Hipparion Datum Implies Miocene Palaeoecological Pattern

Here, we report well–preserved skulls and postcranial specimens of the subgenus Hippotherium from the Linxia Basin, Gansu, China. Based on morphological comparison, the species of subgenus Hippotherium in China, Hipparion weihoense and Hipparion chiai, should be ascribed to the same species, H. weihoense. The species Hipparion prostylum (later Hipparion aff. brachypus) from Maragheh, Iran should also be ascribed to H. weihoense. Phylogenetic analysis shows the subgenus Hippotherium derived from the North American genus Cormohipparion, and as a basal group of Hipparion in Eurasia, representing the Hipparion Datum. Analysis on locomotive ability indicates that H. weihoense likely lived in an open habitat, whereas other species of subgenus Hippotherium likely lived in closed habitats. This result indicates a palaeoecological pattern in the early Late Miocene in Eurasia: inuenced by a series of geological events, aridication of mid–latitude Asia progressed, whereas Europe and North Africa remained relatively humid; as the genus originated from East Asia, Hipparion divided rapidly into different groups with differing functional morphology to occupy diverse niches.


Introduction
The dispersal of Hipparion into the Old World, recognised as the Hipparion Datum, is one of the most signi cant palaeobiological events in the Late Miocene [1][2][3][4] . Hipparion primigenium in Europe is traditionally regarded as the earliest and most primitive Hipparion species in Eurasia 4-6 . Some authors proposed that it should be assigned to a valid genus Hippotherium 6-8 . Qiu et al. 9 indicated that all hipparion species in the Old World should be assigned within one genus, Hipparion, and that the taxon Hippotherium should be regarded as a subgenus. They reviewed the earliest Hipparion species found in China, Hipparion weihoense and Hipparion chiai, and ascribed them to the subgenus Hippotherium. They also ascribed the early species Hipparion africanum found in Bou Hani a, Algeria and Hipparion catalaunicum from Hostalets, Spain to this subgenus. Consequently, the early evolution and distribution of the subgenus Hippotherium have great signi cance regarding the evolution of Hipparion and Late Miocene palaeoecology.
Research on H. weihoense and H. chiai in China remains insu cient. Liu et al. 10 erected these two species based on cranial and dentition material found in Lantian, Shaanxi. Liu 8 described another collection from Lantian. However, known specimens were limited to broken skulls, mandibles, isolated teeth, and metapodials. These two species are almost always found in the same locality. They have many morphological similarities, and H. weihoense specimens are much more abundant than H. chiai. Following the priority principle, the validity of H. chiai is suspect. Consequently, more and betterpreserved specimens are required to evaluate the status of these two species.
information on cranial and postcranial morphology to compare with known specimens of the subgenus Hippotherium to better recognise H. weihoense and H. chiai. This material is also suitable to determine the locomotive ability of subgenus Hippotherium in China. Deng et al. 11 performed comprehensive locomotive analysis of the Tibetan Hipparion zandaense, which provides an ideal template for our research. Based on comparison with the known postcranial material of H. primigenium and H. africanum, we can seek clues regarding the environmental and ecological conditions of the early Late Miocene in Eurasia.

Systematic Palaeontology
The specimens in the present research are described as following: Order Perissodactyla Owen, 1848;

Attribution and revision
The newly described specimens have a characteristic cranial and dentition diagnosis combination, including medium to large size, shallow nasal notch, long POB, developed POF with a posterior pocket, complex fossettes with long and strong folds, and elongated protocone with at labial margin. All of these features are identical to the primitive hipparion species in China, Hipparion (Hippotherium) weihoense.
Liu et al. 10 reported a large Hipparion species discovered from Lantian, Shaanxi and erected the new species Hipparion weihoense. In the same text, they identi ed a smaller skull fragment and some teeth with similar features and stratigraphic position to H. weihoense as another new species Hipparion chiai.
Qiu et al. 9 reviewed hipparion fossils from China and accepted the validity of both species. They ascribed these two species to subgenus Hippotherium, and regarded them as the most primitive hipparion horse in the Old World. Liu 8 described a series of specimens from Lantian, Shaanxi and identi ed parts of these specimens as H. weihoense and H. chiai. Based on the reported specimens from China, there is actually no clear boundary between the cranial features of these two species. Bernor et al. 12 identi ed the hipparion specimens from Maragheh, Iran as Hipparion prostylum. H. prostylum was rst reported in France. Based on observation of the specimens of H. prostylum from the type locality (Luberon, France), now housed in the NMNH, Paris, we determined that the upper cheek teeth from Luberon have rounded protocone, single pli caballine, and simple and robust folds in fossettes. This combination of features, based on personal observation on specimens, is more similar to the subgenus Cremohipparion, such as Hipparion forstenae and Hipparion gracile. Later Bernor et al. 13 ascribed these Maragheh specimens as Hipparion aff. brachypus. H. brachypus is a huge-built form with deep nasal notch and very robust metapodials 14 , which are di cult to correspond to the Maragheh form. Morphologically, specimens from Maragheh, Iran are very similar to H. weihoense in China. Based on measurements of skulls (S Table. 1) the POF positions of these specimens are stably distributed; the difference is ontogenetic. The dimensions of the POF are more variable, but another hipparion species in Eurasia also has variable POFs: Hipparion dermatorhinum, found in Baode, Shanxi, China 9 . The measurements of Bernor et al. 6 show highly variable POF of Hipparion primigenum. Moreover, POF height is easily in uenced by compressional deformation. In the sketches of the skulls of H. weihoense (S Fig. 6), most individuals have clearly or nearly subtriangular POFs; some POFs with signi cantly different shapes show clear evidence of deformation. The dentition was regarded as another important feature to distinguish these two species in previous research. Liu et al. 10 argued that H. chiai had a simpler fossette on the upper cheek tooth than that of H. weihoense. Based on recent ontogenetic sequence analysis, the fossette characters of hipparion largely depend on wear stage 15,16 . Liu et al. 10 also indicated that some large individuals of H. chiai had similar features on upper cheek teeth to those of H. weihoense. They did not discuss the features of lower cheek teeth. In our sketches of reported upper and lower cheek specimens of both H. weihoense and H. chiai, there is no signi cant difference between these two taxa (S Fig. 7-8). Therefore, H. weihoense, H. chiai, and H. prostylum from Maragheh, Iran should be ascribed to the same species. Following the priority principle, all of these specimens belong to H. weihoense.

Phylogenetic analysis
We used characters from Woodburne 4 as our basis, changed a repeated character for the skull, character 36, into a new one on tooth character, added 21 tooth characters from Liu 8 , and added two new characters on the postcranial and size. We partly followed the taxa in the matrix of Woodburne 4

and used
Parahippus leonensis, Merychippus primus, and Merychippus insignis as outgroup taxa. Woodburne 4 erected ve new species of the North American genus Cormohipparion. However, these species overlapped in character, geographic distribution, and age, and are better regarded as synonyms of the type species, Cormohipparion occidentale. We only accept the validity of three known species of this genus: Cormohipparion goorisi, Cormohipparion quinni, and Cormohipparion occidentale. We also added the Eurasian species Hipparion (Hippotherium) weihoense, Hipparion (Hippotherium) africanum, and Hipparion (Hippotherium) catalaunicum, and the subgenera Cremohipparion, Baryhipparion, Sivalhippus, Plesiohipparion, and Proboscidipparion. These 16 taxa and 62 characters constitute a new matrix. One MPT (most parsimonious tree) was obtained. We determined that all Eurasian hipparion subgenera form a monophyly, which fully supports the argument of Qiu et al. 9 on systematic palaeontology, classifying all Chinese hipparion horses as one genus. All other subgenera were derived from the subgenus Hippotherium. This interpretation also supports the phylogenetic relation between Cormohipparion and Old World hipparions proposed by MacFadden 17 . Consequently, subgenus Hippotherium should be regarded as the basal group of Eurasian hipparion horses representing the Hipparion Datum.

Functional morphology
The well-preserved postcranial specimens from the Niugou locality indicate the locomotive ability of H. weihoense. A strong medial trochlear ridge (MTR) of the femur can fasten the medial patellar ligament, or parapatellar cartilage, and the patella when the knee joint is hyperextended 18 , forming a passive stayapparatus to immobilise musculature in the knee extensors during long periods of standing. The femur MTR of H. weihoense is greatly enlarged relative to the lateral trochlear ridge, notably larger than in Hipparion primigenium, but similar to Hipparion zandaense from the Pliocene of the Zanda Basin. The ratio between the maximum depth of the MTR and the maximum length of the femur is 0.27 in H. primigenium 6 , whereas it is 0.32 in H. weihoense and 0.3 in H. zandaense 11 . Gracile limb bone is an indicator of cursorial ability, which is most clearly exhibited in the metapodials of ungulates 19 . The gracility of the metapodial shaft is represented by diminished breadth relative to length. In Fig. 2, above the zero line are the comparatively larger measurements, and below it are the smaller ones. The ratios between the maximum length and the minimum breadth indicate that H. weihoense, H. zandaense, and C. occidentale have relatively slender metapodials (measurement 3 is smaller or slightly larger than measurement 1), but H. primigenium has very robust metapodials (measurement 3 is notably larger than measurement 1), and the subgenus Proboscidipparion (Hipparion sinense and Hipparion pater) and H. houfenense from the North China Plain also show increased robustness. Typically, metapodial robustness of horses has been considered a marker of evolutionary steps. Robust metapodials indicate primitive steps, whereas slender one indicates the opposite 20, 21 . Our results supplement this hypothesis with a trend of increased robustness with increased body size, and with metapodial robustness considerably in uenced by environmental change. A high proportion of distal elements will lengthen the whole limb to keep its centre of mass situated proximally and to reduce its inertia, which allows for a long, rapid stride, as speed is the product of stride length and stride frequency 22 . Lengths of the distal elements of hindlimbs, Mt III, and the rst hind phalange relative to proximal elements of H. weihoense and C. occidentale are signi cantly longer than those of H. primigenium, which indicates the stronger running ability of the former two. Both the advanced H. houfenense and H. sinense have these characteristics (Fig. 3). Consequently, H. weihoense was able to run fast and stand persistently, which is bene cial in open habitats. The running abilities of H. primigenium and H. africanum were weaker and more suited to slower movement in closed habitats 6, 23 , and their locomotive function stands in contrast to the inferred ecosystem and behaviour of H. weihoense.

Discussion
In China, H. weihoense is mainly distributed in the Linxia Basin, Gansu [24]; the Qaidam Basin, Qinghai 25 ; and Lantian 8, 10 24 . We infer that the open, arid habitat to which H. weihoense was adapted in the area surrounding the Tibetan Plateau appeared around 11 Ma. Dettman et al. 32 also con rmed that the period of greatest aridity at the NE margin of the Tibetan Plateau was from 9.6 to 8.2 Ma, which is well consistent with other climate records. This was the age in which H. weihoense thrived in related areas 24,36 . Liu et al. 10 40 argued that Tibetan Plateau uplift and Paratethys retreat occurred at the same time. Retreat of the Paratethys would further reduce vapour delivery to Asia. Thus, habitats of H. weihoense in other localities were likely similar to the Linxia Basin (Fig. 4). Iranotherium morgani was also discovered in the Linxia Basin, China and Maragheh, Iran with H. weihoense. Deng 41 argued that the giant body size and hypsodont cheek teeth with wrinkled enamel and rich cement imply that I. morgani was a grazer. The Maragheh fauna is a bit younger than the Dashengou fauna in Linxia Basin; therefore, I. morgani likely originated in northwestern China and then dispersed westward to central Asia, which had an open habitat similar to that they had adapted to in China. Phylogenetic analysis shows that H. weihoense likely originated from East Asia. Based on the distribution of H. weihoense in China, it likely dispersed into Iran through the Qaidam Basin, along the northern margin of the Tibetan Plateau. Therefore, there was signi cant exchange of taxa between China and Iran in the early Late Miocene.
In comparison, Hipparion africanum and Hipparion primigenium both have robust metapodials, and H. primigenium also has a high proportion of proximal elements of limbs, all of which indicate a closed habitat. Böhme et al. 42 estimated precipitation for Southwest and Central Europe in Miocene and proposed a dry period during 13-11 Ma, end of which is the time Hipparion arrived in Eurasia. Later environment in Europe had generally been humidifying. Fortelius et al. [43] also indicated that the habitat in Europe and North Africa, the respective habitats of H. primigenium and H. africanum, was relatively closed during 11-8 Ma. In the same period, in the habitat of H. weihoense, western China-Central Asia, especially the eastern margin of Tibetan Plateau, high-crowned ungulates were dominant, which indicates an open habitat. Böhme et al. 42 proposed a term "washhouse climate" for analogy of a climate under high precipitation during 10.3-9.8 Ma. This age is consistent with that of Höwenegg (10.3 Ma 4 ) and slightly younger Eppelsheim in Germany, where specimens of H. primigenium were abundant.
North American species C. occidentale also had very slender Mt III and a high proportion of distal elements of hind limbs (Fig. 3-4). The habitat type in the end of the Middle Miocene and the early Late Miocene was likely open, based on the diversity and population of grazers in North America 44,45 .
According to Mihlbachler et al. 46 , hypsodont Equinae species rst occurred in 16 Ma, and later became dominant in 12 Ma. The result of representative SEM photomicrographs of tooth microwear by Hayek et al. 47 showed that C. occidentale were most likely grazers. Based on phylogenetic analysis, the origin of the subgenus Hippotherium was likely in East Asia (not traditionally considered Europe), derived from the grazing C. occidentale. This dispersal route and evolutionary change is also consistent with the parsimony principle of evolution. Based on North American palaeoecology, a dominantly open habitat existed in North America signi cantly earlier than in Eurasia. This habitat led to the emergence of grazing Cormohipparion species, which would later give rise to the Eurasian Hipparion to preadapt for open environments. Hipparion occurred in Eurasia, and rapidly adapted to the widespread open habitat and developed high diversity. This is another typical example of environmental preadaption of late Cenozoic megaherbivores. Deng et al. 48 argued that mammalian fossils found in Pliocene strata in the Tibetan Plateau suggest that some megaherbivores rst evolved in Tibet before the beginning of the famous Ice Age in Pleistocene. The cold winters high in Tibet served as a habituation ground for megaherbivores, which became preadapted for the Ice Age, and successfully expanded to the Eurasian mammoth steppe. The following constitute the ecological pattern of the early Late Miocene: the Tibetan Plateau and Persian Plateau uplifted, and the Neotethys Ocean retreated, which aggravated aridi cation of midlatitude Asia and promoted considerable expansion of grassland. In the meantime, Europe and North Africa still had relatively closed habitats. Hipparion was derived from the North American Cormohipparion and dispersed into Eurasia. They were highly adapted to open environments, widespread over Eurasia in the general environment of aridness. They also responded sensitively to environmental change, showed excellent adaptation ability for humidifying habitat in Europe and arid one in Asia. The taxon divided rapidly into different groups with different functional morphology to occupy diverse niches in the Old World.

Conclusion
(a) Morphologic comparison indicates Hipparion weihoense, Hipparion chiai and Hipparion prostylum in Iran should be the same species, Hipparion weihoense. This also con rmed the distribution area of H. weihoense is Western China-Central Asia.
(b) The phylogenetic analysis shows Hipparion weihoense, Hipparion primigenium and Hipparion africanum constitute a monophyly derived from North American genus Cormohipparion and represented the Hipparion datum in early late Miocene in Eurasia.
(c) The rst complete record of postcranial of H. weihoense revealed the functional morphology and paleoecology of this species. The locomotive comparison among H. weihoense, H. primigenium and H. africanum implies the ecologic pattern in early late Miocene: Hipparion could highly adapt the environment change and live in diverse niches.

Methods
Terminology and measurements.
The terminology of maxilla and mandible structures follows Sisson 49 and Budras et al. 50 , detailed description on cranial and postcranial material is presented in the Supplementary Information (SI). All measurements follow Eisenmann et al. 51 , and were taken using calipers to the nearest 0.1 mm (S Tables  1-5).

Phylogenetic analyses.
We created a new data matrix, which includes 16 taxa, and 62 characters (S Table 6). The phylogenetic analysis was performed using TNT 1.1 with a traditional research method 52 ; 1,000 replications and the trees-bisection-reconnection branch-swapping algorithm (TBR) were applied in our analyses. All characters are equally weighted and non-additive. Gaps are treated as "missing", and multistate taxa are interpreted as polymorphism. The analyses yielded one parsimonious tree, which is presented in Fig. 1. Other results (character list and the data matrix) are presented in the SI.   Ratio diagrams of metapodials of H. weihoense and other equids. Measurement numbers: 1, maximal length; 3, minimal breadth; 4, depth of the shaft; 5, proximal articular breadth; 6, proximal articular depth; 10, distal maximal supra-articular breadth; 11, distal maximal articular breadth; 12, distal maximal depth of the keel; 13, distal minimal depth of the lateral condyle; 14, distal maximal depth of the medial condyle. The y axis is the logarithm (base 10) of ratios between the measurements of each species and the reference species (Asiatic wild ass Equus hemionus onager, zero line).  Artist's reconstruction of Hipparion weihoense in Dashengou fauna in Linxia Basin, Gansu, China, one individual is standing and another one is feeding on grass. In the background, three hyenas