ORIGINAL RESEARCH published: 17 May 2019 doi: 10.3389/fevo.2019.00176 Dietary Adaptations of Early and Middle Pleistocene Equids From the Anagni Basin (Frosinone, Central Italy) Flavia Strani 1,2,3*, Diana Pushkina 4 , Hervé Bocherens 5,6 , Luca Bellucci 2,3,7 , Raffaele Sardella 1,2,3 and Daniel DeMiguel 8,9 1 Dipartimento di Scienze Della Terra, Sapienza Università di Roma, Rome, Italy, 2 Department of Earth Sciences, PaleoFactory, Sapienza Università di Roma, Rome, Italy, 3 Istituto Italiano di Paleontologia Umana, Rome, Italy, 4 Department of Geosciences and Geography, University of Helsinki, Helsinki, Finland, 5 Department of Geosciences, Biogeology, University of Tübingen, Tübingen, Germany, 6 Senckenberg Research Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen, Germany, 7 Museo di Geologia e Paleontologia, Sistema Museale di Ateneo, University of Florence, Florence, Italy, 8 Departamento de Ciencias de la Tierra, Fundación ARAID / Universidad de Zaragoza, Zaragoza, Spain, 9 Institut Català de Paleontologia Miquel Crusafont, Barcelona, Spain The intermontane Anagni Basin (Frosinone, central Italy) is an important region for Italian biochronology and paleoecology due to the presence of two rich fossil assemblages dated to the Early (Coste San Giacomo) and Middle Pleistocene (Fontana Ranuccio). Edited by: Gina Marie Semprebon, These sites have yielded a vast collection of large fossil mammals with a well-documented Bay Path University, United States presence of fossil equids in both localities (represented mostly by isolated teeth). Coste Reviewed by: San Giacomo is dated to around 2.1 Ma, thereby having recorded the effects of the Nikos Solounias, New York Institute of Technology, onset of the Quaternary glacial cycles, which led to a gradual trend toward colder United States and more arid conditions in the Northern Hemisphere. The fossil equids of this site Lindsey Theresa Yann, belong to the first group of grazing stenonid equids of the genus Equus that spread Oklahoma State University, United States to the Italian Peninsula during the so called “Elephant- Equus” event, which marked *Correspondence: the appearance of new large mammals living in herds in open and arid environments. Flavia Strani The site of Fontana Ranuccio is dated to around 400 ka, close to the MIS 12–11 flavia.strani@uniroma1.it; flavia.strani@gmail.com succession (the “Mid-Brunhes event”), which marked the end of the Middle Pleistocene Transition. The fossil horses from Fontana Ranuccio represent one of the oldest caballoid Specialty section: (or “true horses”) populations of the Italian Peninsula. The Anagni Basin, thus, provides This article was submitted to Paleontology, important data to investigate paleoecological adaptations of these groups of equids in a section of the journal response to two critical environmental and climatic shifts of the Pleistocene. We explore Frontiers in Ecology and Evolution their niche occupation by examining long-term dental wear patterns and tooth enamel Received: 29 January 2019 carbon and oxygen stable isotopic composition. Both taxa appear to have exhibited a Accepted: 01 May 2019 Published: 17 May 2019 narrow dietary niche, displaying a clear abrasive (highly specialized) grass-rich diet. In Citation: particular, caballoid equids from Fontana Ranuccio show a more abrasion-dominated Strani F, Pushkina D, Bocherens H, mesowear signature. Stenonid equids from Coste San Giacomo exploited broader and Bellucci L, Sardella R and DeMiguel D (2019) Dietary Adaptations of Early more diverse landscapes during the Early Pleistocene, whereas caballoid horses from and Middle Pleistocene Equids From Fontana Ranuccio appeared to have limited their dietary adaptations to a stricter grazing the Anagni Basin (Frosinone, Central behavior in more closed environments. Italy). Front. Ecol. Evol. 7:176. doi: 10.3389/fevo.2019.00176 Keywords: mesowear, isotopes, equids, Equus, Pleistocene, paleoecology, paleoenvironment Frontiers in Ecology and Evolution | www.frontiersin.org 1 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy INTRODUCTION stehlini from CSG and Equus cf. E. mosbachensis from FR in occlusal surface mesowear, a proxy for dietary abrasiveness in The intermontane Anagni Basin, situated in the central part herbivorous mammals (Fortelius and Solounias, 2000), and in of the Italian Peninsula, developed between the Late Pliocene stable carbon and oxygen isotope abundances, proxies for habitat and the early Middle Pleistocene (Carrara et al., 1995; Galadini openness (Van der Merwe and Medina, 1991) and temperature and Messina, 2004). Two rich vertebrate fossil sites, the Early (Dansgaard, 1964), to shed light on the niche occupation and Pleistocene locality of Coste San Giacomo (CSG) and the Middle resource exploitation mechanisms of stenonid and caballoid Pleistocene site of Fontana Ranuccio (FR), were discovered in equids on central Italian Peninsula. the 1970s, following different surveys carried out by the Italian Our data will allow us to obtain comprehensive information Institute of Human Paleontology (IsIPU) (Figure 1). CSG has on the paleoecological adaptations of the Equus genus in the been dated to around 2.1 Ma based on magnetostratigraphy, region following two major climatic events, and to understand pollen, and its record of micro mammals recovered from alluvial if this group could be more flexible in terms of feeding behavior deposits (Bellucci et al., 2014). It is one of the few Italian and habitat occupation during the Early and Middle Pleistocene. localities that record the terrestrial habitat conditions after the onset of the Pleistocene glaciations that marked the Plio- DENTAL WEAR PATTERNS AND ISOTOPES Pleistocene transition at 2.6 Ma (Lisiecki and Raymo, 2005, 2007). FR fossiliferous layer located within a succession of pyroclastic IN PALEOECOLOGY deposits has been dated to around 0.4 Ma (Pereira et al., 2018), Dental Mesowear close to the climatic transition of the “Mid-Brunhes Event“ (MIS Mesowear reflects the cumulative effects at a macroscopical 12–11) with the definitive association of the glacial cycles ruled by level of the items ingested by herbivores (foods and exogenous a 100 kyr periodicity and the consolidation of the Early-Middle particles such as grit or dust) on tooth morphology that are Pleistocene Transition (Head and Gibbard, 2015). produced in a long period of time compared to the lifespan of Importantly, both localities have yielded abundant fossil the animal (Fortelius and Solounias, 2000). Dental mesowear is a mammal material, among which equid remains are well- direct signal of a species’ diet that varies between browsers, which represented (Segre and Ascenzi, 1984; Bellucci et al., 2012, 2014; feed mostly on soft plant resources (e.g., leaves, twigs, green Strani et al., 2018a). Fossil human teeth were also unearthed stems), and grazers that clip abrasive vegetation (e.g., grasses) at in FR (Rubini et al., 2014). The equid remains from CSG were or near ground level. attributed to Equus stenonis by Biddittu et al. (1979), but a recent Attrition (tooth-to-tooth contact) and abrasion (tooth-to- study (Palombo et al., 2017) points to stronger affinities with food contact) are the main factors that influence the occlusal the middle-size Equus senezensis and proposes a new provisional morphology of the teeth, determining the cusps sharpness and identification of the CSG material as E. senezensis aff. E. sen. the height of the occlusal relief. In browsing animals attrition stehlini. This taxon is among the first groups of stenonid equids mainly determines the tooth wear leading to sharper cusps and (monodactyl horses with a “V” shaped linguaflexid between the a higher occlusal relief. In species that feed on more abrasive metaconid and metasylid of the lower cheek teeth; Forsten, 1988) plant resources the higher level of tooth-to-food contact produces to reach the Italian Peninsula during the so-called “Elephant- blunter cusps and a lower occlusal relief (Fortelius and Solounias, Equus” event with the appearance of new large mammals living 2000). Traditional mesowear is limited to upper second in herds in drier habitats (Azzaroli, 1977; Lindsay et al., 1980). molars (M2), but it can be extended to other tooth positions Equids’ remains from FR have been tentatively classified as Equus (Kaiser and Solounias, 2003; DeMiguel et al., 2010, 2012; cf. E. mosbachensis (Segre and Ascenzi, 1984), belonging to one Strani et al., 2015, 2018a,b). of the first caballoid (or “true horses,” monodactyl equids with a The method has been developed to examine the diet-related “U” shaped linguaflexid; Forsten, 1988) populations recorded on dental wear patterns in ungulates and has been successfully the Italian Peninsula after the disappearance of the early Middle employed to investigate dietary adaptations of different groups of Pleistocene stenonid forms (Caloi, 1997; Alberdi and Palombo, both modern and fossil artiodactyls and perissodactyls (Fortelius 2013). and Solounias, 2000; Schulz et al., 2007; DeMiguel et al., 2008, Both modern and fossil Equus taxa are usually considered 2011, 2018; Bernor et al., 2014; DeMiguel, 2016; Marín-Leyva highly specialized grazers that consistently occupied similar et al., 2016; Maniakas and Kostopoulos, 2017). Dental mesowear habitats through the Pleistocene and Holocene (i.e., open also proved to be an optimal tool to study equid paleoecological grasslands), despite it has been observed that extant species can adaptations, allowing to obtain information about the dietary also include a noticeable amount of browse in their diet (Roeder, change and evolution of this group from the early forms of 1999) and that some fossil Equus populations displayed less the Eocene to the horses of the Pleistocene (Kaiser and Franz- abrasive feeding behaviors (Kaiser and Franz-Odendaal, 2004). Odendaal, 2004; Mihlbachler et al., 2011; Bernor et al., 2017). The material from the Anagni basin represents an exceptional case to investigate the habitat occupation and resource use of Carbon Stable Isotopes two groups of fossil equids and test the hypothesis that both Carbon isotopic (δ13 C) abundance in herbivores reflects the stenonid and caballoid horses exploited exclusively open habitats presence of C3 and C4 vegetation in lower and mid-latitude and fed mostly on grasses in Central Italian Peninsula. In this habitats as well as a degree of canopy closure vs. openness in study we compare dental remains of E. senezensis aff. E. sen. wooded high-latitude habitats. C3 plants, or woody vegetation Frontiers in Ecology and Evolution | www.frontiersin.org 2 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy FIGURE 1 | Geographical location of the Coste San Giacomo and Fontana Ranuccio localities. and grasses that have a cool growing season, have δ13 C values either free drinking water or obtained from food and plants. ranging between −34 and −23‰, with a suggested mean of The δ18 O in the enamel apatite is a proxy for δ18 O values of −27‰ (Calvin and Benson, 1948; Hatch et al., 1967; Smith and meteoric water, which varies due to the source of precipitation Epstein, 1971; Koch, 1998). The canopy effect can result in up to and effects of latitude, altitude, continentality, temperature, 5‰ differences in δ13 C in the same species at one site in pure C3 and evaporation (Dansgaard, 1964; Longinelli, 1984; Luz et al., forest environments between the herbivores feeding in a heavily 1984; Bryant and Froelich, 1995; Bryant et al., 1996; Kohn, enclosed wooded environment or on plants from different tree 1996; Sponheimer and Lee-Thorp, 1999; Levin et al., 2006). elevation, and the herbivores feeding in more open parkland Crudely, δ18 O values of meteoric water are higher in warmer or grassland environments with higher evapotranspiration or dryer environments (Dansgaard, 1964). Thus, as a general (Van der Merwe and Medina, 1991; Bocherens et al., rule, herbivores feeding in open steppe habitat are expected 1999; Feranec and MacFadden, 2006; Drucker et al., 2008; to have more enriched δ18 O values than species from forested Hofman-Kaminska et al., 2018). cooler and more humid habitat (Sponheimer and Lee-Thorp, The δ13 C values of the carbonate fraction of bioapatite in 1999). Obligate or water-dependent drinkers usually have δ18 O large mammalian herbivores are higher than or enriched by 14‰ values lower than non-obligate drinkers, being dependent on compared to their diet: δ13 Cbioapatite = δ13 Cdiet + 14‰ (Cerling water availability and seasonality of rainfall. Seasonal variation et al., 1999). In warm temperate and cold boreal environments in rainwater in mid- and high latitude environments and δ13 C values higher than −13‰ in herbivore enamel indicates species migration can be significant, producing intra-tooth open landscapes, such as grasslands and steppes, in which the variations of 3–4‰, or more (e.g., in high-crowned ungulates, plants have δ13 C values higher than −27‰ in diet or ecosystem horses and bison, Hoppe et al., 2004; Feranec et al., 2009). δ13 C values, whereas tooth enamel δ13 C values lower than −13‰ Oxygen isotopic composition can be affected not only by daily indicate woodlands and denser forests (Bocherens, 2014). ecology and migration but also by life history and physiology of mammals. Oxygen isotopic compositions are higher in Oxygen Stable Isotopes nursing animals during lactation and weaning periods because Oxygen isotopic (δ18 O) abundance of the herbivores’ enamel of milk consumption, which is related to the body water in apatite reflects the source and amount of body-water ingested, the mother, and more enriched in 18 O due to the preferential Frontiers in Ecology and Evolution | www.frontiersin.org 3 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy loss of 16 O through sweat, urine, and expired water vapor (Equus hemionus), and African free ranging donkeys (Equus (Bryant and Froelich, 1995; Kohn, 1996). asinus) (data from Schulz et al., 2007). To distinguish fossil and modern equid taxa based on mesowear variables (percentages MATERIALS AND METHODS of high relief, rounded, and blunt cusps) a hierarchical cluster analysis was performed using the Ward’s method and the The material studied consists of upper cheek teeth belonging to Euclidean distance. E. senezensis aff. E. sen. stehlini dental E. senezensis aff. E. sen. stehlini from CSG and to Equus cf. E. mesowear data were compared to those of E. stenonis from the mosbachensis from FR. All the examined fossil material is housed Early Pleistocene locality of Olivola (∼1.8 Ma, north central Italy) at the Italian Institute of Human Paleontology (IsIPU) (Anagni, (Strani et al., 2018c) and of the Britain sites of Mundesley and Italy). The comparative material was obtained from literature (see East Runton (∼1.8 Ma) (Rivals and Lister, 2016). Equus cf. E. in text). mosbachensis dental wear patterns were also compared to those of the fossil Middle Pleistocene caballoid equid population from Dental Mesowear the central European localities (Steinheim and Heppenloch,MIS Past studies on the dental wear patterns of E. senezensis aff. 11, Germany) (data from Rivals and Ziegler, 2018), from different E. sen. stehlini and Equus cf. E. mosbachensis from the Anagni Britain sites (Clacton, Hoxne and Swanscombe; MIS 11) (Rivals Basin examined both upper and lower cheek teeth (Strani et al., and Lister, 2016) and from Caune de l’Arago (438 ± 31 ka, 2015, 2018a,b). In this study we examined only upper teeth [P4- France) (data from Rivals et al., 2015a). M3, following Kaiser and Solounias (2003)] in their occlusal Discriminant analysis was performed to examine the relief (high or low) and cusp shape (sharp, rounded, or blunt) resolution of mesowear variables applied to the fossil. The of the apex of the paracone or metacone and scored for a percentages of high relief, rounded and blunt cusps were used better comparison with available data of both modern and as independent variables and two dietary (conservative and fossil equids because mesowear analysis is often restricted to radical) classifications of modern ungulate taxa as grouping the upper dentition. Occlusal relief and cusp shape scores were variables (Fortelius and Solounias, 2000). Equus quagga from also converted to a single mesowear score (MWS) following the Rumuruti (Rivals and Semprebon, 2010), Equus hemionus and “mesowear ruler” developed for scoring dental mesowear on Equus asinus (Schulz et al., 2007) were added to the modern taxa fossil equids by Mihlbachler et al. (2011). The method is based with known diets and treated as grazers in both conservative and on seven cusp types (numbered from 0 to 6), ranging in shape radical classifications. All analyses were performed using IBM from high and sharp (stage 0) to completely blunt with no relief SPSS Statistics 24. (stage 6). Additionally, a “stage 7” is given to teeth with a convex cusp apex. A total of 30 specimens were scored using this method (E. senezensis aff. E. sen. stehlini N = 20; Equus cf. E. mosbachensis Stable Carbon and Oxygen Isotope N = 10). Previously unavailable upper teeth of E. senezensis aff. Analyses and Preparation of Teeth Enamel E. sen. stehlini (N = 9) have been added to the original sample Samples analyzed in Strani et al. (2015, 2018b). We obtained stable carbon (δ13 C) and oxygen isotopic (δ18 O) Results were compared with mesowear data of four extant abundances from the upper cheek teeth (upper first or second equids: plains zebras (Equus quagga) (data from Fortelius and molars). In horses the isotopic composition in M1 or M2 would Solounias, 2000 and Rivals and Semprebon, 2010), Grévy’s zebras reflect nutrients from mother’s milk and should be slightly more (Equus grevyi) (data from Fortelius and Solounias, 2000), onagers depleted in 13 C and more enriched in 18 O compared to isotopic TABLE 1 | Summary of dental mesowear analysis. Taxa Locality Age N %High %Low %Sharp %Round %Blunt MWS References Equus cf. E. Fontana ranuccio Middle 10 0.0 100.0 30.0 50.0 20.0 4.8 This work; (Strani et al., mosbachensis pleistocene 2018a) Equus senezensis aff. E. Coste san giacomo Early 20 25.0 75.0 35.0 45.0 20.0 3.8 This work; (Strani et al., sen. stehlini pleistocene 2015, 2018c) Equus quagga Africa Modern 121 0 100 27.0 39.3 33.6 5.0 Fortelius and Solounias, 2000 Equus quagga Africa (Rumuruti, Mount Modern 28 0 100 28.6 64.3 7.1 4.3 Rivals and Semprebon, Kenya) 2010 Equus grevyi Africa Modern 29 0 100 34.4 41.3 24.1 4.9 Fortelius and Solounias, 2000 Equus hemionus Gobi desert Modern 175 47.4 52.6 4 85.7 10.3 / Schulz et al., 2007 Equus asinus Emirate sharjah Modern 184 55.4 44.6 43.5 39.1 17.4 / Schulz et al., 2007 Number of specimens (N); percentage of specimens with high (%High) and low (%Low) occlusal relief; percentage of specimens with sharp (%Sharp); rounded (%Rounded), and blunt (%rBlunt); mesowear score (MWS). Frontiers in Ecology and Evolution | www.frontiersin.org 4 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy composition of subsequently erupting P2, P3, M3, and P4 that would better reflect solid diet of an adult animal (Hoppe et al., 2004). Here we made comparisons with M1 or M2 from German sites and took into account this difference when comparing samples taken from premolars. We used 3 teeth samples of Equus cf. E. mosbachensis from FR and two E. senezensis aff. E. sen. stehlini from CSG. One FR horse sample was from PM4 and exhibited enrichment by 1‰ in 13 C and more enriched in 18 O compared to two FR M2. In German sites only E. hydruntinus had permanent teeth compared and the obtained values were within variation ranges of 1 and 2 ‰ in 13 C and 18 O, respectively. Enamel samples were either mechanically separated and ground to a finer condition in an agate mortar or drilled with a diamond-impregnated rotary tool. The uppermost surface of FIGURE 2 | Mesowear hierarchical cluster diagram based on the percentage the tooth was removed before sampling usually by drilling with a of high occlusal relief, round and blunt cusps of fossil populations with extant dremel© borer. Samples of approximately 20 mg were analyzed. equids. (Data for extant equids from Fortelius and Solounias, 2000; Schulz et al., 2007; Rivals and Semprebon, 2010). Fossil enamel powders were pre-treated following the method described by Bocherens et al. (1996) but using the Continuous Flow (CF) for collecting carbon dioxide (Jacques et al., 2008; Bocherens et al., 2009). Our experience with fossil material from The estimates of the δ18 O values of drinking water limestone cave has shown that the pre-treatment used here is consumed by species were calculated, first, by converting strong enough to remove exogenous carbonates but we are aware the oxygen isotope composition of structural carbonate to of possible slight changes of the oxygen isotopic composition of its equivalent in phosphate, using equations that are robust tooth enamel carbonate. for many different species: δ18 OP = 0.71 × δ18 OW +22.60 Pretreated (Sample /Enamel) carbonate was reacted with (Delgado Huertas et al., 1995). 100% H3 PO4 for 4 h at 70◦ C using a MultiFlow-Geo interfaced Then the oxygen isotope composition of phosphate was with the Elementar IsoPrime 100 IRMS. Final isotopic ratios converted to that of drinking water of different groups of species, are reported per mil (‰) calibrated with international according to formulas below and reformulated based on original standards (IAEA-603 δ13 C: 2.46/δ18 O: −2.37 and NBS-18 data from Skrzypek et al. (2011) based on current relationship δ13 C: −5.014/δ18 O: −23.2), as well as three in-house standards. between air and water. We calculated temperature (◦ Celcius) IonOS software (Version 3.2) by Elementar was used to carry using the formula for continental Europe: out multi-point standard isotope calibration by generating a trend line (y = mx+c) that maps measured vs. expected isotopic Tair = 1.41×δ18 O + 21.63 results, which is then used to calibrate sample results. The measurement uncertainty was monitored using three in-house We used both parametric and non-parametric tests with α = 0.05. standards. The overall analytical precision is higher than 0.1‰ To identify the sources of significant differences we used ANOVA for carbon and better than 0.2‰ for oxygen isotopic values. and among unequal variances we used post hoc Tamhane tests. Stable isotopic results are expressed as the following standard The results were compared with the late middle and late δ-notation: X = [(Rsample/Rstandard)-1]∗ 1,000, where X is Pleistocene horse data from Steinheim, Mauer, Bockstein and referred to δ13 C and δ18 O values and R is equivalent to 13 C/12 C Vogelherd (Pushkina et al., 2014). or 18 O/16 O, respectively. The recorded delta values follow the international reference standards, a “Vienna PeeDee Belemnite” RESULTS (VPDB) for the carbon and oxygen. Additionally, δ18 O values relative to “Vienna Standard Mean Ocean Water” (VSMOW) Dental Mesowear are given. In addition, the carbonate content (CaCO3 %) was E. senezensis aff. E. sen. stehlini upper teeth from CSG showed determined using the ratio between amount of CO2 released by mostly low occlusal relief (75%) and a higher percentage of the reaction, as detected from the peak intensity for mass 44 rounded cusps (45%) over sharp (35%) and blunt cusps (20%) and the weight of pure carbonate used as a standard, with an (Table 1). Low occlusal relief and high occurrence of rounded analytical error of 0.3%, based on multiple analysis of reference cusps E. senezensis aff. E. sen. stehlini from CSG point to a enamel samples. generally abrasive diet. Equus cf. E. mosbachensis teeth also To calculate values for temperature and drinking water displayed low occlusal relief and mostly rounded cusps (50%) from oxygen isotope values, and convert oxygen carbonate (Table 1), a pattern pointing to a dominance of abrasion and phosphate fractions, we used and equation from over attrition. Lécuyer et al. (2010) E. senezensis aff. E. sen. stehlini was clustered with the onager and the free ranging donkeys (cluster B) both displaying a grazing signature but with E. asinus showing a less abrasion-dominated δ 18 Oc = 1.035 δ 18 OP + 8.33 diet (Schulz et al., 2007) (Figure 2). Equus cf. E. mosbachensis Frontiers in Ecology and Evolution | www.frontiersin.org 5 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy FIGURE 3 | Bivariate diagrams based on discriminant analysis: conservative classification (A) and radical classification (B). Minute abraded brachydont species have been excluded. Fossil taxa Ez, Equus senezensis aff. E. sen. stehlini; Em, Equus cf. E. mosbachensis; Eq, Equus quagga; EqR, Equus quagga (Rumuruti population); Eg, Equus grevyi; Eh, Equus hemionus; Ea, Equus asinus. Group centroids: browsers (0); mixed feeders (1); grazers (2). FIGURE 4 | Mesowear results of fossil stenonid equids and extant ungulates. Modern ungulates data from Fortelius and Solounias (2000) (diet information according to the radical classification; minute abraded brachydont species have been included), Rivals and Semprebon (2010). Mundesley and East Runton data from Rivals and Lister (2016). MWS, mesowear score; CSG, Coste San Giacomo; Ez, Equus senezensis aff. E. sen. stehlini; Es, Equus stenonis; Eb, Equus bressanus; FB, fruit browsers; LB, leaf browsers; M, mixed feeders; G, grazers; *, Heterohyrax brucei. fell in cluster A with modern zebras (Figure 2), which are all are classified as grazers by both the conservative and radical characterized by strong abrasion-dominated mesowear patterns classification (Figures 3A,B). (Table 1) (Fortelius and Solounias, 2000; Rivals and Semprebon, E. senezensis aff. E. sen. stehlini displayed an average MWS 2010). Discriminant analysis performed with the mesowear of 3.8, lower than the one recorded in the upper cheek teeth variables provides a satisfactory dietary discrimination with of E. stenonis from Olivola (5.3), from stenonid horses from 71.9% of extant taxa correctly classified according to the East Runton (E. bressanus, 4.5; E. stenonis, 4.0) and from E. conservative classification and 75.4% according to the radical stenonis from Mundesley (Figure 4), pointing to a less strictly one (70.2 and 75.4%, respectively, in cross-validation). Both E. abrasion-dominated feeding behavior in the stenonid equid senezensis aff. E. sen. stehlini and Equus cf. E. mosbachensis from Coste San Giacomo. Equus cf. E. mosbachensis from Frontiers in Ecology and Evolution | www.frontiersin.org 6 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy FIGURE 5 | Mesowear results of fossil stenonid equids and extant ungulates. Modern ungulates data from Fortelius and Solounias (2000) (diet information according to the radical classification; minute abraded brachydont species have been included), Rivals and Semprebon (2010). Caune de l’Arago, Steinheim, Heppenloch, Swanscombe, Hoxne and Clacton data from Rivals et al. (2015a), Rivals and Lister (2016) and Rivals and Ziegler (2018). MWS, mesowear score; CSG, Coste San Giacomo; Ez, Equus senezensis aff. E. sen. stehlini; Es, Equus stenonis; Eb, Equus bressanus; FB, fruit browsers; LB, leaf browsers; M, mixed feeders; G, grazers; *, Heterohyrax brucei. Fontana Ranuccio, showed a mean MWS of 4.8 higher than the equids from Steinheim. Equus cf. E. mosbachensis differed from one recorded in all the other caballoid equids from Germany the late Pleistocene Equus ferus (post hocs Tamhane p = 0.004 (Steinheim, 3.8; Heppenloch, 4.3), France (Caune de l’Arago, δ13 C/ ecosystem). 3.9) and Britain (Swanscome, 3.7; Hoxne, 4.0; Clacton, 4.4) The δ18 O values of FR Equus cf. E. mosbachensis varied (Figure 5), suggesting an overall more abrasive diet for the between −6.96 and −4.24‰ (VPDB), −23.68 and −26.49‰ Middle Pleistocene fossil caballoid equid of the Anagni Basin. (CO3 VSMOW) (Table 2) (Figure 6B), 14.83 and 17.54‰ (PO4 VSMOW), respectively, indicating that water sources used had Stable Carbon and Oxygen Isotope the δ18 Owater values of −10.94 and −7.12‰. Analyses The only peculiar difference between the Italian and German Compared equids E. senezensis aff. E. sen. stehlini from CSG, sites is demonstrated in the temperature calculations, indicating Equus cf. E. mosbachensis from FR and Mauer, E. ferus/ much higher temperatures for both CSG and FR (6.2–12◦ C) than E. hydruntinus/E. sp. from Steinheim and E. caballus from for the German sites (−0–6.8◦ C) (Table 2). Bockstein and Vogelherd varied significantly in δ13 C and δ18 O values (ANOVA [F 5,26 = 8.6, p < 0.001, r2 = 0.55] for DISCUSSION δ13 C / ecosystem; [F 5,26 = 5.7, p < 0.001, r2 = 0.43] for δ18 O VPDB/VSMOW/temperature). Mesowear and stable isotopic analyses suggest that horses in The δ13 C values of E. senezensis aff. E. sen. stehlini from both sites inhabited rather open and dry environments with the Early Pleistocene CSG were −12.49 and −11.94‰, falling abrasive C3 vegetation as a dietary source for the majority. As within the values of the late Pleistocene horses from Bockstein obligate drinkers, however, horses could have used different and Vogelherd and middle late Pleistocene E. hydruntinus from water sources in more humid and closed, or more open and Steinheim (Table 2) (Figure 6A). The δ18 O values of CSG E. arid environments, which could have been at slightly different senezensis aff. E. sen. stehlini were higher than in the compared elevation and subjected to seasonal variation in water availability. German sites −4.02 and −6.19‰ (VPDB), −26.71 and −24.48‰ Both E. senezensis aff. E. sen. stehlini from CSG and Equus cf. (CO3 VSMOW) (Table 2) (Figure 6B), 17.76 and 15.6‰ (PO4 E. mosbachensis from FR display dental mesowear patterns VSMOW), respectively, suggesting that the horses used water highly consistent with a grazing feeding behavior, according sources with δ18 Owater −6.81 and −9.86‰. to the results reported in Strani et al. (2015, 2018a,b,c), where The δ13 C values of Equus cf. E. mosbachensis from the Middle both upper and lower cheek teeth were analyzed. Differences, Pleistocene FR ranged from −13.93 to −12.63‰ (Table 2) however, are observed in terms of niche occupation in the Early (Figure 6A), which is similar to E. mosbachensis from Mauer and Pleistocene stenonid equid, displaying a less abrasion-dominated Frontiers in Ecology and Evolution | www.frontiersin.org 7 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy TABLE 2 | Isotopic results for Equus senezensis aff. E. sen. stehlini from CSG, Equus cf. E. mosbachensis from FR, and for Pleistocene equids from German localities [data from Pushkina et al. (2014)]. Taxon Site d13C (‰ d18O (‰ d18O (‰ Ecosystem Temperature ◦ C References VPDB) VPDB) VSMOW) (central Europe) Equus cf. E. mosbachensis Fontana ranuccio −13.87 −6.43 24.23 −27.87 7.7 This paper Equus cf. E. mosbachensis Fontana ranuccio −12.63 −4.24 26.49 −26.63 13.2 This paper Equus cf. E. mosbachensis Fontana ranuccio −13.93 −6.96 23.68 −27.93 6.33 This paper Equus senezensis aff. E. sen. stehlini Coste san giacomo −12.49 −4.02 26.71 −26.49 13.75 This paper Equus senezensis aff. E. sen. stehlini Coste san giacomo −11.94 −6.19 24.48 −25.94 8.28 This paper Equus caballus Bockstein −11.94 −10.25 20.29 −24.44 −1.93 Pushkina et al., 2014 Equus caballus Bockstein −11.72 −8.66 21.93 −24.22 2.08 Pushkina et al., 2014 Equus caballus Bockstein −11.59 −8.99 21.6 −24.09 1.26 Pushkina et al., 2014 Equus caballus Bockstein −11.39 −9.39 21.18 −23.89 0.23 Pushkina et al., 2014 Equus mosbachensis Mauer −13.91 −7.73 22.89 −26.41 4.44 Pushkina et al., 2014 Equus mosbachensis Mauer −13.1 −9.05 21.53 −25.6 1.1 Pushkina et al., 2014 Equus mosbachensis Mauer −12.83 −6.67 23.98 −25.33 7.11 Pushkina et al., 2014 Equus mosbachensis Mauer −12.72 −9.37 21.2 −25.22 0.28 Pushkina et al., 2014 Equus ferus Steinheim −14.2 −8.9 21.7 −26.72 1.42 Pushkina et al., 2014 Equus ferus Steinheim −13.56 −7.91 22.71 −26.06 3.98 Pushkina et al., 2014 Equus ferus Steinheim −13.43 −6.91 23.74 −25.93 6.51 Pushkina et al., 2014 Equus ferus Steinheim −13.33 −9.39 21.18 −25.83 0.25 Pushkina et al., 2014 Equus ferus Steinheim −13.29 −8.94 24.64 −25.79 1.38 Pushkina et al., 2014 Equus ferus Steinheim −13.03 −9.31 21.26 −25.53 0.43 Pushkina et al., 2014 Equus ferus Steinheim −12.53 −8.97 21.62 −25.03 1.31 Pushkina et al., 2014 Equus ferus Steinheim −12.45 −7.97 22.64 −24.95 3.82 Pushkina et al., 2014 Equus ferus Steinheim −13.6 −7.6 23 −26.1 4.7 Pushkina et al., 2014 Equus ferus Steinheim −12.8 −8.3 22.3 −25.3 2.98 Pushkina et al., 2014 Equus hydruntinus Steinheim −12.7 −7.9 22.7 −25.16 3.98 Pushkina et al., 2014 Equus hydruntinus Steinheim −12.3 −7 23.7 −24.84 6.3 Pushkina et al., 2014 Equus hydruntinus Steinheim −12.2 −7.4 23.3 −24.7 5.35 Pushkina et al., 2014 Equus hydruntinus Steinheim −12 −9.1 21.5 −24.49 1 Pushkina et al., 2014 Equus sp. (ferus) Steinheim −13.8 −7.4 23.2 −26.3 5.16 Pushkina et al., 2014 Equus sp. (ferus) Steinheim −12.7 −7.2 23.4 −25.7 5.67 Pushkina et al., 2014 Equus sp. (ferus) Steinheim −12.5 −7.5 23.2 −25 5.12 Pushkina et al., 2014 Equus sp. (ferus) germanicus Steinheim −13.1 −7.3 23.3 −25.58 5.53 Pushkina et al. (2014) Equus caballus Vogelherd −12.17 −9.49 21.08 −24.67 −0.01 Pushkina et al., 2014 diet in a more open landscape of steppe, shrubland, or light forest from central Europe (Pushkina et al., 2014). Dental mesowear compared to the Middle Pleistocene caballoid species that fed patterns are compatible with a grazing behavior similarly to strictly on abrasive sources in a more closed habitat of woodland E. stenonis populations, e.g., from Sésklo (MN17 biozone, or shrubland. Temperature-wise these localities did not appear Early Pleistocene, Greek Peninsula) (Rivals and Athanassiou, to have differed. These findings allow us to provide information 2008) and from Olivola (∼1.8 Ma, Italian Peninsula) on the paleoecology of the two groups of fossil equids of the (Strani et al., 2018b). Anagni Basin, hereby shedding light on their ecological response E. stenonis is a common species of both Italian and to the major climatic events that marked the Early and Middle Greek Peninsulas associated with fossil ungulates showing Pleistocene: the onset of the Pleistocene glaciation, which led to a generally mixed or grazing dietary adaptations in open and gradual trend toward cooler climatic conditions and subsequent dry environments (Rivals and Athanassiou, 2008; Strani et al., reduction of sub-tropical vegetation in Eurasia (Fortelius et al., 2018b). In contrast, E. senezensis aff. E. sen. stehlini from CSG 2006; Combourieu-Nebout et al., 2015), and strengthening of the is associated with artiodactyls showing a wider range of feeding Early-Middle Pleistocene Transition, which led to an increase behaviors in a more heterogeneous landscape comprised of of interglacial temperatures (Head and Gibbard, 2015). Isotopic wetlands, forests and grasslands (Strani et al., 2015, 2018c), while results obtained for E. senezensis aff. E. sen. stehlini indicate that displaying a less abrasion-dominated dental mesowear pattern this equid occupied open and dry habitats similarly to the Late than E. stenonis from Olivola (Figure 4). Similar conditions Pleistocene horses and middle Late Pleistocene E. hydruntinus are observed in East Runton, where E. bressanus displayed Frontiers in Ecology and Evolution | www.frontiersin.org 8 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy FIGURE 6 | δ13 C (A) and δ18 O (B) values of the studied taxa compared to fossil equids of central Europe (data from Pushkina et al., 2014). The red line represents the boundary between δ13 C values that indicate open (δ13 C > −13‰) or closed landscapes (δ13 C < −13‰) in warm temperate and cold boreal environments (Bocherens, 2014). long-term grazing (according to molar mesowear) and short- Lister, 2016; Rivals and Ziegler, 2018). A similar trend from term browsing diet (according to tooth microwear) and is less abrasion-dominated to strictly grazing diet is observed even grouped with other ungulates with a variety of feeding behaviors in Early and Middle Pleistocene equid populations of Britain (Rivals and Lister, 2016). (Rivals and Lister, 2016). The stenonid Equus altidens from the late Early Pleistocene Isotope analysis points instead to a preference for slightly Southern European site of Vallparadís Estació (layer EVT7) also more closed environments in Equus cf. E. mosbachensis from displayed a certain dietary plasticity with a short-term mixed FR similarly to E. mosbachensis from Mauer and Middle and feeding behavior (Strani et al., 2019). E. senezensis aff. E. sen. Late Pleistocene equids from Steinheim (Pushkina et al., 2014) stehlini is also one of the best represented ungulates from CSG, in comparison to E. senezensis aff. E. sen. stehlini. Long-term whereas fewer individuals have been recorded in Olivola (Strani grazing behavior and short-term less abrasive diets are observed et al., 2018b). This suggests that this equid, while feeding mostly in E. ferus from the MIS 11 German localities of Hoppenloch on grasses, may have also accessed more diverse food items and Steinheim (Rivals and Ziegler, 2018) and from the MIS 9 site leading to a greater intake of softer plant resources compared Schöningen (Rivals et al., 2015b), suggesting that also in central to the similarly aged E. stenonis from Sésklo and to the later Europe Middle Pleistocene horses appeared to have successfully stenonid equids from Olivola. This scenario is consistent with exploited less open habitats. the onset of a gradual trend toward cooler climatic conditions FR Equus cf. E. mosbachensis is associated with ungulates after the initiation at the Plio-Pleistocene boundary (2.6 Ma) displaying a wide range of feeding behavior with a noticeable of the Quaternary glaciations regulated by a 41 kyr periodicity abundance of browsing and mixed feedings cervids (Strani et al., (Lisiecki and Raymo, 2005), which led to the augmentation of 2018a,d). A similar condition is observed in Steinheim, where open habitats and the disappearance of sub-tropical vegetation herbivores with diverse diets in humid and more closed habitats in the European continent (Bertini, 2003; Bertini, 2006, Fortelius are recorded (Pushkina et al., 2014; Rivals and Ziegler, 2018). et al., 2006; Bertini, 2010, 2013; Kahlke et al., 2011; Combourieu- Isotopic analysis from the ungulates of Mauer also suggest warm Nebout et al., 2015). This process probably affected later and densely wooded landscape for this locality (Pushkina et al., Northern Europe as the paleoenvironmental conditions of East 2014). On the contrary, fossil horses from the layer G (MIS Runton appear to be more heterogeneous compared to the coeval 12) of Caune de l’Arago Cave (France) and from Hoppenloch locality of Olivola. are associated with a lower range of dietary traits pointing The more abrasion-dominated mesowear patterns of Equus to drier environmental settings (Rivals et al., 2008; Rivals and cf. E. mosbachensis point to a stricter grazing diet, and are Ziegler, 2018). This suggests that Equus cf. E. mosbachensis comparable to the ones observed in modern zebra populations, from the Anagni Basin occupied an ecological niche similar to which feed almost exclusively on grasses (Bodenstein et al., the fossil horses from Steinheim in comparable environmental 2000; Fortelius and Solounias, 2000; Rivals and Semprebon, conditions, as grazers that feed also in more closed habitats. The 2010; Kartzinel et al., 2015). The same trend is observed higher MWS recorded in the FR population (4.8) compared to also in the Middle Pleistocene caballoid equids from other that of the Steinheim (3.8) (Figure 5), suggests that the fossil European localities, which also display abrasion-dominated horses of central Italian Peninsula had a stricter grazing behavior mesowear signatures (Rivals et al., 2008, 2015a; Rivals and compared to central European populations. Frontiers in Ecology and Evolution | www.frontiersin.org 9 May 2019 | Volume 7 | Article 176 Strani et al. Equus Paleoecology in Central Italy FR archeopaleontological layer with its fossil assemblage is based only on horses, and oxygen stable isotope variation apart dated to around “Mid-Brunhes event” (Biddittu et al., 1979; from higher temperature can be associated with other factors, Muttoni et al., 2009; Pereira et al., 2018), which is associated e.g., water sources from different elevation and subjected to with an increase in the amplitude of the interglacials and seasonal variation in water availability. glacial cycles (Lang and Wolff, 2011; McClymont et al., 2013), Obtained data on the niche occupation of the stenonid Equus and with one of the warmest and longest interglacial periods of the Anagni Basin support the spread of open landscapes in of the last 400 ka, registered during MIS 11 (Weirauch the Italian Peninsula following the initiation of the Pleistocene et al., 2008). According to palynological data from the MIS glacial cycles, suggesting, however, that these equids could 11 Boiano Section (central Italian Peninsula) these warmer have successfully adapted to exploit also more heterogeneous conditions led to the expansion of closed canopy forests resources in the long-term timescale. Data from the caballoid with high summer precipitations (Combourieu-Nebout et al., Equus support the expansion of more closed environments in 2015). The paleoecological information for the fossil horse the region after the MIS 12–11 transition, though horses seemed of FR, suggests that in this scenario the Middle Pleistocene to feed almost exclusively on abrasive vegetation possibly to caballoid equids from central Italian Peninsula adapted to the avoid competition with other large ungulates that exploited softer reduction of open grasslands by exploiting also more closed food resources. habitats in heterogeneous landscapes. A possible competition with other large gregarious ungulates, such as Bos primigenius AUTHOR CONTRIBUTIONS and Stephanorhinus sp., could have also played a key role in the niche occupation of this equid. The higher temperature All authors have provided substantial contribution to the article. gradient recorded by the isotopic analysis of the equid dental FS conceived the ideas and designed the approach. FS collected material compared to those observed in Steinheim, also can imply and elaborated the mesowear data. DP collected and performed warmer conditions that characterized the Anagni Basin during the isotopic analysis with the help of HB, FS, and DP led the this interval. This is concordant with the hypothesis of the Italian writing of the manuscript with the support of HB and DD. RS and Peninsula acting as a southern refugium for mammal taxa and LB provided helpful comments and inputs for the manuscript. human populations during colder periods and extreme glacials All authors have approved the manuscript and agree with its of the Pleistocene (Manzi et al., 2011; Manzi, 2016). submission to the journal. CONCLUSIONS ACKNOWLEDGMENTS This work offers important updated information on the This research has been supported by Avvio alla Ricerca 2017 paleoecological adaptations of Pleistocene equids of the Italian (Ref. AR11715C7CE78D4F to FS) and the Spanish Agencia Peninsula. Overall Early Pleistocene stenonid horses grazed Estatal de Investigación (Ministerio de Economía, Industria in open landscapes but demonstrated less abrasion-dominated y Competitividad)–European Regional Development Fund of diet compared to the later forms, suggesting the existence of the European Union (CGL2016-76431-P, AEI/FEDER EU). We more heterogeneous environmental conditions during the early thank the Italian Institute of Human Paleontology for preserving Early Pleistocene before the Gelasian/Calabrian (middle/late the collection and allowing the study of the material object of Villafranchian) transition. In the same region, Middle Pleistocene this investigation. We are also very grateful to Gina Semprebon caballoid populations displayed more abrasion-dominated long- and Mikael Fortelius for providing the raw mesowear data of term feeding behavior, having occupied less open habitats (at modern equids (Equus quagga and Equus grevyi) and other the edge between grasslands and woodlands) than stenonid extant ungulates We also acknowledge to Raymond L. Bernor for forms of the Early Pleistocene, both in possibly warmer climatic inviting us to contribute to the Examining Evolutionary Trends conditions compared to French and German localities of in Equus and its Close Relatives from Five Continents Research the same age. Temperature calculations, however, should be Topic. Finally, we thank the two reviewers for their helpful considered with caution, since the sample size is quite small and comments and suggestions. REFERENCES Bellucci, L., Mazzini, I., Scardia, G., Bruni, L., Parenti, F., Segre, A. G., et al. (2012). The site of Coste San Giacomo (Early Pleistocene, central Italy): Alberdi, M., and Palombo, M. (2013). The late Early to early Middle palaeoenvironmental analysis and biochronological overview. Quat. Int. 267, Pleistocene stenonoid horses from Italy. Quat. Int. 288, 25–44. 30–39. doi: 10.1016/j.quaint.2011.04.006 doi: 10.1016/j.quaint.2011.12.005 Bernor, R., Göhlich, U., Harzhauser, M., and Semprebon, G. (2017). 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Resource conducted in the absence of any commercial or financial relationships that could and niche differentiation mechanisms by sympatric Early Pleistocene be construed as a potential conflict of interest. ungulates: the case study of Coste San Giacomo. Quat. Int. 481, 157–163. doi: 10.1016/j.quaint.2017.08.064 Copyright © 2019 Strani, Pushkina, Bocherens, Bellucci, Sardella and DeMiguel. Strani, F., Profico, A., Manzi, G., Pushkina, D., Raia, P., Sardella, R., et al. (2018d). This is an open-access article distributed under the terms of the Creative Commons MicroWeaR: a new R package for dental microwear analysis. Ecol. Evol. 8, Attribution License (CC BY). The use, distribution or reproduction in other forums 7022–7030. doi: 10.1002/ece3.4222 is permitted, provided the original author(s) and the copyright owner(s) are credited Van der Merwe, N. J., and Medina, E. (1991). The canopy effect, carbon and that the original publication in this journal is cited, in accordance with accepted isotope ratios and foodwebs in Amazonia. J. Archaeol. Sci. 18 , 249–259. academic practice. No use, distribution or reproduction is permitted which does not doi: 10.1016/0305-4403(91)90064-V comply with these terms. Frontiers in Ecology and Evolution | www.frontiersin.org 13 May 2019 | Volume 7 | Article 176