueled by aquatic macrophytes or phytoplankton. We will now examine how well each of these
criteria works toward identifying aquatic mammals in the fossil record
by applying these proxies to specimens of purported semiaquatic taxa
from the Eocene (Coryphodon), Oligocene (Arsinoitherium, Bothriogenys, Moeritherium), and Miocene (Teleoceras).
METHODS AND MATERIALS
Modeling Oxygen Isotope Fractionation in Semiaquatic Mammals
A model was constructed to determine which physiological, environmental, and behavioral factors most likely account for the difference in
␦18O values between hippopotamids and other ungulates based on the
theoretical work of Kohn (1998). The parameters were adjusted to test
their impact on body water and enamel ␦18O values. Physiological factors
included those identified for hippopotamids—low basal metabolic rate,
no sweating, increased water loss through feces—and others assumed for
aquatic mammals—high water-flux rate, increased water loss via urine or
feces. For calculation of the effect of urine loss on body water and enamel
␦18O values, the range of percent water loss via urine was limited to
those values that maintained positive water balance for all fluxes of oxygen. The effect of nocturnal foraging was modeled by varying the ambient temperature and humidity conditions while holding surface water
␦18O values constant and then calculating the resulting change in plant
water ␦18O values.
Stable Isotope Analysis
Data were collected by either compiling published values for extant
hippopotamids and fossil taxa of interest or by analysis of fossil specimens sampled from collections at the University of California Museum
of Paleontology, the University of Nebraska Museum of Natural History,
the University of Michigan Museum of Paleontology, and the American
Museum of Natural History. When available, a minimum of five specimens was analyzed at each site to provide a robust estimate of the population mean and standard deviation for ␦13C and ␦18O values (Clementz
and Koch, 2001). In addition to Teleoceras, Moeritherium, and Bothriogenys, we analyzed unambiguously terrestrial mammals as controls at
each site. Information on locality age, species sampled, and individual
␦13C and ␦18O values are reported in Supplementary Data 1–21. MacFadden (1998) performed an earlier study for populations of Teleoceras
from the middle to late Miocene of Florida and found no significant
difference between Teleoceras and an associated terrestrial rhinoceros
Aphelops. These data were supplemented by including specimens of Teleoceras from other localities in California, Nebraska, and Oregon and
by augmenting the number of taxa included from the Florida fauna. Bodymass estimates were based on values reported in Damuth and MacFadden
(1990) and Gagnon (1997) and from the Paleobiology Database
(www.pbdb.org) (Table 1).
Stable isotope values for Coryphodon and associated terrestrial fauna
were taken from Koch et al. (1995) and include specimens spanning the
1
www.paleo.ku.edu/palaios
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AQUATIC MAMMAL ISOTOPES
Paleocene-Eocene boundary from 1000 m to 2200 m above the base of
the Willwood Formation in the Clarks Fork Basin, Wyoming. Within this
interval, the body size of Coryphodon species typically is much larger
than that of the rest of the fauna (Table 1). From 1525 m to 1800 m
(interval 2), however, body size is significantly reduced (⬍100 kg) and
approaches that of the other species analyzed from this section (Gingerich, 1990; Uhen and Gingerich, 1995). Taking care to avoid inclusion of
specimens sampled from the interval of intense global warming during
the Paleocene–Eocene Thermal Maximum (i.e., from 1520 m above the
base of Willwood Formation), we separated specimens of the smaller
species of Coryphodon and associated fauna and compared them to those
recovered below (interval 1: 1000–1500 m) and above (interval 3: 1800–
2200 m) interval 2.
Using the criteria outlined in Levin et al. (2006) for identifying species
sensitive (ES) and insensitive (EI) to evaporative effects on plant water,
recent and fossil hippopotamid ␦18O values were compared to the mean
␦18O values for each faunal group. Evaporative-sensitive taxa are defined
as those taxa that show a significant 18O-enrichment between tooth enamel and meteoric water ␦18O values as a response to increasing evaporative
effects on the ␦18O of leaf water. These taxa include giraffids, oryx, dikdik, Grant’s gazelle, and buffalo, which show a positive correlation between enamel ␦18O values and water deficit—the cumulative difference
between potential evapotranspiration and precipitation for a region. In
contrast, EI taxa are defined as those taxa that show no statistically significant 18O-enrichment between enamel and meteoric water ␦18O values.
These taxa include hippopotamus, bush pig, elephant, rhinoceros, warthog, zebra, impala, and baboon, which show a strong correlation between
enamel and meteoric water ␦18O values. For Miocene or younger fossil
communities from North America and Africa where these or related taxa
were available for comparison, designation of EI and ES taxa was relatively straightforward. Identification of these faunal groups from older
fossil communities where behavioral information is unclear, however, was
more complicated. Identification of EI and ES taxa in these cases was
based primarily on morphology, comparison with modern analogs, and
stable isotopic data. The specific criteria used for designating EI and ES
taxa in each case are reported in the Results section.
Approximately 10 mg of enamel powder was collected from each specimen either by drilling directly from the tooth or by grinding enamel
chips in an agate mortar and pestle. Prior to collection, contaminants were
removed by abrading the outer surface of the sample. Powders were transferred to 1-ml microcentrifuge vials, and ⬃0.25 ml of a 1wt%–2wt%
sodium hypochlorite solution was added to remove organic contaminants.
Samples were then agitated on a Vortex Genie for one minute and allowed
to sit for 24 h. The supernatant was removed by aspiration, and the residual powder was rinsed five times with deionized water and then soaked
in ⬃0.25 ml of 1M acetic acid buffered with calcium acetate (pH ⫽ 5.1)
for 24 h. After removing the supernatant via aspiration, the powders were
rinsed five times in deionized water and then lyophilized. Approximately
1.5 mg of powder was reacted with 100% phosphoric acid at 90⬚C for 8
min in a constantly stirred reaction vessel, and the resulting CO2 was
cryogenically purified and analyzed for isotopic composition using an
ISOCARB automated carbonate device attached to an Optima gas source
mass spectrometer in the Stable Isotope Lab at University of California,
Santa Cruz.
All isotope values are reported in standard delta notation, where ␦ ⫽
((Rsample/Rstandard) ⫺ 1) ⫻ 1000 and R is 13C/12C for carbon and 18O/
16O for oxygen. ␦13C values are reported relative to the Vienna Pee Dee
Belemnite standard, and ␦18O values are reported relative to Vienna standard mean ocean water. Uncertainties in analyses were assessed via multiple analyses of an in-house elephant enamel standard calibrated against
NBS-19 (␦13C: SD ⫽ 0.1‰; ␦18O: SD ⫽ 0.2‰; n ⫽ 30 for both).
For species or genus-level differences in mean value within and among
sites, a Student’s t-test was used for comparisons between two species,
and a single-factor analysis of variance was used for comparisons of more
than two species with subsequent pairwise comparisons of taxa evaluated
by a post-hoc Bonferroni test to identify which groups were different.
Significant differences in variance between species and groups were evaluated using a standard F-test. For multiple comparisons that did not meet
the criteria necessary to perform parametric statistics, a nonparametric
Kruskall-Wallis one-way analysis of variance (OW-ANOVA) on ranks
was used and followed by evaluation of pairwise comparisons of taxa
using Dunn’s Method. For all comparisons, the level of statistical significance was set at p ⬍ 0.05. The relationship between hippopotamid and
associated fauna ␦18O values was evaluated through a linear regression
of the data. Box plots of enamel ␦13C and ␦18O values were used to
compare values for fossil herbivores from each site (see Fig. 6). The
dimensions of each box account for 50% of the data (the interquartile
distance; IQD) and is bounded by the upper quartile (UQ) and lower
quartile (LQ). Error bars represent the maximum and minimum values
that fall within the range of UQ ⫹ 1.5· IQD and LQ ⫺ 1.5 ·IQD. Any
points falling outside of this range are plotted as individual circles. A
horizontal line through each box represents median values. All statistical
measurements were performed using Sigmastat v.3.1 or Kaleidagraph
v.3.6 software.
RESULTS
Oxygen Isotope Modeling of Semiaquatic Herbivores
Model results are presented in Figure 3 and illustrate the magnitude
and direction of change in ␦18O values for the hippo ecomorph relative
to a typical fully terrestrial mammal of similar body mass
(␦18Ohippo-terrestrial). Modification of the six selected parameters—(1) basal metabolic rate (BMR), (2) sweating versus panting, (3) water content
of feces, (4) water economy index (WEI ⫽ (water-turnover rate) ·BMR⫺1
⫽ ml· kJ⫺1), (5) water loss via urine, and (6) diurnal variation in leaf
water oxygen isotope composition (i.e., diurnal vs. nocturnal foraging)—
all have a significant impact on the ␦18O composition of mammal body
water. Of these, lowering the basal metabolic rate and reducing the
amount of oxygen lost as sweat was found to increase the ␦18O composition of mammal body water and tooth enamel (Fig. 3A) by increasing
the proportion of oxygen lost through evaporation across the skin and
through panting, respectively. Selected modifications to all other parameters were found to lower body water and tooth enamel ␦18O values by
varying degrees.
Increasing the WEI to values reported for large, fully aquatic herbivores (Ortiz et al., 1999; Ortiz and Worthy, 2006) caused enamel ␦18O
values to drop by more than 2‰ and was associated with an increase in
the proportion of drinking water (from 5% to 80%) ingested relative to
the amount of 18O-enriched water ingested from food (Fig. 3B). Higher
water loss through feces and urine, which are not fractionated relative to
body water, reduced the amount of 18O-depleted water lost through panting or evaporation across the skin and caused body water and enamel
␦18O values to drop by more than 2‰ (Fig. 3C). The effect was exacerbated by varying the WEI from 0.4 ml·kJ⫺1 to 1.3 ml ·kJ⫺1 but varied
in direction and magnitude for different fecal water contents. At 60%
fecal water content, the proportion of total oxygen loss from urine and
feces was low (⬍50%), regardless of the WEI. The amount of 18Odepleted water vapor lost through evaporation across the skin and nose
decreased at higher WEI, causing enamel ␦18O values to decrease. In
contrast, at 90% fecal water content, the proportion of total oxygen loss
from urine and feces decreased significantly when WEI was increased
(from 45% to 74%), resulting in higher enamel ␦18O values. The largest
decrease in ␦18O values (␦18Ohippo-terrestrial 艐 ⫺6.0‰) was produced by
increasing the WEI to 1.3 ml·kJ⫺1, the water content of feces to 60%,
and the percentage of water lost via urine to 90% (Fig. 3C).
Reducing leaf water ␦18O values by increasing relative humidity to
levels expected during nocturnal foraging (Fig. 3D) also caused body
water and enamel ␦18O values to drop by ⬎ 2.0‰. Modeled leaf water
␦18O values varied from surface water ␦18O values (⫺3.5‰) at 100%
578
PALAIOS
CLEMENTZ ET AL.
TABLE 1—Carbon and oxygen isotope data (mean ⫾ 1 SD) and body mass estimates
for possible hippo ecomorphs and associated fauna. VPDB ⫽ Vienna Pee Dee Belemnite; VSMOW ⫽ Vienna standard mean ocean water.
Taxon
Body Mean ␦13C ⫾ 1 s Mean ␦18O ⫾ 1 s
n mass (kg) (VPDB, ‰)
(VSMOW, ‰)
Early Eocene, Bighorn Basin
Coryphodon (1800 to 2200 m)
Phenacodus
Ectocion
Hyracotherium
Coryphodon (1525–1800 m)
Phenacodus
Ectocion
Hyracotherium
Coryphodon (1000–1515 m)
Phenacodus
Ectocion
10
11
1
15
8
7
5
9
9
16
15
210
40
10
20
100
40
10
20
210
40
10
⫺13.0 ⫾ 1.2‰
⫺11.8 ⫾ 0.5‰
⫺9.8‰
⫺11.5 ⫾ 1.3‰
⫺12.2 ⫾ 1.4‰
⫺11.9 ⫾ 1.0‰
⫺10.2 ⫾ 1.0‰
⫺11.3 ⫾ 0.8‰
⫺12.4 ⫾ 1.0‰
⫺10.6 ⫾ 1.2‰
⫺9.7 ⫾ 1.1‰
19.6 ⫾ 1.2‰
20.5 ⫾ 0.9‰
21.6‰
22.1 ⫾ 2.6‰
20.9 ⫾ 1.1‰
20.5 ⫾ 0.9‰
21.8 ⫾ 0.7‰
21.5 ⫾ 1.5‰
19.9 ⫾ 0.9‰
21.3 ⫾ 1.3‰
22.2 ⫾ 2.1‰
Early Oligocene, Fayum, Egypt
Bothriogenys gorringei
Moeritherium trigodon
Moeritherium lyonsi
Arsinoitherium sp.
Phiomia serridens
Palaeomastodon beadnelli
Saghatherium antiquum
Megalohyrax sp.
Geniohyrax sp.
10
4
1
8
12
3
4
1
1
90
810
810
4000
6600
3000
10
180
70
⫺11.1 ⫾ 0.7‰
⫺10.0 ⫾ 0.4‰
⫺9.0‰
⫺10.6 ⫾ 0.6‰
⫺9.6 ⫾ 1.7‰
⫺10.4 ⫾ 0.3‰
⫺11.8 ⫾ 1.3‰
⫺12.1‰
⫺10.8‰
26.8 ⫾ 1.6‰
27.1 ⫾ 1.3‰
27.2‰
28.8 ⫾ 1.1‰
30.9 ⫾ 1.3‰
32.5 ⫾ 1.4‰
27.3 ⫾ 2.3‰
26.6‰
31.6‰
Middle Miocene (11.8 Ma), Nebraska
Teleoceras major
8
Pliohippus sp.
6
Pseudohipparion sp.
6
Cormohipparion sp.
2
Procamelus sp.
3
Longirostromeryx sp.
3
TABLE 1—Continued.
Late Miocene (4.5 Ma), Florida
Teleoceras
Hemiauchenia
Cf. Rhynchotherium
Cf. Catagonus
Cf. Megatylopus
Pseudohipparion
Cormohipparion
Nannipus
Rhynchotherium
Dinohippus
Tapiravus
Neohipparion
⫺9.3
⫺8.4
⫺9.1
⫺6.8
⫺10.1
⫺8.3
⫾
⫾
⫾
⫾
⫾
⫾
0.9‰
0.6‰
1.0‰
3.8‰
0.4‰
0.6‰
25.8
26.4
28.2
24.3
25.8
26.4
⫾
⫾
⫾
⫾
⫾
⫾
0.7‰
0.6‰
2.1‰
2.6‰
1.8‰
2.0‰
Middle–Late Miocene (9.7–5.3 Ma), California
Teleoceras fossiger
8 1000
Pliohippus interpolatus
5
200
Pliohippus sp.
5
200
Gomphotherium productus
7 3200
Procamelus sp.
5
500
⫺12.2
⫺11.9
⫺11.6
⫺12.3
⫺11.6
⫾
⫾
⫾
⫾
⫾
0.7‰
0.5‰
0.3‰
0.3‰
1.1‰
27.2
26.3
25.5
25.9
25.7
⫾
⫾
⫾
⫾
⫾
1.4‰
0.8‰
1.5‰
1.2‰
1.1‰
Middle–Late Miocene (8.4–5.0 Ma), Oregon
Teleoceras fossiger
5 1000
Camelid
1
—
⫺11.8 ⫾ 1.0‰
⫺11.1‰
19.6 ⫾ 0.8‰
21.7‰
Middle Miocene (9.5 Ma), Florida
Teleoceras
Aphelops
Cormohipparion
Procamelus
Tapirus
Prosthenops
Neohipparion
Ambeledon
5
5
7
1
1
1
1
2
635
889
150
500
381
75
150
3440
⫺13.3 ⫾ 0.4‰
⫺12.8 ⫾ 0.3‰
⫺11.5 ⫾ 0.9‰
⫺11.2‰
⫺13.3‰
⫺11.3‰
⫺11.6‰
⫺12.1‰
32.1 ⫾ 0.4‰
31.4 ⫾ 0.8‰
31.7 ⫾ 1.7‰
30.9‰
27.4‰
29.4‰
30.7‰
30.0‰
Middle Miocene (7.5 Ma), Florida
Teleoceras
Aphelops
Hipparion
Ambeledon
Pliohippus
Pseudohipparion
Cormohipparion
Tapirus
4
2
2
1
1
1
1
1
635
889
150
3440
170
61
150
381
⫺13.3 ⫾ 0.7‰
⫺13.2‰
⫺10.4‰
⫺12.3‰
⫺9.6‰
⫺10.5‰
⫺9.7‰
⫺13.1‰
31.8 ⫾ 0.5‰
31.1‰
31.3‰
30.9‰
31.0‰
27.5‰
30.5‰
30.6‰
Late Miocene (7.0 Ma), Florida
Teleoceras
Aphelops
Tapirus
Cormohipparion
Camel
Pliohippus
2
4
1
1
2
2
635
889
381
150
—
170
⫺12.8‰
⫺12.1 ⫾ 0.6‰
⫺13.9‰
⫺9.8‰
⫺10.4‰
⫺9.7‰
31.5‰
31.7 ⫾ 0.6‰
29.7‰
32.1‰
29.8‰
29.5‰
3
1
3
1
1
3
3
3
1
2
1
4
635
110
—
—
1000
61
150
93
—
240
—
150
⫺7.6 ⫾ 1.8‰
⫺14.1‰
⫺4.7 ⫾ 2.2‰
⫺11.8‰
⫺14.8‰
⫺5.0 ⫾ 0.1‰
⫺5.9 ⫾ 2.5‰
⫺5.8 ⫾ 3.4‰
⫺2.5‰
⫺8.3‰
⫺13.3‰
⫺4.2 ⫾ 3.6‰
31.6 ⫾ 0.4‰
30.3‰
30.8 ⫾ 0.5‰
29.8‰
31.4‰
31.7 ⫾ 0.3‰
29.9 ⫾ 0.4‰
30.3 ⫾ 0.3‰
31.6‰
31.2‰
29.1‰
30.3 ⫾ 0.8‰
relative humidity to a high ⬍⫹9.0‰ when relative humidity was reduced to 50%. The effect of these changes in leaf water ␦18O composition
was significant but dampened in modeled enamel ␦18O values; the 12.5‰
change in leaf water ␦18O values translated to a change of ⬃7‰ in
enamel ␦18O values.
Stable Isotope Results
Published ␦ O values for extant hippopotamids were consistently lower than the associated terrestrial fauna by 2.0‰–6.0‰ (Fig. 2). The difference between hippopotamids and ES taxa (4‰–7‰) was found to be
equal to or greater than that observed between hippopotamids and EI taxa
(2‰–4‰) (Fig. 4).
Similar differences are observed between ␦18O values for extinct hippopotamids and coeval fauna (Fig. 4), with most fossil sites falling within
the range reported between living hippopotamids and EI taxa (Bocherens
et al., 1996; Zazzo et al., 2000; Franz-Odendaal et al., 2002; Harris and
Cerling, 2002; Cerling et al., 2003). For these fossil faunas, EI taxa were
identified based on the ES-EI status of closest living relatives or modern
analogues (Levin et al., 2006). A combined bivariate plot of living and
fossil fauna ␦18O values against those for living and fossil hippopotamids
(Fig. 4) shows a strong positive correlation (Spearman rank ⫽ 0.957, p
⬍ 0.01). Assuming that mean ␦18O values for EI taxa reflect surface
water ␦18O values, application of a linear regression through the data
shows that hippopotamid mean ␦18O values are controlled strongly by
surface water ␦18O values and can be predicted from the mean ␦18O
value of EI taxa within the fauna (␦18Ohippopotamids ⫽ 0.96 ⫾
0.09· ␦18Ofauna ⫺ 1.67 ⫾ 2.97; r2 ⫽ 0.886, p ⬍ 0.001, standard error
of the estimate ⫽ 0.72).
Tabulation of variation within modern and fossil hippopotamids
revealed an interesting contrast (see Fig. 1). Variation within fossil populations was much higher (SD ⫽ 1.5 ⫾ 0.5‰) and statistically different
(Student’s t-test, p ⬍ 0.05) compared to living populations of hippopotamids with low levels of variation in ␦18O values among individuals
(SD ⫽ 0.8 ⫾ 0.7‰). A similar, statistically significant increase in variation was detected between living and fossil terrestrial mammal populations but not between living and fossil aquatic mammals (see Fig. 1).
Mean ␦13C values for Coryphodon were significantly lower than those
for Ectocion from all levels (Table 1) and for Phenacodus and Hyracotherium from all levels but those recovered from interval 2 (OWANOVA, interval 1: F ⫽ 17.252; p ⬍ 0.001; interval 2: F ⫽ 3.857, p
⫽ 0.02; interval 3: F ⫽ 6.013, p ⫽ 0.006). No significant difference
was detected between mean ␦13C values for Coryphodon from each interval (OW-ANOVA, F ⫽ 1.239, p ⫽ 0.308). Mean ␦18O values for
Coryphodon were found to be significantly lower than those for fauna
from interval 1 (OW-ANOVA, F ⫽ 6.148, p ⫽ 0.005) and interval 3
(OW-ANOVA on ranks, H ⫽ 9.415, p ⫽ 0.009), but not from interval
18
1000
200
60
150
500
15
Body Mean ␦13C ⫾ 1 s Mean ␦18O ⫾ 1 s
n mass (kg) (VPDB, ‰)
(VSMOW, ‰)
Taxon
PALAIOS
AQUATIC MAMMAL ISOTOPES
579
FIGURE 3—Bivariate plots of the difference between modeled enamel ␦18O values for hippos and the expected value for a typical terrestrial mammal of the same body
mass (hippo-terrestrial ␦18O) against (A) changes in the proportion of water lost via sweating and changes in the magnitude of expected basal metabolic rate; (B) changes
in WEI and the resulting changes in relative contribution of drinking water and food water to ingested oxygen (right y-axis); (C) water loss via urine and feces; and (D)
daily variation in leaf-water oxygen isotope composition with relative humidity and resultant change in enamel ␦18O values. VSMOW ⫽ Vienna standard mean ocean
water; WEI ⫽ water economy index.
2 (OW-ANOVA, F ⫽ 1.754, p ⫽ 0.182). The mean ␦18O value for
Coryphodon from interval 2 (Table 1) was found to be significantly higher
than those for Coryphodon from intervals 1 and 3 (OW-ANOVA, F ⫽
3.393, p ⫽ 0.05). Uncertainties in the inferred ecologies for these early
Eocene mammals make it difficult to differentiate between EI and ES
taxa. Given the lack of large differences in mean ␦18O values among
taxa, the difference in 18O enrichment between EI and ES taxa does not
appear to have been significant for these fauna, and so ␦18O values for
all taxa were combined and used for comparison with mean values for
Coryphodon. Mean ␦18O values for faunas from intervals 1–3 were 21.8
⫾ 1.6‰, 21.2 ⫾ 1.2‰, and 21.4 ⫾ 2.0‰, respectively (Fig. 5B). Mean
␦18O values for large Coryphodon from intervals 1 and 3 are significantly
lower than those of the associated fauna (Student’s t-test, p ⫽ 0.01) and
are similar to expected ␦18O values for hippo ecomorphs using the above
equation and the mean ␦18O value for the whole fauna (18.7‰–19.2‰;
see Fig. 6). In contrast, the mean ␦18O value for small Coryphodon from
interval 2 is indistinguishable from that of the fauna and much higher
than expected for a hippo ecomorph (Fig. 6). Variance in ␦18O values
for Coryphodon was similar to that of Phenacodus and Ectocion but was
significantly lower than that for Hyracotherium from interval 3 (Table 2).
Analysis of ␦13C and ␦18O composition of the proboscidean Moeritherium and the anthracothere Bothriogenys gorringei found statistically
significant isotope differences between these taxa and the associated fauna
(OW-ANOVA, ␦13C: F ⫽ 6.04, p ⬍ 0.001; ␦18O: F ⫽ 12.99, p ⬍
0.001). Moeritherium and the other proboscideans from these deposits
(Phiomia serridens, Paleomastodon beadnelli) had the highest mean ␦13C
values, whereas B. gorringei, Saghatherium antiquum, and Megalohyrax
sp. had the lowest (Table 1). Designation of ES-EI taxa was based on
comparison with living relatives and modern analogues and evaluation of
enamel ␦18O values. Phiomia serridens, and Pa. beadnelli were classified
as EI based on similarities in feeding ecology and relationships with
living proboscideans. Once this designation was set, the remaining fauna
(hyracoids: Saghatherium antiquum, Megalohyrax sp., and Geniohyus
sp.) did not show significant enrichment in 18O relative to the proboscideans and were, therefore, also classified as EI. Mean ␦18O values for
Moeritherium (27.1 ⫾ 1.3‰) and B. gorringei (26.8 ⫾ 1.6‰) were
significantly lower than that of the associated fauna (29.8 ⫾ 2.4‰) and
bracket the expected value for hippo ecomorphs using the above equation
(26.7 ⫾ 0.9‰) (Fig. 6). Arsinoitherium, hypothesized to be semiaquatic
by Court (1993), has values consistent with a terrestrial taxon (28.8 ⫾
1.1‰). Interestingly, Ph. beadnelli (32.5 ⫾ 1.4‰) and Pa. serridens
(30.9 ⫾ 1.3‰) had the highest mean ␦18O values for this locality. No
statistically significant difference in variance was detected between B.
gorringei and Moeritherium and other taxa in the fauna (Table 2)
Teleoceras material from Nebraska, California, and Oregon was compared to that previously sampled from Florida (9.5–4.5 Ma; see MacFadden and Cerling, 1996; MacFadden, 1998). In California and Oregon
few specimens of large ungulates from the same deposits were available
580
CLEMENTZ ET AL.
for comparison with samples from Teleoceras. For the California locality,
large ungulates from the same location but of a slightly earlier age were
sampled and, after statistical analysis showed that ␦13C and ␦18O values
were indistinguishable between time periods (Student’s t-test, ␦13C: t ⫽
⫺1.22, p ⫽ 0.262; ␦18O: t ⫽ 1.04, p ⫽ 0.341), were pooled with the
single species (Pliohippus interpolatus) found with Teleoceras. Mean
␦13C values for Teleoceras sampled from each state and time period were
found to be statistically distinct (OW-ANOVA, F ⫽ 35.663, p ⬍ 0.001)
except between specimens from California, Oregon, and Florida (9.5–7.0
Ma; see Table 3). Mean ␦13C values for Teleoceras and associated fauna
were highest for specimens from Florida (ca. 4.5 Ma) and Nebraska (ca.
11.8 Ma) and lowest for specimens from Florida (ca. 9.5 Ma and 7.5 Ma;
see Fig. 5A).
Enamel ␦18O values for Miocene faunas vary considerably among states
and time periods (Table 1; Fig. 5B). For sample localities in Florida, mean
␦18O values for Teleoceras (OW-ANOVA, F ⫽ 0.645, p ⫽ 0.597) and for
the associated fauna (OW-ANOVA, F ⫽ 0.241, p ⫽ 0.867) did not differ
significantly with age. Except for populations of Teleoceras in California and
Nebraska, differences in the mean ␦18O values among populations from each
location were statistically significant (OW-ANOVA, F ⫽ 199.78, p ⬍ 0.001;
see Table 3), and mean values decrease with increasing latitude. Mean population ␦18O values range from a high in Florida of 32.1 ⫾ 0.4‰ to a low
in Oregon of 19.6 ⫾ 0.8‰ (Fig. 5B). As observed previously by MacFadden
(1998) for Florida populations, enamel ␦18O values for Teleoceras were not
significantly lower than those of coeval terrestrial mammals (Fig. 5B) and
values were much higher than those predicted for a hippo ecomorph from
these sites (Fig. 6). Population-level variance in enamel ␦18O values for Teleoceras at four sites yields conflicting results (Table 2): populations in Nebraska and Florida show low variance in enamel ␦18O values for Teleoceras
relative to coeval fauna, whereas populations on the west coast show much
higher variation that is not significantly different from those of other herbivores at the sites.
DISCUSSION
␦18O and the Hippo Ecomorph
Prior work demonstrated that living and fossil hippopotamids have
lower enamel ␦18O values than that of associated ungulates (Bocherens
et al., 1996). A compilation of these data shows that although the offset
in ␦18O values between hippopotamids and other ungulates may vary
somewhat in magnitude, the trend is consistent and predictable (Fig. 4).
Proposed factors for this difference have included physiological and behavioral differences between hippopotamids and other ungulates that may
or may not be related to aquatic habits. The primary character unrelated
to aquatic habits is nocturnal foraging. Modeling of the fluxes and fractionation of oxygen isotopes within hippopotamids provided a means to
evaluate which of these scenarios could account for the observed difference. Though the reduction in enamel ␦18O values resulting from nocturnal foraging and the associated diurnal changes in leaf water ␦18O
values (D18Oday-night ⬎ ⫹10‰; see Flanagan and Ehleringer, 1991; Cernusak et al., 2002) was sufficient to partially explain the observed difference between hippopotamids and ES taxa (and between EI and ES
taxa, for that matter), the effects of nocturnal foraging did not account
for the observed difference between hippopotamids and EI taxa, which
receive most of their oxygen from water sources with relatively stable
and 18O depleted compositions (i.e., surface water, stem water; see Levin
et al., 2006). Other factors, thus, possibly related to the physiology of
these animals and their mode of life must account for the additional
difference between hippopotamids and EI taxa.
Incorporation of physiological characters specific to living hippopotamids did result in a significant change in predicted enamel ␦18O values
relative to those for ES and EI taxa. Of these characters, increasing the
water flux lost through feces to that measured in living hippopotamids
(Clauss et al., 2004; Schwarm et al., 2006) yielded the greatest drop in
␦18O values and provided the best explanation for this offset. The large
PALAIOS
FIGURE 4—Bivariate plot of mean ␦18O values for living populations and fossil
accumulations of various species of hippopotamids and associated fauna (Kohn et
al., 1996; Leakey et al., 1996; Zazzo et al., 2000; Franz-Odendaal et al., 2002; Harris
and Cerling 2002; Cerling et al., 2003; Levin, et al., 2006). Error bars represent ⫾1
SD from the mean for each group. Dark line represents the linear regression through
the data defined by the equation provided in the graph; long dashed lines represent
the 95% confidence limits for this regression; and short dashed line represents a 1:
1 relationship between enamel ␦18O values for hippos and fauna. VSMOW ⫽ Vienna standard mean ocean water.
body size and expansive foregut of hippopotamids forces a significant
reduction in the size of the lower gastrointestinal tract. Because the large
intestine is the primary site of resorption of water from the feces prior
to defecation, reduction in the size of this organ increases the quantity
of water loss through the feces relative to that observed for smaller artiodactyls and comparably sized perissodactyls with expanded large intestines. Given that this high water loss can only be sustained if these
animals have significant quantities of water available, relatively low ␦18O
values would only be observed when these large animals were associated
with a constant source of water. This unique physiological character of
hippopotamids is, thus, connected to the aquatic habits of this species.
Although this explanation is plausible for large, semiaquatic, foregutfermenting but nonruminating artiodactyls, it raises a question as to
whether perissodactyls and other hindgut fermenters capable of absorbing
a greater quantity of water from their feces would have similarly low
␦18O values relative to the associated fauna. This question was examined
by evaluating the effect of increasing the water loss through other fluxes
(i.e., urine) and augmenting the water economy index to that observed
in fully aquatic herbivorous mammals in fresh-water conditions (i.e., captive Florida manatees, WEI ⫽ 1.3 ml ·kJ⫺1; Ortiz et al., 1999). Increasing
the relative amount of water lost through urine did cause a decrease in
enamel ␦18O values, but this drop was of lower magnitude than that
calculated for increased fecal water loss (Fig. 3C). When the WEI was
increased and combined with an increased rate of water loss through
urine, however, enamel ␦18O values dropped significantly and eventually
matched that achieved through high fecal water loss (Fig. 3C). This mechanism of increased water turnover and loss through urine and feces seems
like a reasonable assumption for most aquatic mammals. When living in
aquatic habitats, large mammals would not need to conserve water but
would most likely need to find ways to expel large quantities of it quickly.
Ingestion or absorption of large amounts of fresh-water could lead
to natremia and would require methods of flushing large quantities of
water out of the body while conserving salts. Examination of the urineconcentrating ability of living hippopotamus supports this suggestion; the
internal structure of the kidneys of living hippopotamus is incapable of
PALAIOS
AQUATIC MAMMAL ISOTOPES
581
FIGURE 5—Box plots of (A) ␦13C values and (B) ␦18O values for five hippo ecomorphs and associated fauna. Hippo ecomorphs ⫽ white boxes, whereas associated
faunas ⫽ gray boxes. Values for Coryphodon and fauna are listed in order from interval 1 (1,000–1,500 m) to interval 3 (1,800–2,200 m). VPDB ⫽ Vienna Pee Dee
Belemnite; VSMOW ⫽ Vienna standard mean ocean water.
producing concentrated urine and would enable this species to flush out
large quantities of water while conserving salts (Beauchat, 1990). This
rapid rate of cycling water through the body would result in body water
␦18O values that closely track that of surface waters and produce enamel
␦18O values that are similar to those observed for hippopotamids.
Given the recent hypothesis of a close relationship between anthracotheres and hippopotamids, the extremely low ␦18O values for Bothriogenys
gorringei are strong evidence that this animal was as aquatic as living
hippopotamids. Anthracotheres were geographically widespread and have
been recovered from Europe, Asia, Africa, and North America. Early forms,
including B. gorringei, typically have been depicted as swinelike and not
as aquatically specialized as later hippopotamids. Through the Cenozoic,
at least one lineage gave rise to taxa (e.g., Kenyapotamus) more hippolike
in form and behavior (Boisserie, 2005; Boisserie et al., 2005a). The discovery of an earlier specialization for fresh-water environments means that
the early ecology of this group should be evaluated in greater detail. Prior
work had also used the expectation of low enamel ␦18O values for fossil
hippopotamids as a measure of the isotopic integrity of fossil materials
from the middle Miocene to the present (Bocherens et al., 1996; Zazzo et
al., 2000). With the detection of a similar relationship in anthracotheres,
this test can now be extended back at least to the early Oligocene African
anthracotheriids and should be investigated in older Eocene taxa and anthracotheriids from faunas on other continents.
Although no living hind-gut fermenters are as morphologically committed to a semiaquatic lifestyle as living hippopotamids, several species
have been described from the fossil record. The three included in this
582
PALAIOS
CLEMENTZ ET AL.
TABLE 3—Statistical results from a post-hoc Bonferroni test of mean ␦13C and ␦18O
for the four populations of Teleoceras. The p-values for each pairwise comparison
between populations are reported for mean ␦13C values (lower-left corner) and mean
␦18O values (upper-right corner).
Middle–Late Miocene Teleoceras localities
Oregon
California
Nebraska
Florida (9.5–7.0 Ma)
Florida (4.5 Ma)
FIGURE 6—Bivariate plot of mean ␦18O values for five hippo ecomorphs and
associated faunas. Solid black line and dashed lines represent the relationships between mean faunal ␦18O values vs. hippopotamid ␦18O values presented in Figure
4. Error bars represent ⫾1 standard error from the mean for each group. Mean ␦18O
value for smaller Coryphodon is in gray. VSMOW ⫽ Vienna standard mean ocean
water.
study (Coryphodon, Moeritherium, and Teleoceras) have been considered
hippo ecomorphs based on morphological similarities with living hippopotamids and were analyzed isotopically to see if the observed difference in ␦18O values between hippopotamids and associated fauna would
hold for hindgut fermenters as well.
Of these three species, only Moeritherium and large species of Coryphodon were found to have low enamel ␦18O values similar to those of
living hippopotamids and the coeval anthracothere, Bothriogenys gorringei. In contrast, enamel ␦18O values for Teleoceras at all sites were statistically indistinguishable from those of the associated fauna. Moeritherium appears to have been the most aquatic, and Teleoceras the least
aquatic, of the three. Coryphodon is interesting because of the difference
in enamel ␦18O values for species of different body size based on this
evidence. A smaller species (⬃100 kg) from the early Eocene does not
meet this criterion and does not appear to have been semiaquatic, whereas
large species from the late Paleocene and early Eocene appear to match
expectations in mean ␦18O values for hippo ecomorphs. Lack of a similar
shift in mean ␦18O values for other taxa sampled within this interval
indicates that this difference cannot be explained by environmental
change and is most likely related to changes in the physiology, or ecology
Oregon
California
—
1.000
0.008
0.538
⬍0.001
⬍0.001
—
⬍0.001
1.000
⬍0.001
Florida
Nebraska (9.5–7.0 Ma)
Florida
(4.5 Ma)
⬍0.001
0.063
—
⬍0.0001
0.007
⬍0.0001
⬍0.0001
⬍0.0001
1.000
—
⬍0.001
⬍0.001
⬍0.001
—
⬍0.001
of Coryphodon, or both. The body masses for large species of Coryphodon are estimated to have been between 200 kg and 400 kg, whereas
those of Phenacodus, Ectocion, and Hyracotherium are predicted to have
been ⬍50 kg (Table 1). As has been shown for living elephants (Bryant
and Froelich, 1995), this order of magnitude difference in body size could
create a small, but significant difference in mean ␦18O values between
Coryphodon and other herbivores. Alternatively, differences in mean
␦18O values are explained by ecological differences based on body size.
Large species of Coryphodon may have spent more time in the water,
whereas smaller species may have favored more time on land. The similarity in enamel ␦13C values would suggest that this habitat separation
did not correlate with a dietary difference between these sizes (Table 1)
and that such factors as predation and thermoregulation may have led to
these differences in aquatic habits. It is difficult to determine whether the
lower ␦18O values for large species of Coryphodon are a result of the
large body mass difference between these species and the associated early
Eocene fauna or an ecological change between large and small species
of Coryphodon. Further empirical work on the effects of body mass and
ecology on oxygen isotope composition is needed to fully address this
question.
Variation in enamel ␦18O values for fossil hippopotamids and the four
hippo ecomorphs was not consistently lower than that of associated terrestrial fauna (Fig. 1; Table 2). This is somewhat surprising given the
low variation typically observed in living hippopotamids and other aquatic mammals (see Fig. 1). For these species, surface waters are the primary
oxygen influx and, as such, constrain the possible range in ␦18O values
among individuals in a population. Any fluctuations in these values over
the course of an individual’s life are most likely to be experienced by all
individuals and therefore are averaged over the entire population. Two
factors that can complicate this interpretation are mixing of individuals
from (1) different geographic locations or (2) different time periods. The
TABLE 2—Results from multiple F-tests between proposed hippo ecomorphs (Teleoceras, Coryphodon, Bothriogenys, and Moeritherium) and coeval species from each locality:
CA ⫽ California; NE ⫽ Nebraska; FL ⫽ Florida. Statistically significant p-values (⬍ 0.05) are listed in bold.
Phenacodus
Ectocion
Hyracotherium
Arsinoitherium
Paleomastodon
Phiomia
Saghatherium
Gomphotherium
Pliohippus
Procamelus
Pseudohipparion
Aphelops
Cormohipparion
Coryphodon
Bothriogenys
Moeritherium
Teleoceras CA
Teleoceras NE
Teleoceras FL
0.105
0.134
0.041
—
—
—
—
—
—
—
—
—
—
—
—
—
0.410
0.665
0.438
0.469
—
—
—
—
—
—
—
—
—
0.770
0.515
0.869
0.346
—
—
—
—
—
—
—
0.868
0.713
0.679
—
—
—
—
—
—
—
—
—
—
—
0.216
0.002
0.001
—
—
—
—
—
—
—
—
—
—
—
—
0.222
0.308
0.025
PALAIOS
583
AQUATIC MAMMAL ISOTOPES
amount of time averaging or spatial averaging affecting a fossil accumulation can vary considerably and be difficult to quantify (Behrensmeyer, 1982). In terrestrial and fresh-water ecosystems, the oxygen isotope composition of precipitation and surface waters can vary greatly
through time or space as a result of changes in temperature, seasonality,
air masses, and a host of other climatic factors. An increase in the amount
of time and space represented in a fossil accumulation may cause the
variation in enamel ␦18O values among individuals of a species to increase as well because of these perturbations. In marine ecosystems, however, seawater is relatively homogeneous (⫾1.0‰) over long time scales,
and mammals living in these waters that differ slightly in age or location
would be expected to show relatively small differences in enamel ␦18O
values. While low population-level variation in ␦18O values is an excellent measure of aquatic habits in marine species (Clementz et al., 2003;
Clementz et al., 2006), greater care must be taken when dealing with
fossil specimens from fresh-water deposits where the effects of time averaging are more pronounced and ␦18O values are potentially more variable.
␦13C and Diets of Fossil Hippo Ecomorphs
Evidence of the dietary preferences of Arsinoitherium, Bothriogenys,
Coryphodon, Moeritherium, and Teleoceras is provided by enamel ␦13C
values. As explained earlier, most of the Cenozoic continental ecosystems
included in this project were dominated by C3 vegetation, which caused
the range in enamel ␦13C values at most sites to be relatively small (Fig.
5A). Even so, significant differences are observed within each fauna that
can be used to interpret dietary differences among species. For instance,
Koch et al. (1995) interpreted the low ␦13C values of Coryphodon relative
to those of the remaining early Eocene fauna as evidence that this early
mammal was foraging more deeply into the forest than other mammals
and probably filled a niche similar to that of living tapirs.
Low ␦13C values for the early Oligocene anthracothere Bothriogenys
gorringei and the hyracoids Saghatherium antiquum and Megalohyrax
sp. may support a similar interpretation of foraging under closed-canopy
conditions. Given the inferred semiaquatic habits of B. gorringei, low
␦13C values could instead indicate it was consuming aquatic macrophytes
growing in fresh-water rivers or streams; the overlap in ␦13C values for
fresh-water macrophytes and terrestrial C3 plants makes it difficult to
distinguish between these two dietary sources. In contrast, the higher
␦13C values of Arsinoitheirum, Moeritherium, and other proboscideans
suggest that these large mammals may have been foraging in more open
conditions. Values for a few individuals of Phiomia serridens are at the
upper extreme for a pure C3 forager. These enamel ␦13C values could
mean that the plants these animals were consuming were environmentally
stressed, possibly from high levels of dissolved salts or limited water.
Fossil plants from these deposits include mangrove specimens, which are
capable of tolerating marine waters and are thought to have formed extensive forests that hemmed the coast of the Tethys Sea (Bown et al.,
1982). The large proboscideans may have foraged more extensively on
these plants and others growing within the forests than other herbivores.
The elevated ␦13C values of mangroves and other salt-stressed plants
would have been passed on to large proboscideans. For Moeritherium in
particular, the possibility that it fed on salt-stressed plants is supported
by the fact that another species of this genus is also found commonly in
the slightly older, nearshore marine deposits of the Qasr el Sagha Formation (Holroyd et al., 1996), suggesting that it also frequented brackish
water or salt water. The lower mean ␦18O value for this genus indicates
it still mostly consumed or inhabited fresh water. The diets of Moeritherium and Bothriogenys were quite distinct and, thus, indicate that the
ecological similarities between these species were limited to their preference for aquatic environments.
Unlike the other fossils analyzed, specimens of Teleoceras were collected from multiple localities that cover a broad temporal and spatial
range. Teleoceras specimens from California, Florida (9.5–7.0 Ma), and
Oregon have enamel ␦13C values consistent with consumption of a pure
C3 diet (Fig. 5A). In Nebraska, the high ␦13C values for Teleoceras and
other herbivores suggest consumption of some C4 grasses (i.e., ⱕ30%
of diet). Recent ␦13C evidence from soil carbonates indicates that C4
grasses were present in the Great Plains at low abundances (12%–34%
of biomass) throughout the Miocene (Fox and Koch, 2003), and this could
account for the high enamel ␦13C values reported at this locality (Fig.
5).
Interestingly, late Miocene and early Pliocene sites in California and
Oregon that date from the time after C4 grass expansion have low ␦13C
values, suggesting that either C4 plants were not present at these localities
or that Teleoceras and the other taxa were not consuming them. The
possession of high-crowned (i.e., hypsodont) molars, as well as the presence of fossil remnants of ingested grasses with Teleoceras remains in
Nebraska, is evidence that Teleoceras was capable of grazing like living
hippos (Voorhies and Thomasson, 1979). If C4 grasses were present at
these localities, Teleoceras would have likely consumed them. The Mediterranean climate (winter rainfall) of the west coast favors the use of C3
over C4 photosynthesis by grasses in this region today. Lack of a C4
signal in herbivore tooth enamel from these localities is evidence that
these climate conditions (i.e., wet winters, dry summers) were in place
in the late Miocene and early Pliocene.
No consistent pattern in ␦13C values or dietary preferences was identified for hippo ecomorphs. The diets of living hippopotamids reflect this
as well. Most dietary studies have focused on the larger species (Hippopotamus amphibius), which has been interpreted as a terrestrial grazer,
favoring a mix of C3 and C4 grasses that grow within a short distance
of the rivers and lakes in which these animals live. Recent stable isotope
and microwear analyses of living individuals found in open and closed
canopy conditions, however, have shown that the relative percentage of
C4 grass in the diet is closely correlated with habitat and can vary from
30% to 100% (Boisserie et al., 2005b). Proximity to aquatic refugia may
be the strongest factor determining the diets of hippo ecomorphs, and
individuals may be capable of varying their diet as the plant life within
their home ranges changes. This finding does suggest that animals in this
role may be more inclined to be dietary generalists rather than specialists,
although this means that enamel ␦13C may not be diagnostic of this ecological niche.
CONCLUSIONS
Analysis of published ␦18O values for living and fossil hippopotamids
revealed a consistent difference between these individuals and the associated terrestrial fauna. Modeling of the oxygen fluxes and fractionations
within hippopotamids showed that increased water loss via urine or feces
may account for this pattern—a physiological character that is likely true
for all large-bodied aquatic herbivores. In contrast, although a significant
difference in intraspecific variation in enamel ␦18O values has been observed between living aquatic and terrestrial mammals, this difference
was not detected consistently for fossil specimens from continental deposits. Extensive time and spatial averaging of continental fossil accumulations may explain this discrepancy and should, therefore, be considered when making ecological interpretations from the geochemical composition of fossil remains.
Morphological characters were not always diagnostic of aquatic preferences based on our results. A small species of the early Eocene pantodont Coryphodon and the Miocene rhinoceros Teleoceras did not have
␦18O values that were low enough relative to the associated fauna to
support aquatic habits for these animals. In contrast, two co-existing species from the early Oligocene of Egypt (the anthracothere Bothriogenys
and the proboscidean Moeritherium) and larger species of Coryphodon
were found to fit predicted values and had ␦18O values 2‰–4‰ lower
than that of the associated fauna. Discovery of this character in Bothriogenys supports an interpretation of aquatic lifestyle for anthracotheres
584
CLEMENTZ ET AL.
and hippopotamids early in their evolutionary history, whereas the presence of low ␦18O values in Moeritherium relative to the coeval fauna
supports the use of this measure to identify aquatic taxa outside of the
anthracothere-hippopotamid clade.
No consistent dietary preference was found to be associated with the
hippo ecomorphs. Enamel ␦13C values for Bothriogenys suggest this species foraged deeper within forests or more heavily on aquatic vegetation
than coeval Moeritherium. Enamel ␦13C values for Coryphodon and Teleoceras populations at most sites were consistent with a pure C3 diet.
Enamel ␦13C values for Teleoceras specimens sampled from California
and Oregon suggest that C4 grasses were not present along the Pacific
Coast by 5 Ma. Significantly higher ␦13C values for Teleoceras in Nebraska, however, support the presence of an appreciable amount of C4
grasses in the Great Plains by at least 11 Ma.
The occurrence and abundance of large, semiaquatic herbivorous mammals today is low, but our findings suggest this may not always have
been the case. The identification of at least two sympatric semiaquatic
species from the Oligocene of Egypt indicates that the diversity and abundance of this ecomorph could have been higher in the past. Development
of a new means of using the stable isotope composition of fossil materials
to identify these species in the fossil record will provide an opportunity
to quantify the relative abundance of these taxa within faunas, examine
what factors might facilitate the evolution of this ecomorph, and evaluate
their role in the structuring of ancient ecosystems.
ACKNOWLEDGMENTS
Funding for this research was provided by National Science Foundation, Division of Earth Sciences grant 0087742. We thank J. Meng at the
American Museum of Natural History and M. Voorhies at the Nebraska
State Museum of Natural History for access to and information on specimens sampled for this project; M.B. Goodwin for assistance with sampling; K. Fox-Dobbs and B. Crowley for assistance with lab work; and
T. Cerling, B. Passey, K. Hoppe, D. Fox and an anonymous reviewer for
comments on this manuscript and early results for this project. This is
University of California Museum of Paleontology contribution no. 1966.
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ACCEPTED APRIL 7, 2008