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JOUJ1WI • I Arch aeoofl oqica SCIENCE J ELSEVIER Journal of Archaeological Science 32 (2005) 567-578 http://www.elsevier.comllocate/jas DNA analysis of archaeological rabbit remains from the American Southwest Dongya Y. Yang'v":", Joshua R. Woiderski'', Jonathan C. Driverb •Ancient DNA Laboratory, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada, V5A 1S6 "Depanment of Archaeology, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia. Canada, V5A IS6 Received 28 October 2004; received in revised form 21 November 2004 Abstract Ancient DNA analysis was carried out on 20 archaeological rabbit remains from an early Pueblo II period site in Colorado (circa 1000 A.D.) to explore the possibility of obtaining accurate rabbit genus and species identifications. The presence of abundant rabbit remains at archaeological sites in the American Southwest indicates the importance of rabbit species in the subsistence economy and ritual activities of early aboriginal populations. The study of these remains is hindered by the difficulty of accurate identification due to the fragmentary nature of the bones and the lack of genus- and species-specific morphological features. A short cytochrome b gene fragment was amplified and sequenced to produce a genetic profile for each bone sample. At the genus level, the DNA identifications were consistent with those based on the analysis of mandible morphology for the majority of specimens. When compared to species-specific reference DNA sequences, Lepus americanus and Lepus californicus samples were easily identified. Identification of an unexpected L. americanus (snowshoe hare) from the remains provided new information concerning hunting ranges or exchange between groups in the region. Sylvilagus nuttallii and Sylvilagus audubonii, however, could not be confidently differentiated at this point due to the difficulty in obtaining accurate species-specific reference sequences. The inability to obtain such reference sequences can be a serious problem for DNA species identification of non-domestic animals that lack population-level genetic data and have few sequences available in GenBank. The lack of the DNA data increases the possibility that inappropriate reference sequences could be applied, resulting in false species identification even when authentic DNA is retrieved and amplified from ancient remains. © 2004 Elsevier Ltd. All rights reserved. Keywords: PCR; Ancient DNA; Species identification; Reference sequences; Rabbits; Lagomorph; mtDNA; American Southwest 1. Introduction Throughout the American Southwest various rabbit species are found in most archaeological sites and often dominate the assemblages. Compilations of data from arid low-elevation areas in Arizona [38,39] and New Mexico [36] or moister upland environments in southern • Corresponding author. Tel.: + I 604 291 4651; fax: +) 604 291 5666. E-mail address:donyang@sfu.ca (D.Y. Yang). 0305-4403/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi: 10.1016/j.jas.2004.II.OIO Utah and Colorado [9] consistently document rabbits as a significant component of the ancestral Puebloan diet. The study of rabbit remains excavated from archae­ ological sites has provided important insights into resource exploitation and habitat modification by early aboriginal populations in the region. However, detailed studies are hindered by the inability to accurately identify these remains using morphological criteria, due to intrinsic problems associated with the lack of genus- and species-specific morphological features and the fragmentary nature of archaeological specimens. 568 D.Y. Yang et al. / Journal of Archaeological Science 32 (2005) 567-578 Most rabbit remains from the American Southwest can only be identified to the genus level based on the size of bone elements. Rabbits have distinctive morpholog­ ical attributes that make separation from other small mammals relatively easy. However, the two genera, Lepus (jackrabbits) and Sylvilagus (cottontails) are morphologically quite similar, and are usually separated on the basis of size. It is assumed that larger specimens are identifiable as Lepus, and smaller specimens as Sylvilagus. Very immature specimens or' highly frag­ mented specimens are identified only as "rabbits". With the advent of ancient DNA techniques [10,33,37], a DNA approach has become an important alternative for accurate identification of ancient remains [3,5,30,44]. In this paper, we report a study using ancient DNA techniques to evaluate the size-based genus identification method that is currently used by zooarch­ aeologists to identify rabbit remains, and to explore the possibility of DNA identification. The results from 20 bone samples demonstrated the reasonable utility of genus identification based on size and the importance of DNA species identification in the study of archaeolog­ ical rabbit remains. This research also showed that for some species the lack of reliable species-specific refer­ ence sequences could be a serious problem for ancient DNA species identifications. 2. Materials and methods 2.1. Archaeological rabbit bones The archaeological specimens used in this study were excavated from an early Pueblo II community in southwest Colorado, at the northern margin of the Southwest culture area. Approximately 10,000 rabbit specimens were identified from a pit structure known as "Kiva H" at the Stix and Leaves Pueblo (5MTlI555) located west of the town of Cortez in Montezuma County, Colorado [4]. The precise location of the site is not given for protection of the site. Kiva H is a rectangular, subterranean feature, lacking the masonry lining that is typical of later kivas. The fill of the kiva consisted of largeamounts of ancient refuse. Rabbit bones" were initially identified to genus, mainly using size to distinguish Lepus from Sylvilagus. The identifications recorded in Fig. I reflect the original identifications made during the first stage of the analysis. As the analysis progressed, we became aware that size criteria were being applied somewhat sub­ jectively. Therefore, once the analysis was complete, we tested our initial identifications with metric data [29] in order to further refine initial genus classifications. Analyses of mature long bones showed very clear differentiation into two size groups. However, measure­ ments of mandibles (Fig. I), revealed two problems. 21.00 E lepus sp. .. 19.00 ' . i'lLA:··+ ~~. =~ u~tlltt. ".r.. ..+ ~: .'. I Group 2 E ;; 17.00 ••A 'Cl ..... ",,' .. '0 .. c: . .....I 13.00 1 SyMtagus sp. ~