How Far into Europe Did Pikas (Lagomorpha: Ochotonidae) Go during the Pleistocene? New Evidence from Central Iberia

This paper reports the first find of pika remains in the Iberian Peninsula, at a site in central Spain. A fragmented mandible of Ochotona cf. pusilla was unearthed from Layer 3 (deposited some 63.4±5.5 ka ago as determined by thermoluminescence) of the Buena Pinta Cave. This record establishes new limits for the genus geographic distribution during the Pleistocene, shifting the previous edge of its known range southwest by some 500 km. It also supports the idea that, even though Europe’s alpine mountain ranges represented a barrier that prevented the dispersal into the south to this and other taxa of small mammals from central and eastern Europe, they were crossed or circumvented at the coldest time intervals of the end of the Middle Pleistocene and of the Late Pleistocene. During those periods both the reduction of the forest cover and the emersion of large areas of the continental shelf due to the drop of the sea level probably provided these species a way to surpass this barrier. The pika mandible was found accompanying the remains of other small mammals adapted to cold climates, indicating the presence of steppe environments in central Iberia during the Late Pleistocene.


Introduction
It is well known that the succession of glacial and interglacial periods of the Pleistocene had a decisive impact on the distribution of mammals [1][2][3][4][5]. The pulsating expansion of the tundrasteppe biome well into Europe during the cold phases of the Pleistocene gave some species the opportunity to expand their previous distributions from central Asia into Europe. Such was the case of pikas (genus Ochotona), a group of small lagomorphs belonging to the family Ochotonidae. Pikas comprise 30 extant species mostly distributed in Asia, except for two species which can be found in North America [6]. According to their ecological preferences, two different groups of pikas can be recognized, rock-dwelling and meadow or steppe-dwelling [6].
The fossil record of Ochotona in Europe comprises at least three fossil taxa [7], recorded between the Late Pliocene and Early Pleistocene, as well as Middle Pleistocene to Recent fossil occurrences assigned to the living species Ochotona pusilla [8]. This last species, known as the steppe pika, was the mostly widespread pika during the Pleistocene, in particular during the cold phases of the Late Pleistocene when it extended its range into western Europe, reaching as far as the British Isles and the northern slopes of the Pyrenees [8][9][10][11][12]. Until now, no pika fossils were known from the Iberian Peninsula. This paper reports the first record for a pika in Spain found in the Buena Pinta Cave at Pinilla del Valle (Madrid Region). The find represents the currently known furthest expansion of the genus towards the southwest in its entire evolutionary history-an important record to accompany the few known for the genus in the peninsulas of the Mediterranean.

Geographic and Geological Setting
The Buena Pinta Cave (one of the Calvero de la Higuera sites) is situated in the narrow Lozoya (or Paular) High Valley. Located 55 km north of Madrid, this SW-NE oriented valley occupies some 300 km 2 within the Spanish Central System (the Sierra de Guadarrama Mountains; Fig  1). The valley floor lies at an altitude of 1000-1100 m. The enclosing peaks can reach over 2000 m; the highest is the Pico de Peñalara (2428 m) at the valley's southwestern end. The pronounced altitudinal gradient and the area's abundant rainfall together allow for a wide diversity of habitats, and consequently a rich flora and fauna. Indeed, the valley has 41 mammalian species (leaving out domestic forms and species that have become extinct in historic times through human action) [13], a figure distinctly higher than the mean for continental Spain [14]. Such exceptional ecological characteristics led the head of the Lozoya Valley to be declared a Natural Park in 1990, and recently in 2013 it was included in the Sierra de Guadarrama National Park. Similar high species richness occurred in the Middle and Late Pleistocene in this valley, as evidence by the fossil record preserved in the Calvero de la Higuera sites [15,16].
The Buena Pinta Cave was discovered in 2003 while conducting a survey in the area as part of a project to search for the earliest human settlements in central Iberia [17]. This small cave lies slightly above the bottom of the valley at an altitude of 1105 m. Its geographic coordinates are 40°55' 23.2" N, 3°48' 29.7" W (datum WGS84). It is completely filled with sediments of complex stratigraphy, and connected to the nearby Des-Cubierta Cave system (another site of karstic origin) through an equally sediment-filled gallery.
The Buena Pinta Cave sediments appear as three distinct units [18]. The upper unit (Layer 1), consists of clay and sand sediments up to 1.80 m thick; these are greyish in colour and contain carbonate clasts. The C14 AMS (2 sigma) values for this unit range from 5740-5610 years cal. BP at the base, to 1940-1800 years cal. BP at the top [19]. Underneath lies a set of sediments at least 2 m thick, consisting of a mixture of orange coloured sands, silt and clay with occasional carbonate clasts. Four approximately horizontal stratigraphic layers have been recognised in this unit, numbered 2-5 from top to bottom (Fig 2). Thermoluminescence dating performed on a sediment sample from Layer 3 has returned an age of 63.4±5.5 ka, placing this set of layers in the middle of the Late Pleistocene, within Marine Isotope Stage (MIS) 4 or the beginning of MIS 3 [18]. At the northwestern wall of the site, clast-supported conglomerates and bone breccias outcrop, which represent the oldest filling of the cavity. Particularly, according to its rodent taxa content (e.g., Microtus brecciensis and Microtus vaufreyi; [20]), the age of the northern wall can be dated to the second half of the Middle Pleistocene.
All the layers of this site are rich in bone remains of both large and small vertebrates. The pika remain described in this paper comes from Layer 3, where it formed part of a rich assemblage containing at least 34   Myotis sp., Myotis myotis/oxygnathus, Rhinolophus ferrumequinum, Rhinolophus hipposideros, and Rhinolophus euryale/mehelyi [16,21]. Microtus arvalis dominates the assemblage, followed by Microtus sp. gr. M. duodecimcostatus-M. lusitanicus and Microtus gr. agrestis; indeed, these three vole species account for some 80% of the small mammal fossils collected from this layer. In addition to these rodents, the layer also contains darker-coloured (probably manganese oxide-stained) remains of the fossil taxa M. brecciensis and M. vaufreyi reworked from the breccias and conglomerates of the northern wall (the oldest unit). These species appear in low proportion (<1%) in this layer, but in the lower part of Layer 5 they reach proportions of 10-40% [16].
The number of small mammal species represented in Layer 3 makes it one of the largest assemblages described for a Late Pleistocene site in Iberia. Similar numbers have only been reported for a few sites of similar age in the Cantabrian region (one of the richest ecological regions of the Iberian Peninsula) [22]. The three vole species (M. arvalis, M. sp. gr. M. duodecimcostatus-M. lusitanicus and M. gr. agrestis) dominating the assemblage indicate that, during the formation of Layer 3, the landscape was mainly one of open spaces and grassland. This interpretation is also supported by the presence in the assemblage of other vole species (M. gregalis, M. oeconomus), the marmot, and the extinct hamster A. bursae. However, patches of forest must also have existed near the cave, as shown by the presence of a few My. glareolus and squirrel remains. Futhermore, the available pollen data [19] also indicate a cold, dry environment dominated by grassland.

Material: Description and Identification
The pika remain found in the Buena Pinta Cave, a 1.1 cm-long fragment of a right mandible, was recovered from grid unit L51 in Layer 3, at a depth of 210-220 cm. The fragment (inventory number MAR 2008/29/CBP/L51/3/175) is currently stored at the Museo Arqueológico Regional de la Comunidad de Madrid and consists of the corpus region of the mandible, preserved from the p4 alveolus through to the beginning of the ascending ramus (Fig 3). All the cheek-teeth except for p3 were standing in their corresponding alveoli. The length of the tooth row, measured on the occlusal view from the anterior part of p4 to the posterior part of m3, is 5.66 mm. The height of the mandibular corpus, taken under m2 is 4.98 mm, and its width at this same point is 2.88 mm. No further measurements could be taken on the mandible, due to its fragmentary state, but its maximal width at p4 has been estimated to be larger than 2.89 mm.
The discovered pika mandible is easy to distinguish from those of recent and Late Pleistocene lagomorphs of the Iberian Peninsula (Fig 4). Those of the Leporidae, represented by the rabbit (Oryctolagus cuniculus) and several hare species (Lepus europaeus, Lepus granatensis, Lepus castroviejoi and Lepus timidus) are considerably larger; the mandibles of early juveniles might be similar in size to that of an adult pika, but the presence of deciduous teeth or unworn permanent teeth in the former make their distinction simple. The lower dentition of leporids also differs from that of ochotonids by the m3 possessing two lobes rather than one (Fig 4). Moreover, in leporids, the two lobes of the lower molariform teeth (excluding m3) are connected by a lingual enamel bridge, whereas in ochotonids they are clearly separate.
The Prolagids are other lagomorphs that inhabited Iberia during the Pleistocene, represented by the single genus Prolagus (it should be noted that some authors consider them to belong to the family Ochotonidae (see [23,24], among others)). The latest records for Prolagus in continental Europe are those of the Cueva del Higuerón (Málaga, Spain) and Cova de Gracia (Barcelona, Spain) sites, both dated to the Middle Pleistocene [25]. Though the lower dental pattern in Prolagus is similar to that of Ochotona, three main differences can be seen: the presence of continuous enamel around both lobes of each tooth in Prolagus (see [26]), the absence of m3 in this same genus, and the presence of a third lobe in the m2 of Prolagus (instead of the typical two lobes of other lagomorphs).
The teeth preserved in the mandible from Buena Pinta Cave here described show the typical morphological pattern observed in ochotonids (see [26]), i.e., they have two lobes (the anteroflexid and postflexid lobes, respectively known as the trigonid and talonid lobes in some papers), except for m3, which has only one. In all teeth the enamel is thinner in the anterior part of the lobes, thicker in the posterior part, and absent on the lingual side. The same pattern can be seen in the Ochotona specimen discussed by Chaline [27] and in O. pusilla [28].
Given the missing p3, an element of high diagnostic value in lagomorphs, the specific identification is more difficult to address. O. pusilla is the only pika species known to have inhabited western Europe during the Late Pleistocene [8]. The size of the mandible from Buena Pinta Cave falls within the range of variation observed in O. pusilla, both in recent and fossil material ( Fig 5). Teeth other than the p3 are usually considered of low diagnostic value, and few biometric data are available for comparisons ( Table 1). Measurements of the teeth of O. pusilla from Mamutowa Cave (Poland) [29], one of the few localities from which these data are known, are very similar to those from the mandible of Buena Pinta Cave. Thus, most of the measurements of the teeth of Buena Pinta Cave fall within the range of variation of the polish sample and only two parameters (length of m2 and width of m3) are slightly over their maximum values. However, these small differences may be due to the wide range of variation O. pusilla displayed during the Late Pleistocene in the length of the lower cheek teeth row as pointed out by Fladerer [30]. Compared to Early Pleistocene European fossil pikas (Table 1), the first and second lower molars of the mandible of Buena Pinta Cave are larger and fall out of the range of variation of both O. polonica from Zamkowa Dolna Cave (the type-locality of this species) [31] and O. dehmi from Schernfeld (the type-locality of this species) [32], although in the last case the sample is so small that little significance can be given to differences found when compared with other samples.
Thus, despite the absence of the p3 in the Buena Pinta Cave mandible, it has been ascribed to O. cf. pusilla since O. pusilla is the only pika species known to have inhabited western Europe during the Late Pleistocene and because both the morphology and the size of the fossil mandible and teeth coincide with those of recent and fossil O. pusilla.

Discussion: the Biogeographical Significance of the Presence of a Pika in the Buena Pinta Cave
Evidence exists that several species of pikas of the genus Ochotona were present in Europe since the Pliocene and, during the Pleistocene, usually coinciding with cold episodes [33]. However, records are quite rare until the Middle Pleistocene, though the largest number of records is for the Late Pleistocene. During this time, O. pusilla extended to occupy most of France (including its Atlantic coast) [8,10,34], and even southern and central Britain, although it never reached Ireland [11,35] (Fig 6). Towards the south, it reached the mountain ranges that form the northern limit of the Mediterranean peninsulas (Table 2). It arrived at the Balkan and Dinaric mountains of the Balkan Peninsula during the Late Pleistocene [10,36] and even reached the Pindus Mountains of Greece, where a record from Arnissa [37] is the southernmost for the species in Europe. In the Italian Peninsula it inhabited the northern and southern  The Buena Pinta Cave finding is the first record for a pika in Iberia. As such, it marks a considerable increase in the southwestern European range of the genus Ochotona during the Pleistocene. Buena Pinta Cave is located at the centre of this Peninsula, over 500 km from the untilnow most southwestern record for the species. There is good evidence that pikas (O. pusilla) were present in southern France at least from the middle of the Middle Pleistocene (records are known from Layers P, L and K of the Caune de l'Arago site dating to MIS14-MIS12 [39,40])   would be worthwhile since pikas may have been taken for juvenile rabbits. Rabbits (Oryctolagus cuniculus) are very common in Iberian sites and well represented throughout the Pleistocene, and though the morphological and biometric differences between adult rabbits and pikas are ample, juvenile rabbits overlap in size with adult pikas, thus enabling misidentification. At the Buena Pinta Cave, the intensive effort undertaken in sampling the small vertebrates of the site led to the recovery of more than 250,000 small vertebrate fossils over 11 excavation campaigns. This allowed even the least abundant taxa in the material to be detected. The finding of the single fossil of O. cf. pusilla was the consequence of the careful analysis of the several thousand lagomorph remains (mostly of O. cuniculus) obtained.
The distribution pattern of pikas in southern Europe during the Late Pleistocene suggests the continent's alpine mountain ranges acted as dispersal barriers; only on a few occasions did pikas succeed in crossing or circumventing them to reach the peninsulas of the Mediterranean. Fig 6 shows that most of the southernmost records of Ochotona in the continent during the Late Pleistocene are located on the northern slopes of the alpine mountain ranges, and only some of them (Arnissa in Greece, Eastern Cave of Brina and Sandalja in Croatia, Riparo Tagliente in Italy and Buena Pinta Cave in Spain) are on their southern slopes or more to the south. The role these mountain ranges had as geographic barriers was probably not strictly due to the mountains themselves, but to the forests that developed around them or on their slopes, that acted as effective ecological barriers to species adapted to open steppe landscapes. During the coldest periods of the Late Pleistocene, however, the combined effect of the reduction of forest cover and the emersion of large areas of the continental shelf due to the drop in sea level, provided steppic small mammals such as the steppe pika (the only pika species known to have inhabited western Europe during the Late Pleistocene) a unique opportunity to avoid these habitats and enter into the mediterranean peninsulas. Concerning the Pyrenees, Fig 7 compares the changes inferred for the sea level of the Mediterranean during the Late Pleistocene [50] and in atlantic forests development of in the northern part of the Iberian Peninsula as Cough's Cave 47 [11] Ochotona pusilla Sun Hole 48 [11] Ochotona pusilla Soldier's Hole 49 [11] Ochotona pusilla Aveline's Hole 50 [11] Ochotona pusilla Bridged Pot 51 [11] Ochotona pusilla Badger Hole 52 [11] Ochotona pusilla Helsfell Cave 53 [11] Ochotona pusilla Merlin's Cave 54 [11] Ochotona pusilla Great Doward Cave 55 [11] Ochotona pusilla King Arthur's Cave 56 [11] Ochotona pusilla Symond's Yat East 57 [11] Ochotona pusilla Cavall's Cave 58 [11] Ochotona pusilla Wolf Den 59 [11] Ochotona pusilla Hutton Cave 60 [11] Ochotona pusilla Wolf's Cave 61 [11] Ochotona pusilla Happaway Cave 62 [11] Ochotona pusilla Broken Cavern 64 [11] Ochotona pusilla Cow Cave 65 [11] Ochotona pusilla  [52]. But it is from MIS4 onwards when more of these taxa start to become frequent and varied in Iberian sites. For instance, to this period belong the earliest records of C. nivalis in the Iberian Peninsula (layers X and XI of Cueva de la Carihuela [53]; south sector in Cueva del Camino [15]); or those of M. gregalis and the single record of O. cf. pusilla referred to in this paper. Records become increasingly abundant in MIS3 and MIS2 [46]. It is unclear why the entry of these species was delayed until the Late Pleistocene and did not happen in any of the previous cold episodes of the Middle Pleistocene during which some of them had already reached southern France. Some authors [54] have suggested that the north of the Iberian Peninsula during the Middle Pleistocene (500-200 ka) was characterized by highly stable ecological conditions during which no relevant changes took place in vegetation landscapes, favouring that the Pyrenees acted then as an effective ecological barrier. Layer 3 of the Buena Pinta Cave, where the pika mandible was found, was deposited some 63.4±5.5 ka ago. Thus it does not belong to MIS2, i.e., the period in which mammalian species well adapted to cold climates were most common in the Iberian Peninsula [55]. Rather, it belongs to an earlier moment within MIS4 or the beginning of MIS3. In this layer, it is accompanied by other cold-adapted small mammals such as M. oeconomus, M. gregalis or Ma. marmota. The cave's records for these species are either the southernmost or among the most southerly for Late Pleistocene Europe. The occurrence of a pika together with other coldadapted small mammals suggests the development of steppe landscapes in the interior of the Iberian Peninsula during the Late Pleistocene. This agrees with the interpretations of pollen analyses for cave sites of similar age in central Spain e.g., Cueva de los Torrejones [56] (although the considerably smaller microvertebrate sample size in this site means cold-adapted small mammals are less well represented).

Conclusions
The present pika mandible fragment found in Layer 3 (age 63.4±5.5 ka) of the Buena Pinta Cave increases by 500 km the known southwestern limit of Ochotona in Europe during the Pleistocene. An important diagnostic dental element in ochotonids, p3, is missing from the fossil found at Buena Pinta Cave, but the morphology and size of its remaining features are all in line with those of O. pusilla, the only pika species known to have inhabited Europe during the Late Pleistocene. The fossil is therefore ascribed to O. cf. pusilla. The absence of pika records from northern Iberia may be a consequence of insufficiently thorough sampling of small mammal assemblages, or of misidentification of its infrequent remains for those of rabbits (which are very common). The co-occurrence of the pika with other cold-adapted species in Layer 3 of the Buena Pinta Cave supports the idea that, during the middle part of the Late Pleistocene, steppe landscapes were present in the interior of the Iberian Peninsula.