Identification of Archaeological Goose Remains Using Ancient DNA Analysis

International Journal of Osteoarchaeology Int. J. Osteoarchaeol. 8: 280–287 (1998)

The Molecular Palaeoecology of Geese: Identification of Archaeological Goose Remains using Ancient DNA Analysis

IAN BARNES*, KEITH M. DOBNEY AND J. PETER W. YOUNG Department of Biology, University of York, PO Box 373, York YO1 5YW, UK

ABSTRACT The remains of six species of geese are commonly recovered from archaeological sites in Britain dating from the Saxon and later periods. However, identification of this material to species level is hampered by a lack of morphological variation and a large overlap in size. To address this issue we obtained DNA sequence data for a section of the mitochondrial cytochrome b gene from modern samples of each species, and successfully identified several DNA markers for Branta species. No markers were found within the cytochrome b gene for the genus Anser. Ancient DNA techniques were then used to recover DNA from goose bones excavated from two archaeological sites. The DNA sequences enabled identification of Barnacle goose (Branta leucopsis) from one site and confirmed the presence of Anser species at another. © 1998 John Wiley & Sons, Ltd.

Key words: ancient DNA ; anser; Branta; geese; mitochondrial DNA ; polymerase chain reaction

Introduction certain times of the year, particularly during the late summer moult which renders them The remains of wild and domestic geese are flightless. among the most frequently recovered bird re- Domestic geese (Anser anser domesticus) are mains from archaeological sites. Six species are derived from Greylags [3]. They are believed to likely to be present in British archaeological have been kept in Britain from the neolithic assemblages on the basis of their present distri- period (BC 3500–2100), although they became bution [1]: Greylag (Anser anser), White Fronted of commercial importance only during the (Anser albifrons), Pink Footed (Anser Saxon and medieval periods (AD 400–1400). At brachyrhynchus), Bean (Anser fabalis), Barnacle this time, geese were exploited for eggs, fat, (Branta leucopsis) and Brent (Branta bernicla). feather quills and down as well as for meat. Other species such as the Lesser White Fronted Work on the archaeozoology of geese in (Anser erythropus), Red Breasted (Branta ruficolis) Britain has been hampered by the difficulties of and Snow (Anser/Chen caerulescens) may have been species identification from skeletal material. Al- occasional visitors. Although today they gener- though methods for differentiating between ally roost near and feed upon grasses, cereals wild and domestic geese have been employed and root crops, in the past they would have on archaeological material [4,5], there is little exploited a more diverse range of habitats in- morphological variation in the post-cranial cluding mudflats, marshes and bogs [2]. For skeleton of the six species of wild geese in- humans living near coastal or wetland areas they volved in this study [6]. While some degree of would have been a useful food resource at metric variation does occur, there is generally a * Correspondence to: Department of Bacteriology, Windeyer In- large degree of overlap between species. Bacher stitute, University College London Medical School, 46 Cleveland Street, London W1P 6DB. Tel.: +44 1904 432611; fax: +44 [7] has studied the skeletal anatomy of eight 1904 432800. species of commonly occurring European goose

CCC 1047–482X/98/040280–08$17.50 Received 28 March 1998 © 1998 John Wiley & Sons, Ltd. Accepted 24 April 1998 Molecular Palaeoecology of Geese 281 species, and has demonstrated that two morpho- Increasingly since the mid-1980s, DNA-based logical criteria might be used to distinguish techniques have been applied to organisms between the genera Anser and Branta: which are traditionally difficult to identify. The development of the polymerase chain reaction (i) Bilateral compression of the spine sterni (PCR) has allowed the amplification of small externa in 80% of Branta.InAnser it is quantities of DNA, which can then be charac- generally pointed, although may resemble terised by sequencing or restriction fragment Branta in rare cases. length polymorphism analysis (RFLP). Species (ii) The furcula of Anser possess a pneumatic level identification has generally been con- foramen. Those of Branta do not. ducted on lower organisms which are difficult to Due to the fragmentary nature of the material identify by morphology alone, for example bac- excavated from archaeological sites, neither of teria and fungi. However, birds have been the these criteria are particularly useful, and even subject of a number of studies on population when they can be applied they can only identify variation, employing similar techniques [14– material to generic level. Similarly with metric 17]. criteria, fragmentation often renders measure- The extraction and amplification of DNA ment impossible. When measurements can be from archaeological material is a relatively new made, material tends to fall into one of two size field. The majority of work has been conducted groups: a large group containing Pink Footed, on human remains, to answer questions about Domestic, Greylag and Bean geese and a smaller population movements [18–22], status and kin- ship [23] and gender [24,25]. There is less group representing White Fronted and Barnacle published work on archaeozoological material, geese. Some even smaller bones may represent which has addressed questions about the phy- the remains of Brent geese. To further compli- logeny of extinct species and identification of cate matters, the small size of most modern bone and other materials [26–30]. reference collections means that the full size Ancient DNA research can be most clearly range of modern individuals may not be repre- characterised by its technical difficulties, rather sented. In addition, the size range of modern than any broader methodological framework individuals may not represent the full range of [31–33]. These include the degraded nature of sizes possible for that species, due to the alter- the endogenous target DNA within the sample, ations in habitat and diet outlined above. the presence of humic acids and other soil In archaeozoological reports this problem is components which will inhibit the PCR, and dealt with in a variety of ways. Most workers do perhaps most problematically, the presence of not apply the morphological criteria identified modern contaminant DNA. by Bacher [7]. While some attempt to make In this paper we describe the development species identifications based on metric variation and application of an ancient DNA based proto- [8–10], others simply identify the largest indi- col for the identification to species level of viduals as domestic and all others as wild [11] or skeletal remains of geese from archaeological identify all remains as those of domestic birds sites. In order to achieve this, it was necessary [12,13]. This situation is unsatisfactory for two to identify a short region of the mitochondrial reasons. First, because no uniform criteria for genome of the genera Anser and Branta which is species identification exists, the intersite com- sufficiently variable to allow species level identi- parison of wildfowl exploitation in a given pe- fication. Primers were designed which amplify riod is extremely difficult. Second, due to the all species, and these were used to amplify DNA vagaries of metric identification outlined above extracted from archaeological material. The re- much identification is likely to be incorrect. A sulting data can be used to make inferences standardised, accurate identification protocol about wildfowl exploitation in the past. In addi- would significantly improve our understanding tion, the project was concerned with broader of past distributions, wildfowling strategies and questions of ancient DNA preservation from a domestication. variety of burial environments, and the large

© 1998 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 8: 280–287 (1998) 282 I. Barnes et al. scale application of ancient DNA techniques to again, dryed and resuspended in 100, mLofTE archaeozoological projects. buffer (10 mM Tris pH 8.0, 1 mM EDTA). DNA was extracted from feathers using a method derived from that described by Ellegren [35]. Using a clean, flame sterilised scalpel Materials and methods blade, 5–10 mm of the basal section of the rachis (the calamus) were removed and finely Origin of samples chopped. The feather fragments were added to 200 mL of 5% Chelex-100 Resin (Bio-Rad Labo- Ethanol preserved muscle tissue samples of indi- ratories) in sterile distilled water. This mixture viduals from four species (greylag (western was vortexed and incubated at 54°C for 2 h race), brent (dark bellied race), bean (Russian with 5 mLofa10mg/mL solution of proteinase race), and pink footed) were supplied by Martin K (Sigma). The sample was then vortexed, Brown of the Wetland and Wildfowl Trust, boiled for 8 min, spun at 13000 rpm for 2 min Slimbridge. Fresh primary flight feathers were and 150 mL of supernatant removed. DNA was obtained from greylag, barnacle and pink footed extracted from bone using the method of Boom geese from York. Three Greylag goose pha- et al. [36], following the modifications given by langes from the Environmental Archaeology Ho¨ ss and Pa¨a¨bo [37] and Ho¨ ss [38]. This Unit, University of York reference collection method proved to be effective and rapid, there- were also tested. This material was between 3–5 fore making it suitable for a project such as this years old and had been prepared either by where a large throughput of samples is required. soaking in Neutrase (a commercial proteolytic All bone extraction took place in a laboratory treatment) and Lipolase (a lipolytic enzyme; where no PCR work is conducted, to minimise both Novo Nordisk) or by boiling and bleach- the possibility of contamination. All extractions ing with H2O2. were repeated from a different part of the same Archaeological goose bone material was ob- bone. DNA extraction was conducted as fol- tained from a post medieval (17–19th century lows: The outer layer of the bone fragment was AD) kitchen waste deposit from Vicars Court, removed using a clean, flame sterilised scalpel Lincoln [34] (n=3) and a Romano–British and discarded. The remaining bone was crushed (1st century AD) site at Ulrome, East Yorkshire in a percussion mortar (cleaned by soaking in (n=1). 1% sodium hypochlorite for 15 min) and 0.1– 0.5 g of the resulting powder was added to 1 DNA Extraction mL of bone extraction buffer (10 M guanidinium isothiocyanate, 0.1 M Tris–HCl pH 7.4, DNA was extracted from tissue samples as fol- 0.02 M EDTA pH 8.0, 1.3% Triton X-100) and lows. Each sample was finely minced with a incubated at 60°C for 60 min. After centrifuga- m clean, flame sterilised scalpel blade and added to tion for 5 min at 5000 rpm, 500 Lofthe 500 mL of Hirt Buffer (10 mM Tris pH 8, 10 supernatant was recovered and added to 500 mL mM EDTA pH 8.0, 0.5% SDS) and 15 mLof of extraction buffer and 40 mL of coarse fraction a10mg/mL solution of proteinase K (Sig- of silica suspension. This mixture was incubated ma). The sample was incubated for 4–6 h at for 10 min at 23°C with gentle agitation. The 57°C and then extracted twice with phe- silica was pelleted by centrifugation at 2000 rpm nol:chloroform:isoamyl alcohol (25:24:1) and for 1 min and the supernatant discarded. The once with chloroform:isoamyl alcohol (24:1). silica pellet was washed twice with a bone wash The DNA in this sample was then precipitated buffer consisting of 10 M guanidinium isothio- with 50 mL of 3 M sodium acetate and 500 mL cyanate and 0.1 M Tris–HCl pH 7.4 and once of 80% isopropanol at 4°C by centrifugation at with 70% ethanol. After drying the pellet at 15000 rpm for 20 min at 4°C. The pellet was 50°C for 10 min, DNA was eluted at 65°C with washed once with 500, mL of 70% ethanol, spun 60 mL of TE buffer.

PCR amplification TE buffer. The beads were resuspended in 5 mL of 98.2 mM NaOH and incubated at 23°C for 5 Avian specific PCR primers were designed on min. The beads were placed in the magnetic the basis of sequence data present in the EMBL particle concentrator, and the supernatant re- and GENBANK databases. BSP1 (ATACACTA- moved to be sequenced separately. The beads CACCGCAGACAC) and BSP2 (AAGAAAC- were then washed with 30 mL of 98.2 mM CTGAACACAGGA) amplify a short (192 bp) NaOH, 30 mL of binding buffer, 30 mLofTE section of the cytochrome b gene, homologous buffer and finally resuspended in 5 mL of SDW. to bases 15055 to 15243 of the human whole The non-biotinylated strands removed during mitochondrial genome [39] Under the condi- the alkali denaturation step were neutralised to tions described below, these primers have pH 7 using a volume of 50 mM HCl previously proved to be avian specific. A biotinylated ver- calculated by pH titration. The sequencing sion of BSP1 was manufactured for use in direct primer was annealed to the template DNA in a sequencing. mixture containing 4 mL of bead DNA, 3.3 mL PCR amplifications were done in 50 mlof of SDW, 4.7 mL of a modified annealing buffer Promega amplification buffer (1×concentra- (120 mM Tris–HCl pH 7.5, 75 mM MgCl2,50 tion) with 0.5 U of Taq polymerase (Promega), mM NaCl, 30% DMSO) and 2 mL of non-bi- m 2.5 mM of MgCl2, 1 mM of each dNTP, 0.2 otinylated primer (40 pmol/ L). This mixture mM of each primer and 10–100 ng of template was heated to 60°C for 10 min and allowed to DNA. Approximately 10 mL of mineral oil over- cool to room temperature over a period of 15 layed the reaction mixture, which was subjected min. Sequencing reactions were carried out us- to an initial 2 min period of denaturation at ing the T7 sequencing kit (Pharmacia Biotech) 93°C followed by 40 cycles of annealing at and [h-35S]-dATP (Amersham) following the 50°C for 1 min, extension at 72°C for 2 min manufacturer’s instructions. After the extension and denaturing at 93°C for 45 s. After the last reaction was complete, the supernatant was re- cycle, a 7 min extension at 72°C was added. moved and 5 mL of stop solution were added. Following amplification, 5 mL of product was Products of all sequencing reactions were elec- electrophoresed in a 2% agarose gel (Sigma) trophoresed on 5% Sequagel polyacrylamide and visualised with ethidium bromide staining. gels (National Diagnostics). Sequence data was aligned with published sequences using the CLUSTALW program [41]. DNA sequencing

Direct sequencing using Dynabeads M-280 Results and analysis streptavidin beads (Dynal) was carried out following the manufacturers instructions with modifications suggested by Thomas et al. [40]. A Sequencing of modern specimens bulk suspension of 50 mL of Dynabeads was washed twice with 100 mL of binding buffer (10 The results of the sequence analysis of modern mM Tris–HCl pH 7.5, 1 mM EDTA, 2.0 M specimens are shown in Figure 1. This analysis NaCl). The beads were then resuspended in 100 has identified a large degree of variation in the mL of fresh binding buffer. A 30 mL aliquot of amount of sequence difference between differ- bead suspension was added to an equal volume ent species. While species within the genus of PCR product (the amplification having been Branta are readily differentiated using this sec- conducted with biotinylated primers) and incu- tion of cytochrome b, the Anser species tested all bated at 23°C for 15 min with gentle agitation. share the same DNA sequence. The Branta spe- The beads were placed in the Dynal magnetic cies (Brent, Barnacle, Canada) are differentiated particle concentrator and the supernatant re- from each other by 11–13 base pair differences, moved. The beads were then washed once with with the exception of the Barnacle/Canada se- 30 mL of binding buffer and once with 30 mlof quences which differ by only two bases. Some

© 1998 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 8: 280–287 (1998) 284 I. Barnes et al. subspecies of Branta can be differentiated by a that of the divergence of Canada goose subspe- similar amount (e.g. Brent/Pacific Black Brant, cies. More extensive phylogenetic analysis using Taverners Canada/other Canada Goose subspe- this data is not possible, due to the limited cies). The lack of variation between Anser spe- amount of variation detected. cies suggests a very recent divergence of these Comparison of this section of cytochrome b species, potentially within a similar time scale to with other sections of the gene which have have

Figure 1. Alignment of partial cytochrome b sequence for selected Goose and Swan species. Numbering refers to the complete human mitochondrial genome [39], dots represent identity with the Anser spp. sequence, N indicates an unresolved base. Broken lines indicate the position of primers BSP-1 and BSP-2 and underscoring indicates the position of the Aci I restriction enzyme recognition site in Barnacle geese. Anser spp.=Bean (A. fabalis), Greylag (A. anser), Pinkfoot (A. brachyrynchus), White Front (A. albifrons) sequenced by the authors and Domestic Goose (A. anser) from EMBL database entry L07522. Emperor goose=Chen canagica [43]. Black brant=Branta bernicla nigricans [43]. Brent=Branta bernicla bernicla sequenced by the authors. Barnacle=Branta leucopsis sequenced by the authors. L. Canada=Large bodied Canada goose species (Brant canadensis occidentalis, B. c. parvipes, B. c. moffitti, B. c. fulva) [43] and sequence obtained by the authors. Taverners=Branta canadensis taverneri [43]. Black Swan=Cygnus atratus [44].

© 1998 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 8: 280–287 (1998) Molecular Palaeoecology of Geese 285 been sequenced for Branta and Chen species indi- amplification protocols can be successfully ap- cate that this region is typical in its level of plied to archaeological bone of recent date. The variability. This suggests that further sequencing identification of this material as Anser spp. has of other parts of cytochrome b will not yield been confirmed by DNA sequencing. any useful variation among the Anser species. In While at present the identification of separate order to fully address the original question the Anser species is not feasible, the protocol can search for species specific genetic markers will still be useful in selected situations. A single need to be extended to other parts of the goose humerus recovered from the Iron Age site mitochondrial genome which are known to be at Ulrome, North Yorkshire had been tenta- more variable. However, cytochrome b does tively identified as either barnacle or brent, on allow the accurate identification of Branta spe- the basis of metric criteria. As sequence varia- cies and and separation of Anser and Branta and tion has been identified between these two we have tested the viability of using it with species, this material seemed suitable for use in both archival and archaeological material. a trial analysis in order to establish the effi- ciency of the procedure. Two separate extractions were performed, and these were subjected Extraction and amplification from archival and to PCR amplification. Both extractions yielded archaeological material positive PCR products of the expected size and in both cases negative controls were blank. Successful DNA amplifications have been made These products were digested with the restric- from both archival and archaeological bone. tion enzyme Aci I. On the basis of the sequence Samples EAU 604 and 607, prepared using Neu- data (Figure 1), this enzyme is expected to trase and Lipolase have consistently yielded cleave the amplified DNA from Barnacle but not strong amplification products. However, sample from Brent goose. The presence of this site in EAU 321, prepared by boiling and bleaching both of the PCR products allowed a preliminary with H2O2, has failed to give any amplification identification of this specimen as Barnacle products. As both samples are of similar age, goose. Subsequent DNA sequencing of one of this result suggests that while enzymatic pre- the PCR products has confirmed that identifica- treatment of bone still allows recovery of tem- tion. This is, to our knowledge, the first defini- plate from the sample, even brief treatment with tive identification of this species from the H2O2 will seriously reduce the quantity of tem- archaeological record. plate suitable for amplification. If there is any intention to use bone reference material as a source of DNA, then extensive treatments with Conclusion harsh compounds (particularly bleaching agents) need to be avoided. This point needs to be Species identification is one of the most communicated to the archaeozoological com- straightforward applications of ancient DNA munity who use a variety of treatments for technology, in terms of the interpretation of preparing, and in particular degreasing, refer- results. The potential for applying these tech- ence material [42]. niques to archaeological bird remains is particu- Work on material from archaeological sites larly good, as the species identification of these has principally concentrated on a set of well remains is often problematic [45]. In this paper preserved bird remains from a seventeenth cen- we have developed and applied a system for the tury kitchen waste deposit at Vicars Court, identification of archaeological Anser and Branta Lincoln (VC93/178). The geese bones recov- remains. Our results demonstrate that the iden- ered have been identified as domestic, on the tification procedure, although not fully estab- basis of their large size and date. A series of lished, has the potential to address a range of amplification attempts have given products of questions. Further sequence analysis of modern the expected size from two of the three samples individuals should identify a more variable re- tested, demonstrating that the extraction and gion to amplify from the archaeological mate-

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