IDENTIFYING AQUATIC HABITS OF HERBIVOROUS …pkoch/pdfs/Koch papers/2008/Clementz et 08 Palaios...

Copyright � 2008, SEPM (Society for Sedimentary Geology) 0883-1351/08/0023-0574/$3.00

PALAIOS, 2008, v. 23, p. 574–585

Research Article

DOI: 10.2110/palo.2007.p07-054r

IDENTIFYING AQUATIC HABITS OF HERBIVOROUS MAMMALS THROUGH STABLEISOTOPE ANALYSIS

MARK T. CLEMENTZ,1* PATRICIA A. HOLROYD,2 and PAUL L. KOCH3

1University 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, USAe-mail: [email protected]

ABSTRACT

Large-bodied, semiaquatic herbivorous mammals have been a recur-ring component of most continental ecosystems throughout the Ce-nozoic. Identification of these species in the fossil record has largelybeen based on the morphological similarities with present-day hip-popotamids, leading to the designation of this pairing of body typeand ecological niche as the hippo ecomorph. These morphologicalcharacters, however, may not always be diagnostic of aquatic habits.Here, enamel �13C and �18O values from living hippopotamuses wereexamined to define an isotopic signature unique to the hippo eco-morph. Although �13C values do not support unique foraging habitsfor this ecomorph, living and fossil hippopotamids typically have lowmean �18O values relative to associated ungulates that fit a linearregression (�18Ohippopotamids � 0.96 � 0.09 ·�18Ofauna � 1.67 � 2.97;r2 � 0.886, p � 0.001). Modeling of oxygen fluxes in large mammalssuggests that high water-turnover rates or increased water lossthrough feces and urine may explain this relationship. This relation-ship was then used to assess the aquatic adaptation of four purportedhippo ecomorphs from the fossil record: Coryphodon (early Eocene),Moeritherium and Bothriogenys (early Oligocene), and Teleoceras(middle–late Miocene). Only fossil specimens of Moeritherium, Both-riogenys, and large species of Coryphodon had �18O values expectedfor hippo ecomorphs; �18O values for Teleoceras and a small speciesof Coryphodon were not significantly different from those of the as-sociated fauna. These results show that the mean �18O value of fossilspecimens is an effective tool for assessing the aquatic habits of ex-tinct species.

INTRODUCTION

The purpose of this paper is to assess the validity of isotopic methodsfor identification of aquatic and semiaquatic taxa in the fossil record andto determine what conditions must be met before these methods may beused. Although prior work has applied oxygen isotope analysis to hip-popotamids (Bocherens et al., 1996), no study has adequately addressedwhat factors generate characteristic oxygen isotope values in hippopotam-ids and, therefore, whether this approach is applicable to mammals out-side of this clade.

Using published isotope data from living and fossil hippopotamids, weconstruct a quantitative model for the hippo ecomorph that accounts forenvironmental and physiological influences on oxygen isotope values.This model is then applied to three test cases from the fossil record. Thefirst looks at the early Oligocene anthracothere Bothriogenys gorringei,the early proboscidean Moeritherium, both of which have been hypoth-esized as semiaquatic mammals based on morphology and occurrence offossil remains in fluvial environments (Osborn, 1909; Simons, 1964; Cop-pens and Beden, 1978; Pickford, 1983, 1991; Ducrocq, 1997; Boisserie,

* Corresponding author.

2005), and the co-occurring embrithopod Arsinoitherium from the JebelQatrani Formation, Egypt, interpreted as both a terrestrial grazer andsemiaquatic form (Thenius, 1969; Sen and Heintz, 1979; Court, 1993).The second case reexamines published isotope values for the North Amer-ican pantodont Coryphodon and associated early Eocene fauna from theBighorn Basin, Wyoming (Koch et al., 1995), to look for evidence ofaquatic adaptations to support previous interpretations based on postcra-nial and dental morphology. The third case applies our model to theextinct rhinoceros Teleoceras, a genus whose aquatic adaptations havebeen questioned (Prothero, 1992; MacFadden, 1998; Mead, 2000; Mihl-bachler, 2003). Analysis of the �13C and �18O values of each of thesetaxa and comparison with our hippo ecomorph model will enable us toassess whether this model is appropriate for taxa outside of the hippo-potamid clade and better document the recurrence of large, semiaquaticmammals in terrestrial ecosystems.

Large-bodied, semiaquatic herbivores are an interesting ecological mor-photype (i.e., ecomorph) best exemplified by two living artiodactyls—theAfrican river hippopotamus (Hippopotamus amphibius) and the pygmy hip-popotamus (Choeropsis liberiensis; see Gatesy, 2002). Both hippopotamidshave 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. Hippo-potamids have a relatively short fossil record (middle Miocene–recent), butlatest phylogenetic analyses nest this group within the anthracotheres—anextinct group of artiodactyls—which have a fossil record dating back to thelate middle Eocene (Boisserie, 2005; Boisserie et al., 2005a) and which aresuggested to be semiaquatic in their habits.

Several Cenozoic mammalian clades are thought to have independentlyevolved semiaquatic lineages, based on morphological characters shared withextant hippopotamids (e.g., Meehan and Martin, 2003). Aside from anthra-cotheres and hippopotamids in Africa and Eurasia, at least one species ofproboscidean may have been a semiaquatic herbivore. Moeritherium was anearly relative of elephants that had a long body with short limbs and elevatedorbits, and it inhabited river channels and estuaries in northern and westernAfrica from late Eocene to early Oligocene (Osborn, 1909; Simons, 1964).In North America, the hippo ecomorph may have been filled convergentlyby taxa from three separate orders—Pantodonta, Perissodactyla, andArtiodactyla—from the late Paleocene into the late Miocene. If so, the hippoecomorph was a recurring component of North American ecosystems forover 55 million years (Meehan and Martin, 2003). Two of the most oftencited examples of this body type in the North American fossil record are thepantodont Coryphodon from the early to middle Eocene (Lucas, 1998) andthe rhinoceratid Teleoceras from the middle Miocene to early Pliocene (15–4.5 Ma; see Webb, 1983). Both were stout-bodied, short-legged herbivoresthat were geographically widespread and abundant within continental eco-systems. 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 de-posits, both of which support interpretations of their aquatic habits (Lucas,1998; Prothero, 1998).

PALAIOS 575AQUATIC MAMMAL ISOTOPES

FIGURE 1—Bar graph depicting the mean levels of standard deviation (here de-noted by s) for living populations (gray) and fossil accumulations (black) of aquaticmammals, hippopotamids, and terrestrial mammals (Kohn et al., 1996; Leakey et al.,1996; Zazzo, et al., 2000; Clementz and Koch, 2001; Cerling et al., 2003; Franz-Odendaal et al., 2002; Harris and Cerling, 2002; MacFadden et al., 2004; Levin etal., 2006). Error bars reflect 1 standard deviation from the mean, and numbers inparentheses above each bar indicate the number of populations or accumulationsincluded in each. Statistically significant differences were detected among mean val-ues (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).

How well these morphological criteria actually diagnose ecologicalpreferences is debated, and it is particularly difficult to determine whetherthe few modern taxa can be used effectively as morphologic analogs fororganisms not closely related. Even the definition of aquatic or semi-aquatic behavior in modern mammals is unclear. Most uses of the termsare undefined. Where defined, the criteria are not necessarily ones thatcan be readily applied to fossils. Fish and Stein (1991, p. 340) recognizeaquatic mammals as those ‘‘(1) that exhibit obvious external modifica-tions of both the limbs and body which allow them to live successfullyin water, (2) that forage primarily in water, and (3) that use water as aprimary means to escape predation.’’ They characterized semiaquaticmammals by their second and third criteria, but not the first. These cri-teria, however, are not directly observable in the fossil record, and thereare few clear-cut morphological characters that permit reliable inferencesto be made about the use of aquatic habitats by fossil organisms.

For these reasons, hypotheses about semiaquatic behavior should betested using alternative methods that are independent of morphology. Thestable isotope composition of fossil material has been shown to be anexcellent source of ecological information, making it a prime candidatefor independently testing ecomorphologic hypotheses. The oxygen iso-tope composition of fossil materials has been exploited as a proxy forpaleotemperature and salinity, but, more important, it has been used todistinguish between terrestrial and aquatic species (Bocherens et al., 1996;MacFadden, 1998; Clementz and Koch, 2001). Both the mean and thevariance in the oxygen isotope composition of individuals within fossilpopulations have proven effective in identifying aquatic species and couldbe used to do the same for hippolike species within ancient faunas.

Interestingly, despite their morphological adaptations to a semiaquaticlife and the fact that they spend a great deal of time in the water, modernhippopotamuses obtain the bulk of their food outside of the water. Of thetwo living species, the larger common hippo primarily grazes on terres-trial grasses at night (Field, 1970; Scotcher et al., 1978), whereas thesmaller pygmy hippopotamus is primarily a browser of semiaquatic veg-etation, wild fruits, and low-growing ferns and herbs (Nowak and Para-diso, 1983). Carbon isotope (�13C) values are already widely used inpaleodietary research to distinguish grazing and browsing behavior forfossil hippopotamids (Bocherens et al., 1996; Zazzo et al., 2000; Boisserieet al., 2005b) and may also be useful for assessing whether semiaquaticspecies 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)] isdictated by the �18O value of an animal’s body water and the temperatureat which the mineral forms. As mammal body temperature is relativelyconstant, variations in enamel �18O values are controlled by variationsin body water �18O values that, in turn, are controlled by the �18O valueof environmental oxygen sources as well as fluxes and fractionationsassociated with physiological functions (Bryant and Froelich, 1995;Kohn, 1996). Mean enamel �18O values typically correlate with surfacewater �18O (�18OSW) values (Luz and Kolodny, 1985; Delgado Huertaset al., 1995), but within-population variance is high for published �18Odata from terrestrial species (Fig. 1; 1 SD � 1.0‰) as a result of temporaland geographic variations in the �18O values of environmental oxygensources as well as individual differences in physiological response (Cle-mentz and Koch, 2001). Published �18O data for aquatic species—animals spending �50% of their time in the water—show a similar cor-relation with �18OSW, but �18O values among individuals within a pop-ulation typically vary much less (Yoshida and Miyazaki, 1991; Clementzand Koch, 2001). The major oxygen flux for aquatic species comes fromthe water that they inhabit (Hui, 1981; Andersen and Nielsen, 1983;Kohn, 1996), which can result in low within-population variance (Fig. 1;1 SD � 0.5‰) for species living in such isotopically homogeneous wa-ters as seawater, large lakes, or rivers. Population-level variation in �18O

values for extant hippopotamids typically falls within the expected rangefor aquatic and semiaquatic mammals with a few exceptions (Fig. 1).

Aquatic habits may not be the only cause for low �18O variation inpopulations, however. At large body sizes (�4,500 kg), environmentalwater is the dominant oxygen flux for mammals (�80% of total influxfor mammals; see Bryant and Froelich, 1995). For environments withlittle seasonal or spatial variation in surface water �18O values, this mayreduce the variance for populations of extremely large mammals to thatof aquatic or semiaquatic species. Elephants are the only mammal speciesto reach these extreme body sizes today (Silva and Downing, 1995). Thetypical value for most elephant populations is on par with that of otherterrestrial species (mean SD � 1.1 � 0.3‰) and much higher than thatfor aquatic species (Cerling et al., 2007), though there is a significantrange 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 dampenthe enamel �18O variation for a population, these values should be dis-tinguishable from those of aquatic and semiaquatic species.

Published �18O values for modern hippopotamids are consistently lowerthan those for associated fauna (Fig. 2; see Bocherens et al., 1996; Kohn etal., 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 theyare thought to relate to semiaquatic habits that lead to reduced evaporativeloss of 16O-enriched water from the body; nocturnal foraging that leads toreduced evaporative water loss across the skin and consumption of plantwater that has not experienced 18O-enrichment due to evapotranspiration; orthe unique physiology of hippopotamids (Bocherens et al., 1996). This patternhas 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 usefulfor defining the hippo ecomorph.

The �13C composition of the structural carbonate in tooth enamel ap-atite is directly related to that of an animal’s diet (Ambrose and Norr,

576 PALAIOSCLEMENTZ ET AL.

FIGURE 2—Mean �18O values for one species of hippopotamid (Hippopotamusamphibius) 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 andCerling 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 SDfrom the mean for each group. VSMOW � Vienna standard mean ocean water.

1993; Cerling and Harris, 1998). For herbivorous mammals, vegetationis the primary carbon source. The �13C value of dietary plants is depen-dent on the photosynthetic pathway used by the plant (O’Leary, 1988;Farquhar et al., 1989) and the �13C composition of the environmentalcarbon source (Bunn and Boon, 1993; Boon and Bunn, 1994; Raven etal., 2002; Cerling et al., 2004). For terrestrial vegetation, the metabolicpathway used for carbon fixation during photosynthesis creates the largestdifferences in �13C values for such plant types as C3 trees, shrubs, andcool-climate grasses (mean �13C � �27 � 3‰; C4 warm-climate grass-es: mean �13C � �13 � 2‰), but environmental stress (i.e., aridity,salinity) and variation in the �13C composition of CO2 (closed canopyforests: mean �13C � �31.2 � 2.3‰;) can also create significant dif-ferences in plant �13C values (O’Leary, 1988; Farquhar et al., 1989;Cerling et al., 2004).

In fresh-water environments, primary producers use the same photo-synthetic pathways as those used by terrestrial plants, but their �13C com-positions are also strongly controlled by the physical conditions associ-ated with growth in an aquatic environment. Variation in the �13C com-position, concentration, and source of dissolved inorganic carbon as wellas a significant reduction in the rate of diffusion of CO2 in water versusair can cause the �13C values for fresh-water primary producers to sig-nificantly 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 evenwith the inherent variation imposed by the environment. Mean �13C val-ues for fresh-water phytoplankton (temperate: �28.6 � 1.3‰; tropical:�33.3‰), however, are much lower (Hamilton et al., 1992; Forsberg etal., 1993; Cloern et al., 2002) and suggest that enamel �13C values—offset from plant �13C values by �14.1‰ for large herbivorous mam-mals (Cerling and Harris, 1998)—can discriminate among consumers for-aging on different primary producers in terrestrial and fresh-water eco-systems. Even with the absence of significant quantities of C4 grassesfrom terrestrial ecosystems until the middle Miocene (Cerling et al., 1997;Fox and Koch, 2003), differences in �13C values for terrestrial and aquat-ic C3 plants are still sufficient to distinguish among consumers foragingin open, arid terrestrial habitats (higher �13C values), humid terrestrialhabitats or aquatic food webs fueled by fresh-water macrophytes (low�13C values), and deep forest or aquatic food webs fueled by fresh-waterphytoplankton (very low �13C values; see Clementz and Koch, 2001;Darimont et al., 2007).

Semiaquatic mammals may be recognized by two stable isotope signalsthat also characterize living hippopotamids. First, mean �18O values forhippopotamids are significantly lower than those of the associated fauna.The ability to apply this feature to taxa other than hippopotamids is ex-amined in this study. Second, population-level variance in �18O valuesfor hippopotamids is generally lower than that of the associated faunawhen local surface waters are isotopically homogeneous. This character-istic has already been identified for marine mammal species (Clementzand Koch, 2001) and should be useful when interpreting the aquatic hab-its of extinct species. In conjunction with the information provided by�18O values, enamel �13C values can be used to determine whether semi-aquatic, herbivorous mammals were feeding on land in open habitats orclosed forests and in fresh-water food webs fueled by aquatic macro-phytes or phytoplankton. We will now examine how well each of thesecriteria works toward identifying aquatic mammals in the fossil recordby applying these proxies to specimens of purported semiaquatic taxafrom the Eocene (Coryphodon), Oligocene (Arsinoitherium, Bothrio-genys, Moeritherium), and Miocene (Teleoceras).

METHODS AND MATERIALS

Modeling Oxygen Isotope Fractionation in Semiaquatic Mammals

A model was constructed to determine which physiological, environ-mental, and behavioral factors most likely account for the difference in�18O values between hippopotamids and other ungulates based on thetheoretical work of Kohn (1998). The parameters were adjusted to testtheir impact on body water and enamel �18O values. Physiological factorsincluded those identified for hippopotamids—low basal metabolic rate,no sweating, increased water loss through feces—and others assumed foraquatic mammals—high water-flux rate, increased water loss via urine orfeces. 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 tothose values that maintained positive water balance for all fluxes of ox-ygen. The effect of nocturnal foraging was modeled by varying the am-bient temperature and humidity conditions while holding surface water�18O values constant and then calculating the resulting change in plantwater �18O values.

Stable Isotope Analysis

Data were collected by either compiling published values for extanthippopotamids and fossil taxa of interest or by analysis of fossil speci-mens sampled from collections at the University of California Museumof Paleontology, the University of Nebraska Museum of Natural History,the University of Michigan Museum of Paleontology, and the AmericanMuseum of Natural History. When available, a minimum of five speci-mens was analyzed at each site to provide a robust estimate of the pop-ulation mean and standard deviation for �13C and �18O values (Clementzand Koch, 2001). In addition to Teleoceras, Moeritherium, and Bothri-ogenys, we analyzed unambiguously terrestrial mammals as controls ateach site. Information on locality age, species sampled, and individual�13C and �18O values are reported in Supplementary Data 1–21. Mac-Fadden (1998) performed an earlier study for populations of Teleocerasfrom the middle to late Miocene of Florida and found no significantdifference between Teleoceras and an associated terrestrial rhinocerosAphelops. These data were supplemented by including specimens of Te-leoceras from other localities in California, Nebraska, and Oregon andby augmenting the number of taxa included from the Florida fauna. Body-mass 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 faunawere taken from Koch et al. (1995) and include specimens spanning the

1 www.paleo.ku.edu/palaios


Paleocene-Eocene boundary from 1000 m to 2200 m above the base ofthe Willwood Formation in the Clarks Fork Basin, Wyoming. Within thisinterval, the body size of Coryphodon species typically is much largerthan 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) andapproaches that of the other species analyzed from this section (Ginger-ich, 1990; Uhen and Gingerich, 1995). Taking care to avoid inclusion ofspecimens sampled from the interval of intense global warming duringthe Paleocene–Eocene Thermal Maximum (i.e., from 1520 m above thebase of Willwood Formation), we separated specimens of the smallerspecies of Coryphodon and associated fauna and compared them to thoserecovered 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 speciessensitive (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 definedas those taxa that show a significant 18O-enrichment between tooth enam-el and meteoric water �18O values as a response to increasing evaporativeeffects on the �18O of leaf water. These taxa include giraffids, oryx, dik-dik, Grant’s gazelle, and buffalo, which show a positive correlation be-tween enamel �18O values and water deficit—the cumulative differencebetween potential evapotranspiration and precipitation for a region. Incontrast, EI taxa are defined as those taxa that show no statistically sig-nificant 18O-enrichment between enamel and meteoric water �18O values.These taxa include hippopotamus, bush pig, elephant, rhinoceros, wart-hog, zebra, impala, and baboon, which show a strong correlation betweenenamel and meteoric water �18O values. For Miocene or younger fossilcommunities from North America and Africa where these or related taxawere available for comparison, designation of EI and ES taxa was rela-tively straightforward. Identification of these faunal groups from olderfossil communities where behavioral information is unclear, however, wasmore complicated. Identification of EI and ES taxa in these cases wasbased primarily on morphology, comparison with modern analogs, andstable isotopic data. The specific criteria used for designating EI and EStaxa in each case are reported in the Results section.

Approximately 10 mg of enamel powder was collected from each spec-imen either by drilling directly from the tooth or by grinding enamelchips in an agate mortar and pestle. Prior to collection, contaminants wereremoved by abrading the outer surface of the sample. Powders were trans-ferred 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 allowedto sit for 24 h. The supernatant was removed by aspiration, and the re-sidual powder was rinsed five times with deionized water and then soakedin �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 wererinsed five times in deionized water and then lyophilized. Approximately1.5 mg of powder was reacted with 100% phosphoric acid at 90C for 8min in a constantly stirred reaction vessel, and the resulting CO2 wascryogenically purified and analyzed for isotopic composition using anISOCARB automated carbonate device attached to an Optima gas sourcemass 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 DeeBelemnite standard, and �18O values are reported relative to Vienna stan-dard mean ocean water. Uncertainties in analyses were assessed via mul-tiple analyses of an in-house elephant enamel standard calibrated againstNBS-19 (�13C: SD � 0.1‰; �18O: SD � 0.2‰; n � 30 for both).

For species or genus-level differences in mean value within and amongsites, a Student’s t-test was used for comparisons between two species,and a single-factor analysis of variance was used for comparisons of morethan 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 eval-uated using a standard F-test. For multiple comparisons that did not meetthe criteria necessary to perform parametric statistics, a nonparametricKruskall-Wallis one-way analysis of variance (OW-ANOVA) on rankswas used and followed by evaluation of pairwise comparisons of taxausing Dunn’s Method. For all comparisons, the level of statistical signif-icance was set at p � 0.05. The relationship between hippopotamid andassociated fauna �18O values was evaluated through a linear regressionof the data. Box plots of enamel �13C and �18O values were used tocompare values for fossil herbivores from each site (see Fig. 6). Thedimensions of each box account for 50% of the data (the interquartiledistance; IQD) and is bounded by the upper quartile (UQ) and lowerquartile (LQ). Error bars represent the maximum and minimum valuesthat fall within the range of UQ � 1.5 · IQD and LQ � 1.5 · IQD. Anypoints falling outside of this range are plotted as individual circles. Ahorizontal line through each box represents median values. All statisticalmeasurements were performed using Sigmastat v.3.1 or Kaleidagraphv.3.6 software.

RESULTS

Oxygen Isotope Modeling of Semiaquatic Herbivores

Model results are presented in Figure 3 and illustrate the magnitudeand direction of change in �18O values for the hippo ecomorph relativeto a typical fully terrestrial mammal of similar body mass(�18Ohippo-terrestrial). Modification of the six selected parameters—(1) bas-al metabolic rate (BMR), (2) sweating versus panting, (3) water contentof feces, (4) water economy index (WEI � (water-turnover rate) ·BMR�1

� ml·kJ�1), (5) water loss via urine, and (6) diurnal variation in leafwater oxygen isotope composition (i.e., diurnal vs. nocturnal foraging)—all have a significant impact on the �18O composition of mammal bodywater. Of these, lowering the basal metabolic rate and reducing theamount of oxygen lost as sweat was found to increase the �18O com-position of mammal body water and tooth enamel (Fig. 3A) by increasingthe proportion of oxygen lost through evaporation across the skin andthrough panting, respectively. Selected modifications to all other param-eters were found to lower body water and tooth enamel �18O values byvarying degrees.

Increasing the WEI to values reported for large, fully aquatic herbi-vores (Ortiz et al., 1999; Ortiz and Worthy, 2006) caused enamel �18Ovalues to drop by more than 2‰ and was associated with an increase inthe proportion of drinking water (from 5% to 80%) ingested relative tothe amount of 18O-enriched water ingested from food (Fig. 3B). Higherwater loss through feces and urine, which are not fractionated relative tobody water, reduced the amount of 18O-depleted water lost through pant-ing or evaporation across the skin and caused body water and enamel�18O values to drop by more than 2‰ (Fig. 3C). The effect was exac-erbated by varying the WEI from 0.4 ml ·kJ�1 to 1.3 ml ·kJ�1 but variedin direction and magnitude for different fecal water contents. At 60%fecal water content, the proportion of total oxygen loss from urine andfeces was low (�50%), regardless of the WEI. The amount of 18O-depleted water vapor lost through evaporation across the skin and nosedecreased at higher WEI, causing enamel �18O values to decrease. Incontrast, at 90% fecal water content, the proportion of total oxygen lossfrom urine and feces decreased significantly when WEI was increased(from 45% to 74%), resulting in higher enamel �18O values. The largestdecrease in �18O values (�18Ohippo-terrestrial � �6.0‰) was produced byincreasing 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 tolevels expected during nocturnal foraging (Fig. 3D) also caused bodywater and enamel �18O values to drop by � 2.0‰. Modeled leaf water�18O values varied from surface water �18O values (�3.5‰) at 100%


TABLE 1—Carbon and oxygen isotope data (mean � 1 SD) and body mass estimatesfor possible hippo ecomorphs and associated fauna. VPDB � Vienna Pee Dee Bel-emnite; VSMOW � Vienna standard mean ocean water.

Taxon nBody

mass (kg)Mean �13C � 1 s

(VPDB, ‰)Mean �18O � 1 s

(VSMOW, ‰)

Early Eocene, Bighorn BasinCoryphodon (1800 to 2200 m) 10 210 �13.0 � 1.2‰ 19.6 � 1.2‰Phenacodus 11 40 �11.8 � 0.5‰ 20.5 � 0.9‰Ectocion 1 10 �9.8‰ 21.6‰Hyracotherium 15 20 �11.5 � 1.3‰ 22.1 � 2.6‰Coryphodon (1525–1800 m) 8 100 �12.2 � 1.4‰ 20.9 � 1.1‰Phenacodus 7 40 �11.9 � 1.0‰ 20.5 � 0.9‰Ectocion 5 10 �10.2 � 1.0‰ 21.8 � 0.7‰Hyracotherium 9 20 �11.3 � 0.8‰ 21.5 � 1.5‰Coryphodon (1000–1515 m) 9 210 �12.4 � 1.0‰ 19.9 � 0.9‰Phenacodus 16 40 �10.6 � 1.2‰ 21.3 � 1.3‰Ectocion 15 10 �9.7 � 1.1‰ 22.2 � 2.1‰

Early Oligocene, Fayum, EgyptBothriogenys gorringei 10 90 �11.1 � 0.7‰ 26.8 � 1.6‰Moeritherium trigodon 4 810 �10.0 � 0.4‰ 27.1 � 1.3‰Moeritherium lyonsi 1 810 �9.0‰ 27.2‰Arsinoitherium sp. 8 4000 �10.6 � 0.6‰ 28.8 � 1.1‰Phiomia serridens 12 6600 �9.6 � 1.7‰ 30.9 � 1.3‰Palaeomastodon beadnelli 3 3000 �10.4 � 0.3‰ 32.5 � 1.4‰Saghatherium antiquum 4 10 �11.8 � 1.3‰ 27.3 � 2.3‰Megalohyrax sp. 1 180 �12.1‰ 26.6‰Geniohyrax sp. 1 70 �10.8‰ 31.6‰

Middle Miocene (11.8 Ma), NebraskaTeleoceras major 8 1000 �9.3 � 0.9‰ 25.8 � 0.7‰Pliohippus sp. 6 200 �8.4 � 0.6‰ 26.4 � 0.6‰Pseudohipparion sp. 6 60 �9.1 � 1.0‰ 28.2 � 2.1‰Cormohipparion sp. 2 150 �6.8 � 3.8‰ 24.3 � 2.6‰Procamelus sp. 3 500 �10.1 � 0.4‰ 25.8 � 1.8‰Longirostromeryx sp. 3 15 �8.3 � 0.6‰ 26.4 � 2.0‰

Middle–Late Miocene (9.7–5.3 Ma), CaliforniaTeleoceras fossiger 8 1000 �12.2 � 0.7‰ 27.2 � 1.4‰Pliohippus interpolatus 5 200 �11.9 � 0.5‰ 26.3 � 0.8‰Pliohippus sp. 5 200 �11.6 � 0.3‰ 25.5 � 1.5‰Gomphotherium productus 7 3200 �12.3 � 0.3‰ 25.9 � 1.2‰Procamelus sp. 5 500 �11.6 � 1.1‰ 25.7 � 1.1‰

Middle–Late Miocene (8.4–5.0 Ma), OregonTeleoceras fossiger 5 1000 �11.8 � 1.0‰ 19.6 � 0.8‰Camelid 1 — �11.1‰ 21.7‰

Middle Miocene (9.5 Ma), FloridaTeleoceras 5 635 �13.3 � 0.4‰ 32.1 � 0.4‰Aphelops 5 889 �12.8 � 0.3‰ 31.4 � 0.8‰Cormohipparion 7 150 �11.5 � 0.9‰ 31.7 � 1.7‰Procamelus 1 500 �11.2‰ 30.9‰Tapirus 1 381 �13.3‰ 27.4‰Prosthenops 1 75 �11.3‰ 29.4‰Neohipparion 1 150 �11.6‰ 30.7‰Ambeledon 2 3440 �12.1‰ 30.0‰

Middle Miocene (7.5 Ma), FloridaTeleoceras 4 635 �13.3 � 0.7‰ 31.8 � 0.5‰Aphelops 2 889 �13.2‰ 31.1‰Hipparion 2 150 �10.4‰ 31.3‰Ambeledon 1 3440 �12.3‰ 30.9‰Pliohippus 1 170 �9.6‰ 31.0‰Pseudohipparion 1 61 �10.5‰ 27.5‰Cormohipparion 1 150 �9.7‰ 30.5‰Tapirus 1 381 �13.1‰ 30.6‰

Late Miocene (7.0 Ma), FloridaTeleoceras 2 635 �12.8‰ 31.5‰Aphelops 4 889 �12.1 � 0.6‰ 31.7 � 0.6‰Tapirus 1 381 �13.9‰ 29.7‰Cormohipparion 1 150 �9.8‰ 32.1‰Camel 2 — �10.4‰ 29.8‰Pliohippus 2 170 �9.7‰ 29.5‰

TABLE 1—Continued.

Taxon nBody

mass (kg)Mean �13C � 1 s

(VPDB, ‰)Mean �18O � 1 s

(VSMOW, ‰)

Late Miocene (4.5 Ma), FloridaTeleoceras 3 635 �7.6 � 1.8‰ 31.6 � 0.4‰Hemiauchenia 1 110 �14.1‰ 30.3‰Cf. Rhynchotherium 3 — �4.7 � 2.2‰ 30.8 � 0.5‰Cf. Catagonus 1 — �11.8‰ 29.8‰Cf. Megatylopus 1 1000 �14.8‰ 31.4‰Pseudohipparion 3 61 �5.0 � 0.1‰ 31.7 � 0.3‰Cormohipparion 3 150 �5.9 � 2.5‰ 29.9 � 0.4‰Nannipus 3 93 �5.8 � 3.4‰ 30.3 � 0.3‰Rhynchotherium 1 — �2.5‰ 31.6‰Dinohippus 2 240 �8.3‰ 31.2‰Tapiravus 1 — �13.3‰ 29.1‰Neohipparion 4 150 �4.2 � 3.6‰ 30.3 � 0.8‰

relative humidity to a high ��9.0‰ when relative humidity was re-duced to 50%. The effect of these changes in leaf water �18O compositionwas significant but dampened in modeled enamel �18O values; the 12.5‰change in leaf water �18O values translated to a change of �7‰ inenamel �18O values.

Stable Isotope Results

Published �18O values for extant hippopotamids were consistently low-er than the associated terrestrial fauna by 2.0‰–6.0‰ (Fig. 2). The dif-ference between hippopotamids and ES taxa (4‰–7‰) was found to beequal to or greater than that observed between hippopotamids and EI taxa(2‰–4‰) (Fig. 4).

Similar differences are observed between �18O values for extinct hip-popotamids and coeval fauna (Fig. 4), with most fossil sites falling withinthe range reported between living hippopotamids and EI taxa (Bocherenset al., 1996; Zazzo et al., 2000; Franz-Odendaal et al., 2002; Harris andCerling, 2002; Cerling et al., 2003). For these fossil faunas, EI taxa wereidentified based on the ES-EI status of closest living relatives or modernanalogues (Levin et al., 2006). A combined bivariate plot of living andfossil 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 surfacewater �18O values, application of a linear regression through the datashows that hippopotamid mean �18O values are controlled strongly bysurface water �18O values and can be predicted from the mean �18Ovalue of EI taxa within the fauna (�18Ohippopotamids � 0.96 �0.09 ·�18Ofauna � 1.67 � 2.97; r2 � 0.886, p � 0.001, standard errorof the estimate � 0.72).

Tabulation of variation within modern and fossil hippopotamidsrevealed an interesting contrast (see Fig. 1). Variation within fossil pop-ulations was much higher (SD � 1.5 � 0.5‰) and statistically different(Student’s t-test, p � 0.05) compared to living populations of hippopo-tamids with low levels of variation in �18O values among individuals(SD � 0.8 � 0.7‰). A similar, statistically significant increase in var-iation was detected between living and fossil terrestrial mammal popu-lations but not between living and fossil aquatic mammals (see Fig. 1).

Mean �13C values for Coryphodon were significantly lower than thosefor Ectocion from all levels (Table 1) and for Phenacodus and Hyra-cotherium from all levels but those recovered from interval 2 (OW-ANOVA, 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 differencewas detected between mean �13C values for Coryphodon from each in-terval (OW-ANOVA, F � 1.239, p � 0.308). Mean �18O values forCoryphodon were found to be significantly lower than those for faunafrom 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


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 bodymass (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) changesin 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 oceanwater; WEI � water economy index.

2 (OW-ANOVA, F � 1.754, p � 0.182). The mean �18O value forCoryphodon from interval 2 (Table 1) was found to be significantly higherthan those for Coryphodon from intervals 1 and 3 (OW-ANOVA, F �3.393, p � 0.05). Uncertainties in the inferred ecologies for these earlyEocene mammals make it difficult to differentiate between EI and EStaxa. Given the lack of large differences in mean �18O values amongtaxa, the difference in 18O enrichment between EI and ES taxa does notappear to have been significant for these fauna, and so �18O values forall taxa were combined and used for comparison with mean values forCoryphodon. 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 significantlylower than those of the associated fauna (Student’s t-test, p � 0.01) andare similar to expected �18O values for hippo ecomorphs using the aboveequation 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 frominterval 2 is indistinguishable from that of the fauna and much higherthan expected for a hippo ecomorph (Fig. 6). Variance in �18O valuesfor Coryphodon was similar to that of Phenacodus and Ectocion but wassignificantly lower than that for Hyracotherium from interval 3 (Table 2).

Analysis of �13C and �18O composition of the proboscidean Moerith-erium and the anthracothere Bothriogenys gorringei found statisticallysignificant 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 �13Cvalues, whereas B. gorringei, Saghatherium antiquum, and Megalohyraxsp. had the lowest (Table 1). Designation of ES-EI taxa was based oncomparison with living relatives and modern analogues and evaluation ofenamel �18O values. Phiomia serridens, and Pa. beadnelli were classifiedas EI based on similarities in feeding ecology and relationships withliving proboscideans. Once this designation was set, the remaining fauna(hyracoids: Saghatherium antiquum, Megalohyrax sp., and Geniohyussp.) did not show significant enrichment in 18O relative to the probosci-deans and were, therefore, also classified as EI. Mean �18O values forMoeritherium (27.1 � 1.3‰) and B. gorringei (26.8 � 1.6‰) weresignificantly lower than that of the associated fauna (29.8 � 2.4‰) andbracket the expected value for hippo ecomorphs using the above equation(26.7 � 0.9‰) (Fig. 6). Arsinoitherium, hypothesized to be semiaquaticby 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. Nostatistically 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 com-pared to that previously sampled from Florida (9.5–4.5 Ma; see Mac-Fadden and Cerling, 1996; MacFadden, 1998). In California and Oregonfew specimens of large ungulates from the same deposits were available


FIGURE 4—Bivariate plot of mean �18O values for living populations and fossilaccumulations of various species of hippopotamids and associated fauna (Kohn etal., 1996; Leakey et al., 1996; Zazzo et al., 2000; Franz-Odendaal et al., 2002; Harrisand Cerling 2002; Cerling et al., 2003; Levin, et al., 2006). Error bars represent �1SD from the mean for each group. Dark line represents the linear regression throughthe data defined by the equation provided in the graph; long dashed lines representthe 95% confidence limits for this regression; and short dashed line represents a 1:1 relationship between enamel �18O values for hippos and fauna. VSMOW � Vi-enna standard mean ocean water.

for comparison with samples from Teleoceras. For the California locality,large ungulates from the same location but of a slightly earlier age weresampled and, after statistical analysis showed that �13C and �18O valueswere 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 thesingle species (Pliohippus interpolatus) found with Teleoceras. Mean�13C values for Teleoceras sampled from each state and time period werefound to be statistically distinct (OW-ANOVA, F � 35.663, p � 0.001)except between specimens from California, Oregon, and Florida (9.5–7.0Ma; see Table 3). Mean �13C values for Teleoceras and associated faunawere 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 statesand 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 forthe associated fauna (OW-ANOVA, F � 0.241, p � 0.867) did not differsignificantly with age. Except for populations of Teleoceras in California andNebraska, differences in the mean �18O values among populations from eachlocation were statistically significant (OW-ANOVA, F � 199.78, p � 0.001;see Table 3), and mean values decrease with increasing latitude. Mean pop-ulation �18O values range from a high in Florida of 32.1 � 0.4‰ to a lowin Oregon of 19.6 � 0.8‰ (Fig. 5B). As observed previously by MacFadden(1998) for Florida populations, enamel �18O values for Teleoceras were notsignificantly lower than those of coeval terrestrial mammals (Fig. 5B) andvalues were much higher than those predicted for a hippo ecomorph fromthese sites (Fig. 6). Population-level variance in enamel �18O values for Te-leoceras at four sites yields conflicting results (Table 2): populations in Ne-braska and Florida show low variance in enamel �18O values for Teleocerasrelative to coeval fauna, whereas populations on the west coast show muchhigher variation that is not significantly different from those of other herbi-vores at the sites.

DISCUSSION

�18O and the Hippo Ecomorph

Prior work demonstrated that living and fossil hippopotamids havelower enamel �18O values than that of associated ungulates (Bocherenset al., 1996). A compilation of these data shows that although the offsetin �18O values between hippopotamids and other ungulates may varysomewhat in magnitude, the trend is consistent and predictable (Fig. 4).Proposed factors for this difference have included physiological and be-havioral differences between hippopotamids and other ungulates that mayor may not be related to aquatic habits. The primary character unrelatedto aquatic habits is nocturnal foraging. Modeling of the fluxes and frac-tionation of oxygen isotopes within hippopotamids provided a means toevaluate which of these scenarios could account for the observed differ-ence. Though the reduction in enamel �18O values resulting from noc-turnal foraging and the associated diurnal changes in leaf water �18Ovalues (D18Oday-night � �10‰; see Flanagan and Ehleringer, 1991; Cer-nusak et al., 2002) was sufficient to partially explain the observed dif-ference between hippopotamids and ES taxa (and between EI and EStaxa, for that matter), the effects of nocturnal foraging did not accountfor the observed difference between hippopotamids and EI taxa, whichreceive most of their oxygen from water sources with relatively stableand 18O depleted compositions (i.e., surface water, stem water; see Levinet al., 2006). Other factors, thus, possibly related to the physiology ofthese animals and their mode of life must account for the additionaldifference between hippopotamids and EI taxa.

Incorporation of physiological characters specific to living hippopotam-ids did result in a significant change in predicted enamel �18O valuesrelative to those for ES and EI taxa. Of these characters, increasing thewater 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

body size and expansive foregut of hippopotamids forces a significantreduction in the size of the lower gastrointestinal tract. Because the largeintestine is the primary site of resorption of water from the feces priorto defecation, reduction in the size of this organ increases the quantityof water loss through the feces relative to that observed for smaller ar-tiodactyls and comparably sized perissodactyls with expanded large in-testines. Given that this high water loss can only be sustained if theseanimals have significant quantities of water available, relatively low �18Ovalues would only be observed when these large animals were associatedwith a constant source of water. This unique physiological character ofhippopotamids is, thus, connected to the aquatic habits of this species.

Although this explanation is plausible for large, semiaquatic, foregut-fermenting but nonruminating artiodactyls, it raises a question as towhether perissodactyls and other hindgut fermenters capable of absorbinga greater quantity of water from their feces would have similarly low�18O values relative to the associated fauna. This question was examinedby evaluating the effect of increasing the water loss through other fluxes(i.e., urine) and augmenting the water economy index to that observedin fully aquatic herbivorous mammals in fresh-water conditions (i.e., cap-tive Florida manatees, WEI � 1.3 ml ·kJ�1; Ortiz et al., 1999). Increasingthe relative amount of water lost through urine did cause a decrease inenamel �18O values, but this drop was of lower magnitude than thatcalculated for increased fecal water loss (Fig. 3C). When the WEI wasincreased and combined with an increased rate of water loss throughurine, however, enamel �18O values dropped significantly and eventuallymatched that achieved through high fecal water loss (Fig. 3C). This mech-anism of increased water turnover and loss through urine and feces seemslike a reasonable assumption for most aquatic mammals. When living inaquatic habitats, large mammals would not need to conserve water butwould most likely need to find ways to expel large quantities of it quickly.Ingestion or absorption of large amounts of fresh-water could leadto natremia and would require methods of flushing large quantities ofwater out of the body while conserving salts. Examination of the urine-concentrating ability of living hippopotamus supports this suggestion; theinternal structure of the kidneys of living hippopotamus is incapable of


FIGURE 5—Box plots of (A) �13C values and (B) �18O values for five hippo ecomorphs and associated fauna. Hippo ecomorphs � white boxes, whereas associatedfaunas � 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 DeeBelemnite; VSMOW � Vienna standard mean ocean water.

producing concentrated urine and would enable this species to flush outlarge quantities of water while conserving salts (Beauchat, 1990). Thisrapid 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 anthracoth-eres and hippopotamids, the extremely low �18O values for Bothriogenysgorringei are strong evidence that this animal was as aquatic as livinghippopotamids. Anthracotheres were geographically widespread and havebeen recovered from Europe, Asia, Africa, and North America. Early forms,including B. gorringei, typically have been depicted as swinelike and notas 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 dis-covery of an earlier specialization for fresh-water environments means thatthe early ecology of this group should be evaluated in greater detail. Priorwork had also used the expectation of low enamel �18O values for fossilhippopotamids as a measure of the isotopic integrity of fossil materialsfrom the middle Miocene to the present (Bocherens et al., 1996; Zazzo etal., 2000). With the detection of a similar relationship in anthracotheres,this test can now be extended back at least to the early Oligocene Africananthracotheriids and should be investigated in older Eocene taxa and an-thracotheriids from faunas on other continents.

Although no living hind-gut fermenters are as morphologically com-mitted to a semiaquatic lifestyle as living hippopotamids, several specieshave been described from the fossil record. The three included in this


FIGURE 6—Bivariate plot of mean �18O values for five hippo ecomorphs andassociated faunas. Solid black line and dashed lines represent the relationships be-tween mean faunal �18O values vs. hippopotamid �18O values presented in Figure4. Error bars represent �1 standard error from the mean for each group. Mean �18Ovalue for smaller Coryphodon is in gray. VSMOW � Vienna standard mean oceanwater.

TABLE 3—Statistical results from a post-hoc Bonferroni test of mean �13C and �18Ofor the four populations of Teleoceras. The p-values for each pairwise comparisonbetween populations are reported for mean �13C values (lower-left corner) and mean�18O values (upper-right corner).

Middle–Late Miocene Teleoceras localities

Oregon California NebraskaFlorida

(9.5–7.0 Ma)Florida

(4.5 Ma)

Oregon — �0.001 �0.001 �0.001 �0.0001California 1.000 — 0.063 �0.001 �0.0001Nebraska 0.008 �0.001 — �0.001 �0.0001Florida (9.5–7.0 Ma) 0.538 1.000 �0.0001 — 1.000Florida (4.5 Ma) �0.001 �0.001 0.007 �0.001 —

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.

Coryphodon Bothriogenys Moeritherium Teleoceras CA Teleoceras NE Teleoceras FL

Phenacodus 0.105 — — — — —Ectocion 0.134 — — — — —Hyracotherium 0.041 — — — — —Arsinoitherium — 0.410 0.770 — — —Paleomastodon — 0.665 0.515 — — —Phiomia — 0.438 0.869 — — —Saghatherium — 0.469 0.346 — — —Gomphotherium — — 0.868 — —Pliohippus — — 0.713 0.216 —Procamelus — — 0.679 0.002 —Pseudohipparion — — — 0.001 0.222Aphelops — — — — 0.308Cormohipparion — — — — 0.025

study (Coryphodon, Moeritherium, and Teleoceras) have been consideredhippo ecomorphs based on morphological similarities with living hip-popotamids and were analyzed isotopically to see if the observed differ-ence in �18O values between hippopotamids and associated fauna wouldhold for hindgut fermenters as well.

Of these three species, only Moeritherium and large species of Cory-phodon were found to have low enamel �18O values similar to those ofliving hippopotamids and the coeval anthracothere, Bothriogenys gorrin-gei. In contrast, enamel �18O values for Teleoceras at all sites were sta-tistically indistinguishable from those of the associated fauna. Moerith-erium appears to have been the most aquatic, and Teleoceras the leastaquatic, of the three. Coryphodon is interesting because of the differencein enamel �18O values for species of different body size based on thisevidence. A smaller species (�100 kg) from the early Eocene does notmeet this criterion and does not appear to have been semiaquatic, whereaslarge species from the late Paleocene and early Eocene appear to matchexpectations in mean �18O values for hippo ecomorphs. Lack of a similarshift in mean �18O values for other taxa sampled within this intervalindicates that this difference cannot be explained by environmentalchange and is most likely related to changes in the physiology, or ecology

of Coryphodon, or both. The body masses for large species of Corypho-don are estimated to have been between 200 kg and 400 kg, whereasthose of Phenacodus, Ectocion, and Hyracotherium are predicted to havebeen �50 kg (Table 1). As has been shown for living elephants (Bryantand Froelich, 1995), this order of magnitude difference in body size couldcreate a small, but significant difference in mean �18O values betweenCoryphodon 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 sim-ilarity in enamel �13C values would suggest that this habitat separationdid not correlate with a dietary difference between these sizes (Table 1)and that such factors as predation and thermoregulation may have led tothese differences in aquatic habits. It is difficult to determine whether thelower �18O values for large species of Coryphodon are a result of thelarge body mass difference between these species and the associated earlyEocene fauna or an ecological change between large and small speciesof Coryphodon. Further empirical work on the effects of body mass andecology on oxygen isotope composition is needed to fully address thisquestion.

Variation in enamel �18O values for fossil hippopotamids and the fourhippo ecomorphs was not consistently lower than that of associated ter-restrial fauna (Fig. 1; Table 2). This is somewhat surprising given thelow variation typically observed in living hippopotamids and other aquat-ic mammals (see Fig. 1). For these species, surface waters are the primaryoxygen influx and, as such, constrain the possible range in �18O valuesamong individuals in a population. Any fluctuations in these values overthe course of an individual’s life are most likely to be experienced by allindividuals and therefore are averaged over the entire population. Twofactors that can complicate this interpretation are mixing of individualsfrom (1) different geographic locations or (2) different time periods. The


amount of time averaging or spatial averaging affecting a fossil accu-mulation can vary considerably and be difficult to quantify (Behrens-meyer, 1982). In terrestrial and fresh-water ecosystems, the oxygen iso-tope composition of precipitation and surface waters can vary greatlythrough time or space as a result of changes in temperature, seasonality,air masses, and a host of other climatic factors. An increase in the amountof time and space represented in a fossil accumulation may cause thevariation in enamel �18O values among individuals of a species to in-crease as well because of these perturbations. In marine ecosystems, how-ever, seawater is relatively homogeneous (�1.0‰) over long time scales,and mammals living in these waters that differ slightly in age or locationwould be expected to show relatively small differences in enamel �18Ovalues. While low population-level variation in �18O values is an excel-lent measure of aquatic habits in marine species (Clementz et al., 2003;Clementz et al., 2006), greater care must be taken when dealing withfossil specimens from fresh-water deposits where the effects of time av-eraging are more pronounced and �18O values are potentially more var-iable.

�13C and Diets of Fossil Hippo Ecomorphs

Evidence of the dietary preferences of Arsinoitherium, Bothriogenys,Coryphodon, Moeritherium, and Teleoceras is provided by enamel �13Cvalues. As explained earlier, most of the Cenozoic continental ecosystemsincluded in this project were dominated by C3 vegetation, which causedthe range in enamel �13C values at most sites to be relatively small (Fig.5A). Even so, significant differences are observed within each fauna thatcan be used to interpret dietary differences among species. For instance,Koch et al. (1995) interpreted the low �13C values of Coryphodon relativeto those of the remaining early Eocene fauna as evidence that this earlymammal was foraging more deeply into the forest than other mammalsand probably filled a niche similar to that of living tapirs.

Low �13C values for the early Oligocene anthracothere Bothriogenysgorringei and the hyracoids Saghatherium antiquum and Megalohyraxsp. may support a similar interpretation of foraging under closed-canopyconditions. Given the inferred semiaquatic habits of B. gorringei, low�13C values could instead indicate it was consuming aquatic macrophytesgrowing in fresh-water rivers or streams; the overlap in �13C values forfresh-water macrophytes and terrestrial C3 plants makes it difficult todistinguish between these two dietary sources. In contrast, the higher�13C values of Arsinoitheirum, Moeritherium, and other proboscideanssuggest that these large mammals may have been foraging in more openconditions. Values for a few individuals of Phiomia serridens are at theupper extreme for a pure C3 forager. These enamel �13C values couldmean that the plants these animals were consuming were environmentallystressed, possibly from high levels of dissolved salts or limited water.Fossil plants from these deposits include mangrove specimens, which arecapable of tolerating marine waters and are thought to have formed ex-tensive forests that hemmed the coast of the Tethys Sea (Bown et al.,1982). The large proboscideans may have foraged more extensively onthese plants and others growing within the forests than other herbivores.The elevated �13C values of mangroves and other salt-stressed plantswould have been passed on to large proboscideans. For Moeritherium inparticular, the possibility that it fed on salt-stressed plants is supportedby the fact that another species of this genus is also found commonly inthe slightly older, nearshore marine deposits of the Qasr el Sagha For-mation (Holroyd et al., 1996), suggesting that it also frequented brackishwater or salt water. The lower mean �18O value for this genus indicatesit still mostly consumed or inhabited fresh water. The diets of Moerith-erium and Bothriogenys were quite distinct and, thus, indicate that theecological similarities between these species were limited to their pref-erence for aquatic environments.

Unlike the other fossils analyzed, specimens of Teleoceras were col-lected from multiple localities that cover a broad temporal and spatialrange. Teleoceras specimens from California, Florida (9.5–7.0 Ma), and

Oregon have enamel �13C values consistent with consumption of a pureC3 diet (Fig. 5A). In Nebraska, the high �13C values for Teleoceras andother herbivores suggest consumption of some C4 grasses (i.e., �30%of diet). Recent �13C evidence from soil carbonates indicates that C4grasses were present in the Great Plains at low abundances (12%–34%of biomass) throughout the Miocene (Fox and Koch, 2003), and this couldaccount for the high enamel �13C values reported at this locality (Fig.5).

Interestingly, late Miocene and early Pliocene sites in California andOregon that date from the time after C4 grass expansion have low �13Cvalues, suggesting that either C4 plants were not present at these localitiesor that Teleoceras and the other taxa were not consuming them. Thepossession of high-crowned (i.e., hypsodont) molars, as well as the pres-ence of fossil remnants of ingested grasses with Teleoceras remains inNebraska, is evidence that Teleoceras was capable of grazing like livinghippos (Voorhies and Thomasson, 1979). If C4 grasses were present atthese localities, Teleoceras would have likely consumed them. The Med-iterranean climate (winter rainfall) of the west coast favors the use of C3over C4 photosynthesis by grasses in this region today. Lack of a C4signal in herbivore tooth enamel from these localities is evidence thatthese climate conditions (i.e., wet winters, dry summers) were in placein the late Miocene and early Pliocene.

No consistent pattern in �13C values or dietary preferences was iden-tified for hippo ecomorphs. The diets of living hippopotamids reflect thisas well. Most dietary studies have focused on the larger species (Hip-popotamus amphibius), which has been interpreted as a terrestrial grazer,favoring a mix of C3 and C4 grasses that grow within a short distanceof the rivers and lakes in which these animals live. Recent stable isotopeand microwear analyses of living individuals found in open and closedcanopy conditions, however, have shown that the relative percentage ofC4 grass in the diet is closely correlated with habitat and can vary from30% to 100% (Boisserie et al., 2005b). Proximity to aquatic refugia maybe the strongest factor determining the diets of hippo ecomorphs, andindividuals may be capable of varying their diet as the plant life withintheir home ranges changes. This finding does suggest that animals in thisrole may be more inclined to be dietary generalists rather than specialists,although this means that enamel �13C may not be diagnostic of this eco-logical niche.

CONCLUSIONS

Analysis of published �18O values for living and fossil hippopotamidsrevealed a consistent difference between these individuals and the asso-ciated terrestrial fauna. Modeling of the oxygen fluxes and fractionationswithin hippopotamids showed that increased water loss via urine or fecesmay account for this pattern—a physiological character that is likely truefor all large-bodied aquatic herbivores. In contrast, although a significantdifference in intraspecific variation in enamel �18O values has been ob-served between living aquatic and terrestrial mammals, this differencewas not detected consistently for fossil specimens from continental de-posits. Extensive time and spatial averaging of continental fossil accu-mulations may explain this discrepancy and should, therefore, be consid-ered when making ecological interpretations from the geochemical com-position of fossil remains.

Morphological characters were not always diagnostic of aquatic pref-erences based on our results. A small species of the early Eocene pan-todont Coryphodon and the Miocene rhinoceros Teleoceras did not have�18O values that were low enough relative to the associated fauna tosupport aquatic habits for these animals. In contrast, two co-existing spe-cies from the early Oligocene of Egypt (the anthracothere Bothriogenysand the proboscidean Moeritherium) and larger species of Coryphodonwere found to fit predicted values and had �18O values 2‰–4‰ lowerthan that of the associated fauna. Discovery of this character in Bothri-ogenys supports an interpretation of aquatic lifestyle for anthracotheres


and hippopotamids early in their evolutionary history, whereas the pres-ence of low �18O values in Moeritherium relative to the coeval faunasupports the use of this measure to identify aquatic taxa outside of theanthracothere-hippopotamid clade.

No consistent dietary preference was found to be associated with thehippo ecomorphs. Enamel �13C values for Bothriogenys suggest this spe-cies foraged deeper within forests or more heavily on aquatic vegetationthan coeval Moeritherium. Enamel �13C values for Coryphodon and Te-leoceras populations at most sites were consistent with a pure C3 diet.Enamel �13C values for Teleoceras specimens sampled from Californiaand Oregon suggest that C4 grasses were not present along the PacificCoast by 5 Ma. Significantly higher �13C values for Teleoceras in Ne-braska, however, support the presence of an appreciable amount of C4grasses in the Great Plains by at least 11 Ma.

The occurrence and abundance of large, semiaquatic herbivorous mam-mals today is low, but our findings suggest this may not always havebeen the case. The identification of at least two sympatric semiaquaticspecies from the Oligocene of Egypt indicates that the diversity and abun-dance of this ecomorph could have been higher in the past. Developmentof a new means of using the stable isotope composition of fossil materialsto identify these species in the fossil record will provide an opportunityto quantify the relative abundance of these taxa within faunas, examinewhat factors might facilitate the evolution of this ecomorph, and evaluatetheir role in the structuring of ancient ecosystems.

ACKNOWLEDGMENTS

Funding for this research was provided by National Science Founda-tion, Division of Earth Sciences grant 0087742. We thank J. Meng at theAmerican Museum of Natural History and M. Voorhies at the NebraskaState Museum of Natural History for access to and information on spec-imens sampled for this project; M.B. Goodwin for assistance with sam-pling; K. Fox-Dobbs and B. Crowley for assistance with lab work; andT. Cerling, B. Passey, K. Hoppe, D. Fox and an anonymous reviewer forcomments on this manuscript and early results for this project. This isUniversity of California Museum of Paleontology contribution no. 1966.

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