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Osteological identifi cation criteria for Capra pyrenaica and Capra hircus validated with DNA

The taxonomic identifi cation of Capra hircus and Capra pyrenaica from bone remains is essential in order to be able to infer a prehistoric society’s subsistence strategy. Although osteometry is the criterion that is typically used to distinguish both of these species, Sarrión (1988) also defi ned morphological characteristics for distinguishing them. The objective of this work is to validate these morphological criteria with the genetic identifi cation of ovicaprine bone remains from Palaeolithic and Neolithic levels of the Chaves site (Sierra de Guara, Huesca). The preliminary results indicate that the astragalus and calcaneus are more adequate for identifying Capra pyrenaica and Capra hircus and the discrimination criteria of mandibles and metacarpals are called into question.


INTRODUCTION
The discrimination of Iberian ibex (Capra pyrenaica) and domestic goat (Capra hircus) (Artiodactila; Mammalia) (Wilson & Reeder, 2005) is essential for inferring the subsistence strategy or economy of a population: hunter, livestock breeder or mixed economy. Therefore, an adequate taxonomic classification of goats at an archaeopalaeontological site is necessary in order to gain an understanding of prehistoric societies' way of life.
The differentiation between Iberian ibex (Capra pyrenaica) and domestic goat (Capra hircus) at a peninsular site is based on their difference in size: the Iberian ibex is bigger than the domestic goat. Nevertheless, the osteometric overlap among the big females and the small males must be considered (Altuna, 1978). On few occasions, the morphology of the bone remains, defi ned by Sarrión (1988), is used by zooarchaeologists for their differentiation. Sarrión (1988) observed morphological differences in anatomical elements, such as mandibles, astragali, calcanei, metapodials (basically metacarpals) and phalanges. This study focuses on all of these anatomical elements.
The fi rst objective is to carry out a detailed study of the diagnostic characteristics, which morphologically distinguish the bones of Iberian ibex and domestic goats from the Iberian Peninsula. The next objective is to validate the currently proposed morphological criteria, through genetic samples extracted from the bone remains of goats from Palaeolithic and Neolithic levels of the Chaves site. A complete morphological review, which distinguishes between wild and domestic goats, has been carried out. Some characteristics have been validated and some of them have been called into question.

Material
The material is composed of 23 bone remains (five mandibles, 11 astragali, two calcanei, three metapodials and two phalanxes are included) of Iberian ibex and domestic goats from Palaeolithic and Neolithic levels of the Chaves site, according to zooarchaeological (osteometric and morphological methodology) criteria. Genetic information was recovered from 18 of the 23 bone remains using ancient DNA techniques. The results indicated that 18 bone remains belong to the genus Capra (Capra hircus or Capra pyrenaica). These 18 bone remains are the elements that have been used for the morphological analysis, because both genetics and zooarchaeological criteria that indicated that they belong to wild or domestic goats.
The Chaves cave is located in the Sierra de Guara (Huesca, Aragón). It comprises Upper Palaeolithic and Neolithic levels, clearly separated by a speleothem (Utrilla, 1992). The high frequency of wild goats, which were identifi ed within Magdalenian (level IIa: 12,020 BP and level IIb: 12,800 BP), is noteworthy, and the site served as an area for hunting bucardos (Castaños, 1993). Bucardo is the Spanish common name of Capra pyrenaica pyrenaica: subspecies of Iberian wild goat from the Pyrenees which was recently extinct. In Neolithic levels, the Iberian goat constituted the most abundant wild ungulate followed by the deer (Castaños, 2004). The ovicaprines (Ovis aries and Capra hircus) are the best represented group from the Neolithic with 6,163 identifi ed bone remains and a ratio (Ovis aries/Capra hircus) of 1.34 in level Ia and 1.76 in level Ib (Castaños, 2004).

Zooarchaeological method
In order to conduct palaeontological discrimination (Capra hircus versus Capra pyrenaica), metric data, considering the range of values between wild and domestic form, according to Altuna (1978) and morphological criteria following Sarrión (1988) were used.

Genetic method
All the DNA extractions were performed at the ancient DNA laboratory (Centro Mixto UCM-ISCIII, Madrid and EBC, Uppsala). Between 150 and 200 mg of bone/tooth powder were used to conduct DNA extractions following a silica extraction method (Yang et al., 1998). For every 10 samples, 2 water negative controls were used. Each extract was eluted in a fi nal volume of 60 μl Elution Buffer).
In order to identify the samples, a short fragment of the mitochondrial DNA was amplifi ed. The primers Mbos L1269 and Mbos H1346 (Rollo et al., 2002) were used to amplify a 117 bp fragment of the mitochondrial gene 12S).
The PCR amplifi cation was conducted in 25 μl using 1 X Smart-Taq Hot 10X PCR Buffer (Naxo), 1mM Smart-Taq Hot 25mM MgCl2 solution (Naxo), 0.1 mM dNTPs (Naxo), 0.2 μM of each tagged primer (Biomers), one unit of Smart Taq Hot Polymerase 10U/μl (Naxo) and 2 μl of DNA extract. One PCR water control was prepared for every 10 samples. Thermal cycling conditions were 95ºC for 15 min and 52 cycles of 94ºC for 30 sec, 51-54ºC for 30 sec, 72ºC for 30 sec, and 72ºC for 10 min. Amplicons were purifi ed using ExoStarTM and sequenced at Centro Nacional de Investigaciones Oncológicas (the National Cancer Research Centre) or at Macrogen. The results obtained were compared using BLAST with the available sequences in the GenBank for the different species of mammals.

RESULTS
DNA recovery and genetic identifi cation were possible for 18 of the 23 samples: two of them were identifi ed as Capra hircus, 16 as Capra pyrenaica.
The zooarchaeological identifi cation (Table 2) of the metacarpal CH1 (Neolithic) suggested that it belonged to Capra pyrenaica; nevertheless, the genetic analysis indicated that this element belonged to Capra hircus (Table 6).
Nine out of the 11 astragali that were studied gave positive results from a DNA analysis. Eight of these nine were identified as Iberian ibex or domestic goat by zooarchaeological and genetic criteria: seven were identified as Capra pyrenaica (Tables 3, 6) (CH76, CH77, CH80, CH82, CH83, CH10, CH20) and one as Capra hircus (CH98) ( Table 6). The astragalus CH93 was identifi ed as Ovis aries by osteometric and morphological criteria; nevertheless, the genetic criteria indicate that it belonged to Capra pyrenaica (Tables 3, 6).
The two analysed calcanei (CH18 from the Palaeolithic level and CH8 from the Neolithic) were identified as Capra pyrenaica, based on their large size (Table 4) and morphology. The genetic analysis indicated the same taxonomical identifi cation ( Table 6).
The second phalanx (CH6) was identifi ed as Capra pyrenaica by zooarchaeological (Table 5 for osteometry) and genetic analysis (  Therefore, 18 anatomical cranial and postcranial elements of Capra pyrenaica and Capra hircus have been analysed (* in Table 6): nine astragali, one metacarpal, two calcanei, fi ve mandibles and one second phalanx, which they were identifi ed as either Iberian ibex or as domestic goats, by both zooarchaeological and genetic analysis.
Next, the morphological description of some samples is indicated.
On the left astragalus CH10 (Fig. 1). From plantar aspect, the junction between the proximo-plantar and lateral trochlea is rounded. The surface from medial to lateral side (c-d), if the astragalus were cut transversally, it    would be slightly concave (a). Nevertheless, this surface is fl at in domestic goats. The cavity (b) located among plantar and proximal trochlea (i.e., the contact point between it and the coracoid process of the calcaneus) is deep and sharpened. From a lateral aspect, the distal articular surface (e) is elongated and forms a kidney shape. All morphological criteria (according to Sarrión, 1988) and genetic analysis indicate that this astragalus belongs to Capra pyrenaica.
These diagnostic criteria for Capra pyrenaica have been observed on six additional astragali (CH20, CH76, CH77, CH80, CH82, CH83), which are consistent with the taxonomic identifi cation achieved through DNA analysis. The zooarchaeological and genetic criteria indicate that one of the astragalus (CH98) belongs to Capra hircus. Just one of the astragalus was identifi ed as Ovis aries with zoorchaeological criteria, and the genetic results indicate that it belongs to Capra pyrenaica (CH93 in Table 6). Thus, seven of nine astragali (77.7 %) were identifi ed as Capra pyrenaica through the use of diagnostic morphological and genetic criteria. Nevertheless, just one of the nine samples (11.11 %) that belongs to Capra hircus as identifi ed using morphological criteria (Zeder & Lapham, 2010), was identifi ed as Capra hircus through genetic criteria. One of the nine astragali was identifi ed as Ovis aries by morphological criteria and as Capra pyrenaica with DNA identifi cation.
In the case of the calcaneus CH8 (Fig. 2), the junction between the coracoid process and the articular facet for the os malleolare is rounded (a) (the negative of the astragalus morphology) and the side of the coracoid is curved (c). On the cavity's articular surface for the trochlea of the astragalus, the surface is joined in proximal parts (b). Both morphological and genetic criteria indicate the calcaneus CH8 belongs to Capra pyrenaica.
The calcaneus CH18 presents diagnostic morphological characteristics of Capra pyrenaica so that, through the use of palaeontological criteria like genetics, it concurs with the taxonomic identifi cation.
The mandible CH7 (Fig. 3) includes the almost complete cheektooth row (P 3 -M 3 ), missing P 2 . The lack of  Sarrión (1988). This missing second premolar is due to lack of entity in the roots, which causes its loss from the alveoli (Sarrión, 1988). The index that indicates this characteristic has been calculated: Index (CH7) = (LP 2 *100)/LP 3 = (3.5*100)/5.5 = 63.63 where LP: alveolar length of premolar; P2: second premolar; P3: third premolar. This result is closer to the average value from the Capra hircus index than to the Capra pyrenaica index average value, according to Sarrión (1988).
The mandibles CH40 and CH41 have no P 2 , and the alveoli are reabsorbed, so the metric data of the alveoli length have not been taken and the index could not be calculated. The genetic analysis indicates that these mandibles belong to Capra pyrenaica.
The P 2 is missing in mandible CH25, which is a typical characteristic of Capra pyrenaica, and the index = (2.8*100)/4.5 = 62.22 indicates a value closer to Capra belong to C. pyrenaica. The missing P 2 is observed in all mandibles. The index could be calculated in three of the fi ve mandibles. The three mandibles have an index closer to C. hircus than C. pyrenaica. The DNA analysis conducted on the mandibles indicates that they belonged to Capra pyrenaica.
The morphological criteria of the metacarpal CH1 (Fig. 4) have been described in order to check the taxonomic identification. In dorsal aspect, the distal third of the diaphysis is fl at (b) without epiphysis protuberances and links to distal articular surface without (or with slight) cavities above (a). The cavities, located above the distal articular surfaces, are slightly marked, opposite of the Capra hircus metacarpal, which has very marked cavities. In lateral or medial aspect, if a line was extended from the anterior surface of the diaphysis to the distal articular surface, the line should cut the distal articular surface (c). All morphological characteristics indicate that this metacarpal belongs to Capra pyrenaica according to Sarrión (1988). However, the DNA analysis showed haplotypes of Capra hircus.   hircus than the index of Capra pyrenaica. The genetic analysis indicates that this mandible belongs to Capra pyrenaica.
The P 2 is missing in the mandible CH27 and the index = (3.9*100)/6 = 65 indicates that the value is closer to Capra hircus than the index of Capra pyrenaica. The genetic analysis indicates that this mandible belongs to Capra pyrenaica.
The size of the mandibles and the chronology of the level (Palaeolithic) indicate that the fi ve mandibles The morphological and genetic criteria are in agreement with regard to the second phalanx (CH6) in that it belongs to Capra pyrenaica.

DISCUSSION
The morphological and genetic criteria gave the same taxonomical identifi cation of goat (wild or domestic) in 91.67 % (11 of 12 anatomical elements) of the analysed samples of astragalus, calcaneus and second phalanx. Therefore, these anatomical elements are considered appropriate for their identifi cation.
A missing P 2 is observed in 100 % of the analysed mandibles and thus, we consider it to be a valid criterion for taxonomic identifi cation of Capra pyrenaica (Table 7). Nevertheless, the calculation of the dental index does not match the expected rate in any of the analysed mandibles. Therefore, this criterion is not valid for distinguishing C. hircus and C. pyrenaica. In the case of the metacarpal, the criteria according to Sarrión (1988) for identifying C. pyrenaica does not concur with genetic results, so these must be used with caution.

CONCLUSIONS
The morphological criteria of the anatomical elements described by Sarrión (1988) for distinguishing between Capra pyrenaica and Capra hircus have been assessed using DNA analysis. The zooarchaeological analysis is consistent with the genetic analysis on astragalus, calcaneus and second phalanges from Palaeolithic and Neolithic levels at the Chaves site. Some morphological criteria of mandibles and metacarpals are not consistent with the genetic analysis for their identifi cation, so these criteria must be used with caution.
Despite the fact that this analysis is preliminary and more samples are necessary in order to assess the morphological criteria, the criteria considered a valid link to osteometry are suffi cient to identify Capra pyrenaica without DNA analysis.