Journal of Archaeological Science 35 (2008) 27422750

Contents lists available at ScienceDirect

Journal of Archaeological Science

journal homepage: http://www.elsevier.com/locate/jas

Evaluating protein residues on Gainey phase Paleoindian stone tools

Mark F. Seeman a, *, Nils E. Nilsson b, Garry L. Summers b, Larry L. Morris b, Paul J. Barans b, Elaine Dowd b, Margaret E. Newman c, d
a

Department of Anthropology, Kent State University, Kent, OH 44242, USA Nobles Pond Excavation Program, Department of Anthropology, Kent State University, Kent, OH 44242, USA c Department of Archaeology, The University of Calgary, 2500 University Drive, N.W., Calgary, Alberta T2N 1N4, Canada d Department of Biological Sciences, The University of Calgary, 2500 University Drive, N.W., Calgary, Alberta T2N 1N4, Canada
b

a r t i c l e i n f o
Article history: Received 3 December 2006 Received in revised form 30 April 2008 Accepted 1 May 2008 Keywords: Cross-over immunoelectrophoresis Blood protein Gainey phase Nobles Pond Paleoindian

a b s t r a c t
Blood protein analysis provides a method for acquiring and interpreting archaeological data bearing on humananimal relationships. The present study makes use of cross-over immunoelectrophoresis (CIEP) and a large sample of stone tools (N 130) from an early Paleoindian site pertaining to the Gainey phase (ca. 11,200 BP) of the Midwestern USA. Results are used to interpret toolkit organization and site structure, and indicate that hafted tools with longer use-life potentialities are more likely to be associated with preserved residues than are tools of a more ad hoc or situational character. Protein residues derived from cervids are the largest category of identied samples, a result consistent with an interpretation that these animals were important rst-line resources of Gainey phase populations. The identication of caribou (Rangifer sp.) is notable. Interpretations of subsistence importance, however, must be tempered by an appreciation of the possible confounding effects of tool manufacture and maintenance in early Paleoindian contexts. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Settlement and technological data indicate that the Late Pleistocene societies of Midcontinental North America were oriented towards terrestrial hunting rather than gathering, shing, or alternative subsistence strategies. To date, more specic inferences on this score have been strongly and negatively affected by poor preservation. Most occupations of the period provide no preserved bone, and in those few cases where bone is present, sample sizes are too small to provide realistic estimates of regional subsistence and/or the cultural relationship between human action and animal bone accumulation (Cannon and Meltzer, 2004). Assuming the basic character of Paleoindian sites in the Midwest will not change in the future, alternative methods for developing inferences on regional foraging practices must be developed. The analysis of protein residues is one such method. Protein residue analysis has been used in medical, pharmaceutical, industrial, biological, and forensic contexts for nearly a century. It has been incorporated into archaeological investigations for over 25 years, mainly in conjunction with the analysis of stone tools (Loy, 1983). Proteins can provide species-specic signatures; they accumulate on stone tools more quickly than detectable polishes

* Corresponding author. Tel.: 1 330 672 2705; fax: 1 330 672 2999. E-mail address: mseeman@kent.edu (M.F. Seeman). 0305-4403/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2008.05.001

and are likely to identify a broader range of species than is often present in the preserved faunal inventory itself (Shanks et al., 2001: 968; Shanks et al., 2004: 664). Protein residues have been shown to adhere to stone tools for prolonged periods, both experimentally ` , 2002; Loy and Dixon, 1998; and in archaeological contexts (Hortola Shanks et al., 2004). Several different diagnostic tests have been developed and utilized in archaeological analyses, notably: crossover immunoelectrophoresis (CIEP); enzyme-linked immunosorbant assay (ELISA); radioimmune assay (RIA), and Western blot. Each has its advantages and disadvantages (Downs and Lowenstein, 1995; Kooyman et al., 1992; Newman et al., 1996: 679681; Shanks et al., 2004: 664; Barnard et al., 2007a). The Downs and Lowenstein, 1995 study is perhaps the most widely cited comparative assessment of these methods, despite the small sample sizes for both their controls (N 3) and archaeological cases (N 4). The prospect of false positives due to cross-over reactions, poor preservation, biased sampling, and a lack of antisera to detect proteins from archaeologically important species are all important caveats (Lambert et al., 2000: 401402.; Loy and Dixon, 1998: 26; Tuross et al., 1996: 289290; Shanks et al., 1999: 11831184, see also Petraglia et al., 1996: 128). As with any analytical procedure, protein residue analysis must be approached critically. An additional consideration in assessing the viability of protein testing as it pertains to archaeological materials is sample size; the majority of protein residue studies involve a small number of tested tools per site, thus negatively affecting the ability to comment on

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750

2743

general humananimal relationships. Large samples are useful in identifying patterning in any data set, and protein residue is certainly no exception. The present study focuses specically on testing 130 tools for preserved protein residues. These tools represent several different classes of hafted and unhafted tools and were recovered from a single Gainey phase Paleoindian site in northeastern Ohio, Nobles Pond (33ST357).

for prey selection. The present study makes use of a large sample of tools with an emphasis on toolkit organization and site structure. 2. Methods One hundred and thirty tools from the Nobles Pond assemblage were selected for protein residue analysis, emphasizing those tools deemed most likely to be: (1) in direct contact with blood, specifically end scrapers and uted points; (2) untouched and unwashed; (3) not heat damaged; and (4) associated with specic Paleoindian loci. The breakdown by tool type is: 17 uted points; 96 end akes; 1 scrapers; 10 side scrapers; 2 akeshavers; 2 outre passe perforator; 1 single-edged knife; and 1 graver. Of the 130 tools in the study, 115 were untouched and unwashed (115/130 88%). The remaining 15 were washed in tap water only (15/130 12%). The majority of the tools selected for testing showed no evidence of being heated (120/130 92%) and it has been shown that protein residues heated to temperatures greater than 100  C will not give positive reactions (Newman et al., 1996: 678). Heat alteration of int or chert typically takes place at temperatures above 250  C (Luedtke, 1992: 92). Regarding spatial distribution, tools were selected from seven contexts, specically: (1) nine tools from A Block; (2) one tool from B Block; (3) 32 tools from C Block; (4) seven tools from D Block; (5) ve tools from E Block; (6) 28 tools from F Block; and (7) 48 tools from elsewhere on the site and unassociated with any lithic concentration. Fifty-six soil samples directly associated with submitted stone tools also were analyzed as controls. The South Field Block at the site, that area that thus far has seen the most extensive analytical attention, was not sampled in the present investigation; the South Field Block was the earliest area excavated at Nobles Pond and all materials were washed, catalogued, analyzed, and extensively handled before the present study was initiated. The testing method used in this analysis is cross-over immunoelectrophoresis (CIEP). The specic substances tested for in CIEP are immunoglobulins, or antibodies, a group of glycoproteins present in the serum and tissue uids of all mammals. Initially, each tool was placed in a plastic dish with 0.5 ml ammonium hydroxide (5%) and sonicated for 5 min. This has been shown to be an effective extractant for old and denatured bloodstains and does not interfere with subsequent testing (Dorrill and Whitehead, 1979). Both dish and contexts were then placed in a rotating mixer for 30 min. The resulting solution was transferred into a sterile plastic vial and refrigerated below 0  C prior to testing. In the case of soil tests, 2 ml of Tris buffer (pH 8.0) was added to 1 g of each soil sample, mixed well and allowed to extract for 24 h on a shaking mixer at 4  C. The resultant supernatant uids were removed, refrigerated below 0  C, and subsequently tested together with samples obtained from the lithics. About 3 ml of each ammonia solution was then transferred into an agarose gel in a Shandon electrophoresis chamber containing a barbital buffer with a pH of 8.6 and paired to a second well with 3 ml of antiserum. The application of an AC current of 100 V was applied to the gel for 45 min causing the test sample and the antiserum to migrate, with antigens towards the anode and antibodies (antiserum) towards the cathode. As they come into contact and if the test sample contains protein corresponding to the species antiserum against which it is being tested, an antigenantibody reaction occurs which results in the formation of an extended lattice as the protein precipitates. Appropriate positive and negative controls, prepared in 5% ammonia solution were run with each gel. These were: positive, proteins of species being tested for; and negative, proteins of the species in which the antiserum was raised. After the test run was completed the gel was pressed and dried. The dry gel was immersed in a Coomassie Blue R250 stain for 3 min and then destained in a solution of ethanol, distilled water and acetic acid (5:5:1, v/v) until the background was clear and any positive

1.1. Nobles Pond Nobles Pond (33ST357) is a multi-component site located in Stark County, Ohio 16 km north of the southern extent of Wisconsinan glaciation (site datum: 40 5102900 N; 81 290 1200 W). The predominant component is early Paleoindian with diagnostics attributable to the Gainey phase ca. 11,20010,800 BP (Seeman, 1994; Seeman et al., 1994). Nearly 6000 m2 have been excavated at the site, and over 55,000 akes and tools have been recovered, mainly from plow-disturbed contexts. Many of the hafted tools were brought to the site in a depleted condition and show evidence of extreme curation before discard. The scarcity of manufacturing debris and early-stage rejects indicates that few of the hafted tools, including the 94 uted points and 1765 end scrapers, were made at Nobles Pond. Altogether, the site extends over approximately 90,600 m2 (8.9 ha). Field investigations at Nobles Pond resulted in the recognition of 14 distinct loci of Paleoindian lithic debris largely resulting from the curation and resharpening of unifacial tools. These concentrations are situated on a series of slight ridges on a glacial outwash terrace and immediately southeast of a large pond (Fig. 1). Plow disturbance has affected the shape and size of the individual clusters, most notably by elongating them somewhat in a northsouth perspective, the predominant direction of plowing as identied from plow scars and aerial photographs. Analysis of raw material distributions and retted specimens indicate that six of these roughly 10 15 m concentrations were organized in a semi-circle and were probably contemporaneous. Our current interpretation is that these loci were strongly redundant with regard to human activities on the land. For the South Field area which contains four of these loci, 45% of the rets are less than 5 m apart and 76% are less than 10 m apart (N 530). As expected, there is some elongation in the dominate direction of plowing between ret pieces (EW mean 3.70 m; NS; mean 6.35 m; N 530), but not enough to negatively affect cluster integrity. Rets in other portions of the site are predicted to show similar relationships, but analyses of these patterns are still in progress. The over 970 rets from the site represent one of the most successful retting programs for an eastern North American Paleoindian site, and carry considerable utility for the study of manufacture, use, tool curation and discard patterns. Overall, the closest structural comparisons of the Nobles Pond site appear to be to Shoop (Witthoft, 1952) and Bull Brook (Grimes et al., 1984) to the east. The purpose of the present protein residue investigation is to better characterize the functionality of end scrapers and uted points, the two most common classes of hafted tools at the site and components of a common early Paleoindian toolkit. Regarding the functional link between humans and animals, it can be assumed that most uted points were used to kill and butcher game and that at least some end scrapers were used to process the hides of these animals. The incidence, location, and species-association of protein residues with these tool classes, therefore, should bear a direct relationship to the link between point and scraper: the greater the functional dependence between these classes, the greater should be the similarity with regard to any protein residues present. To date, most protein residue analyses have focused strictly (and descriptively) on the identication of species and their implications

2744

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750

33 33
330

33

0
32 7
339

321

342

32 4

32

32

241

Block H

330
33

33

Block G
31 8 32

Block I

Nobles Pond
el. 324 m

333
34 2
9

33

Block D Block 1987 Block C

327

Block E

Block F

South Field
33 33 3 33

Block A Block B N
327

330

333

50

100

m.
336

Fig. 1. Map of the Nobles Pond site showing location of excavation blocks and lithic concentrations.

responses visible. Duplicate testing was carried out on all positive reacting specimens to insure the reliability of results. Sterile equipment was used throughout the analysis. The antisera used in this study were obtained from commercial sources that were developed specically for use in forensic medicine (Table 1). Commercial antisera manufacturers provide protocol sheets listing cross reactions; additionally, each batch of antiserum was tested against a full slate of known animal blood as a control for cross-reactivity. None were detected. False positives typically exhibit weak reactivity when compared to the target species, and have been detected in previous studies (Gerlach et al., 1996: 8; Shanks et al., 1999: 1189; Newman et al., 1996: 678). If cross-reactivity had been encountered, the antiserum would have been diluted to a point where only strongly positive results were obtained (Newman et al., 1996: 678). It should be noted that despite controlling for cross-reactivity, most antisera recognize epitopes shared by closely related species within a given biological family. For example, elephant antiserum, raised against serum from modern African elephant, will elicit positive reactions to extant (and presumably extinct) species of the Elephantidae family. Nineteen antisera were polyclonal, and therefore reactive among closely related species. The antisera for three species, elk, moose, and caribou, were raised against serum from modern species, and were puried by absorption to block epitopes that react with closely related species. These tests were developed at the University of Calgary, are considered species-specic, and have been used in other investigations. For the Calgary antisera ELISA was used to

test antibody titer (antibody strength) then PAGE and Western blot to assess the specicity of antibodies (Newman et al., 1996: 678). Tools in the Nobles Pond sample were tested as they became available from ongoing excavations. It should be noted that although 22 animal antisera were utilized in this analysis, not all tools were evaluated against all antisera since some of the latter may not have been available at the time of a particular test. All but 13 tools were tested as whole analytical units. The exceptions were tested only on areas specically identied, and included: nine end scrapers tested on the distal bit only, one end scraper tested only in a presumed hafting notch; one side scraper tested on the one lat passe ake tested only on the distal eral margin only; one outre margin, and one akeshaver tested on one lateral margin. Descriptions of the methods utilized in the present parallel those used in previous research (e.g., Newman et al., 1998: 555; Barnard et al., 2007b: 32). Although there is yet no single, generally accepted method for the use of CIEP on archaeological materials, that used in the present study is the most generally accepted protocol (Barnard et al., 2007a: 223). Tests were conducted at the California State University, Bakerseld, and later at the Department of Biological Sciences, University of Calgary in the laboratory of M.E. Newman.

3. Results Results of CIEP tests are described as positive or negative. Negative results can indicate that: (1) no protein was present; (2)

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750 Table 1 Animal antisera used and most probable species identied Antisera Bear Bovine Camel Cat Dog Horse Rabbit Sheep Guinea pig Deer Chicken Elk Caribou Moose Rat Mouse Elephant Duck Pigeon Human Trout Pig Source of antisera Organon Teknika (Cappel) Organon Teknika Sigma Scientic Organon Teknika Organon Teknika Organon Teknika Organon Teknika Organon Teknika Organon Teknika (Cappel) (Cappel) (Cappel) (Cappel) (Cappel) (Cappel) (Cappel) Most probable speciesa Black bear, grizzly, polar, short-faced Bison, cow, musk ox Camel Bobcat, lynx, puma, cat Coyote, wolf, dog, fox Horse, donkey, kiang, etc. Rabbit, snowshoe hare, pika Sheep, goat Guinea pig, beaver, porcupine, squirrel Deer, elk, moose, caribou, stag-moose, pronghorn Chicken, grouse, turkey, quail and other gallinaceous fowl Elk Caribou Moose All mouse and rat species All mouse and rat species Elephant, mammoth, mastodon Duck, goose pigeon, passenger pigeon Human Trout, salmon Pig, possibly peccary

2745

for rabbits, 6 were for other small mammals, and 3 (the cats) related to a group that might include large and/or small animals. 3.3. Tool size Larger tools, because they have more potential surfaces subject to micro-cracking, might be expected to preserve protein residues more frequently than smaller tools. The distribution of tool size (measured to the nearest 0.1 g) for positive and negative results shows no such tendency (positive: mean 9.32 g, SD 12.487, N 40; negative: mean 8.57 g, SD 6.649, N 90). Side scrapers, physically one of the larger early Paleoindian tool types, produced very few positive results; of the ten side scrapers tested, only one produced a positive reaction (#39564:bear). 3.4. Laboratory cleaning Twenty-nine percent (33/115) of the unwashed tools yielded positive results for the presence of protein residue, whereas 47% (7/ 15) of the tools washed in tap water yielded positive results (Table 2). On average, the washed tools were larger than the unwashed tools, which together with the much smaller sample size, may help to explain the notably higher percentage of positive results for washed specimens. Shanks et al. (2004) indicate that repeatedly washed stone tools still yield identiable DNA and protein residues, and other researchers have achieved good success rates with cleaned and curated materials (Loy and Dixon, 1998). 3.5. Heat damage Of the ten heat-altered tools in the analysis, three (3/10 30%) yielded positive results, a percentage that is equivalent to that obtained for the unheated tools (37/120 31%). As noted in Section 2 of this investigation, protein residues should not survive in most contexts where temperatures were sufcient to thermally alter cryptocrystalline rock. These results therefore could represent false positives of unknown cause, sample size effects, the possibility that not all portions of heated tools reached temperatures sufcient to destroy protein residues, and/or the prospect that blood or other sources of animal protein were in contact with these tools after they were heated-altered. Retting at Nobles Pond has demonstrated that a number of tool fragments at the site were salvaged and/or recycled after heating events, thereby strengthening this last alternative. 3.6. Raw material Tools made of exotic raw materials were more likely to test positive for protein residues than those of more locally derived materials. Of the 130 tools tested, 61 were manufactured of Upper Mercer int, 55 of Flint Ridge Flint, 6 of Wyandotte int, 2 of Attica int, and 6 of other or unidentied materials. Combining Attica and Wyandotte, the two materials derived more than 300 km from the site, tools made of exotic materials have more than twice the chance of testing positive for protein residue as all those other raw materials combined (5/8 62% versus 35/122 29%). Petraglia et al. (1996: 133) found that ne-grained materials had a greater chance of yielding protein residues than course-grained materials, but that study appears to have been focusing on differences between cherts and quartzites. At Nobles Pond this kind of distinction does not explain the greater reactivity of tools made of exotic materials since all materials are of similar cryptocrystalline structure. Other studies have shown that Paleoindians not only preferred high quality raw materials, but that a minority of these materials came from distances that are difcult to explain as a result of the movement of individual Paleoindian bands (e.g., Frison,

Organon Teknika (Cappel) Organon Teknika (Cappel) University of Calgary University of Calgary University of Calgary Organon Teknika (Cappel) Organon Teknika (Cappel) University of Calgary Nordic Immunological Nordic Immunological Organon Teknika (Cappel) University of Calgary Organon Teknika (Cappel)

a Immunological relationships do not necessarily bear a linear relationship to the Linnaean system, although they usually do (Gaensslen, 1983). Probable species for Late Pleistocene Ohio contexts are informed by community data from the Hiscock locality, Genesee Co., New York (Steadman, 1988); Carter Bog locality, Darke Co., Ohio (McDonald, 1994), Big Boone Lick locality, Boone Co., Kentucky (Jillson, 1936; Schultz et al., 1963); and Sheriden Cave, Wyandot Co., Ohio (McDonald, 1994).

the extraction method failed to remove enough protein for detection; or (3) the antiserum for a specic animal was not part of the test. Positive results can indicate: (1) the presence of the target protein; (2) cross-reactivity; or (3) a non-specic immune reaction (see Shanks et al., 1999: 1188).

3.1. Pre-test reactivity No positive results were obtained in testing any extracts from artifacts or associated soil samples against pre-immune serum of the sort expected if contaminants in the soil such as bacteria, tannic acid or iron chlorates were affecting results. False positives from soil contamination are rare (Gerlach et al., 1996: 8).

3.2. Species reactivity A total of 45 positive reactions were obtained on 40 of the 130 tools in the study (Table 2). These results are comparable to those obtained by similar methods in grassland and boreal forest environments (Gerlach et al., 1996: 6). Cervids, including generic deer/elk/moose/caribou/stag-moose (N 13), elk (N 2), and caribou (N 2) represent the largest general category of positive identications, followed by rabbit/hare (N 11), bear (N 5), and smaller numbers for bovine, cat, dog, chicken, guinea pig, and mouse antisera. Specimens 45321, 48001, 53980, and 51500, to the extent that they tested positive for deer, but negative for specic tests to caribou, elk, and moose antisera make an identication of white-tailed deer (Odocoileus virginianus) more likely. Specimens 1862 and 48135, to the extent that they yielded positive results for both the generic antiserum for deer and the species-specic antiserum for elk could represent both elk and another cervid species on the same tool or simply elk. Grouped more broadly, 25 of the positive reactions were for cervids or other large mammals, 11 were

2746

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750

Table 2 Tools with positive blood protein results, Nobles Pond (33ST357)a,b Catalog Tool number class 1725 1862 27461 27476 31084 31130 32009 38888 39564 39591 39591c,d 39888 41418 41536 41540 41540c 41582 41607 41607c 42084 42174 42213 42575 44434 44870 45306f 45321f 45445 46086 47110 47346 47766 48001 48135 48580 49547 51297 51500 51605 52376 53980 54332 54334
a b c d e f

Excavation Heat Soil Wt. block Touched Washed damaged testedd (g) C A A D D C F F C A F F Yes Yes Yes Yes Yes Yes Yes No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Yes No No No Yes No No No No No No No No No No No No No No No No No No No No No No No Yes No No No No No No No No No No No No No No No No No No Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes No No No No No Yes No Yes No Yes No No No No No No No No No 3.9 79.3 4.5 9..3 2.4 5.2 7.4 10.6 7.9 7.7 6.9 7.9 9.2 6.4 3.2 7.3 12.4 30.7 18.8 4.9 8.4 5.5 4.3 2.5 7.4 4.1 6.8 1.9 3.3 11.2 9.0 4.6 4.2 6.5 16.7 1.7 10.7 5.7 5.1 7.3

Raw Guinea materiale Deer Caribou Elk Bear Bovine pig Dog Cat Rabbit Mouse Chicken FR FR UM FR FR UM FR UM UM WY FR UI FR FR UM FR UM UM FR FR FR FR FR FR UI WY AT AT UM UM FR UM UM UM UM FR FR WY FR UM / / / / / / / / / / / / / / / / / / / / / / / / / /

Fluted point Knife End scraper End scraper End scraper End scraper Fluted point Fluted point Side scraper End scraper End End End End scraper scraper scraper scraper

/ / /

End scraper F End scraper F End scraper Fluted point Fluted point End scraper End scraper End scraper End scraper End scraper End scraper End scraper Fluted point End scraper Fluted point End scraper End scraper End scraper End scraper End scraper Fluted point End scraper End scraper End scraper End scraper End scraper

F C C C C C

C C A

Positive test (); antiserum not available for test (/). Additional species tested with no positive results are: pig, trout, camel, horse, sheep, duck, pigeon human, moose, rat, and elephant. Resubmission of previously tested tool. All soil samples tested with associated tools yielded negative results for blood protein. Raw materials are: Upper Mercer (UM); Flint Ridge (FR); Attica (AT); Wyandotte (WY); and Unidentied (UI). #45306 and #45321 were tested separately, but are the distal and proximal fragments, respectively, of a single ret end scraper.

1987; Gramly, 1988: 275; Seeman et al., 1994: 88). Trade and migration have both been suggested as possible explanations (Meltzer, 1989; Tankersley, 1994a). For our purposes, we simply note that if distance-to-source can be seen in part as a function of time, then these exotic materials may have had longer use-lives than those made closer to the point of deposition, and thus a greater chance of accumulating protein residues. 3.7. Tool classes Major tool classes were not equally reactive. Hafted tools were about three times as reactive as unhafted tools, although differences in sample size should be noted (38/115 33% vs. 2/15 13%; N 130). Within the hafted tool category, 8 of the 17 uted points tested were reactive (8/17 47%), compared to 31% (30/96 31%) of the end scrapers and none of the akeshavers (0/2 0%). Hafted tools can be expected to have longer use-lives than unhafted tools and thus an increased opportunity for the accumulation of protein residues. Based on curation potential and multifunctionality, it is

assumed that uted points should have the greatest longevity of the hafted tool classes and of course uted points are assumed to be more directly involved in penetrating the bloody parts of animals (see Tankersley, 1989: 133135; Kay, 1996). Conversely and based on microwear analysis, at least some of the hafted end scrapers at Nobles Pond were used to work wood and other non-animal materials (M. Pope, personal communication, 2005). One unanticipated result was a positive test for bear on the distal portion of an end scraper (#45306) and a positive test for deer on the ret proximal portion of the same tool (#45321). This result is consistent, however, with actualistic ndings that show that protein residues sometimes can accumulate on tools in quixotic fashion as a result of routine use and maintenance activities (Rots and Williamson, 2004). Four of the eight reactive Nobles Pond uted points showed positive results for large mammals and one of these points was reactive for two different large mammal groups, cervids and bovines. High reactivity for projectile points has been reported in other studies, but not explicitly emphasized (e.g. Petraglia et al.,

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750

2747

1996: 133; Loy and Dixon, 1998; Gerlach et al., 1996: 5). Small game protein residues present on uted points related to a single antiserum, rabbit. An additional consideration in explaining the differences in reactivity of hafted and unhafted tools is the hafting itself. Hafting, to the extent that it makes use of hide-, blood-, or bone-based glues, and/or sinews provides a proximate source of protein residues in addition to the blood of targeted animals. Glues derived from hides, blood or bone are especially useful in hafting tools because they strengthen the connections between stone point, foreshaft, and lashings, yet subsequent heating allows the easy removal of broken or exhausted components for replacement or realignment. The use of organic glues of this type is well documented in the ethnographic record and such materials continue to be used by modern primitive weapons enthusiasts for hafting stone projectile points (e.g., Balikci, 1970: 46; Birket-Smith, 1929: 27; Grinnell, 1923: 175176; Lowie, 1935: 85; Mails, 1972: 418; McPherson, 2006: 39; see also Tankersley, 1994b). Rabbit hide produces an especially good quality product with high viscosity, and rabbit protein residue has been reported consistently in other Paleoindian contexts (Cannon, 1995: 16; Dumais, 2000: 106; Gramly, 1991: 7; Gerlach et al., 1996: 5; Downs and Lowenstein, 1995: 15: see also Storck and Spiess, 1994: 128). A recently tested Cody Complex knife showed a positive test for rabbit on the haft element and sheep on the blade (Shortt, 2002: 238). Results of the present study were more ambiguous; reactivity to rabbit antiserum was equivalent for proximal and distal fragments of hafted tools at Nobles Pond (complete 5; proximal 3; distal 3). Rabbit (Lagomorpha) blood proteins are not known to cross-react with those of other orders (Gerlach et al., 1996: 8). It should be noted that snowshoe and/or arctic hare (Lepus sp.) were identied at Udora, the only Gainey phase site that has thus far produced identied mammalian bone (Storck and Spiess, 1994: 128). Rabbits are typically not an important group in the faunal inventories of later archaeological sites in the Midwest. 3.8. Spatial patterning The reactivity of tools by spatial and cultural context provides an additional opportunity to examine variability (Fig. 2). Although Paleoindian materials occur across a considerable area at Nobles Pond, the 14 Paleoindian lithic concentrations or hotspots were located in a more limited area to the southwest of the pond. These were hand-excavated as a series of macroblocks. Most relevant for the present study are investigations in Blocks A, B, C, D, E, and F. These blocks contained one or more Paleoindian lithic concentrations and were excavated after our recovery methods were modied to accommodate protein residue analysis. Test results pertaining to spatial patterning indicate an association of larger mammals with the main concentrations of Paleoindian activity in the southern portion of the site (Tables 2 and 3). Much of this results from the fact that most of the positive results for cervids (deer, caribou, elk) were associated with the southern block excavations. In contrast, positive tests for smaller animals were less likely to occur in these concentrated activity zones. This spatial patterning is consistent with the interpretation that lithic concentrations at Nobles Pond relate in part to the processing of large mammals and/ or the selective discard of tools associated with such efforts. 4. Conclusions The sample of reactive protein residues from Nobles Pond is one of the largest available for any archaeological site. Results indicate a wide range of taxa, and it is likely that if these tools were tested against a wider range of antisera that the range would be even broader (see Grayson and Cole, 1998). This pattern contrasts with

the results from other Late Pleistocene studies suggesting a relatively small number of targeted species and notable large mammal associations (e.g., Loy and Dixon, 1998; Kooyman et al., 2001; Gramly, 1991). At Nobles Pond there do seem to be notable large mammal association with the main lithic concentrations at the site. The protein residue data from Nobles Pond support an interpretation that large cervids were important rst-line resources in lower Great Lakes Paleoindian economies. This view is consistent with our current knowledge of regional ecosystems, as well as other archaeological data pointing to similar conclusions. Caribou hunting in particular has gured importantly in discussions of regional hunting strategies (Deller and Ellis, 1992: 8; Jackson, 1998: 13; Shott, 1993: 13; Storck, 1997: 273). Caribou bone has been recovered from several Midwestern Paleoindian sites, and also from related sites to the east (Storck and Spiess, 1994; see also Nova Scotia Museum, 1996). Terminal Pleistocene occurrences of caribou in non-archaeological contexts in the region include Sheriden Pit (McDonald, 1994: 30; and Hiscock (Steadman, 1988). The presence of caribou protein residue on two hafted end scrapers at Nobles Pond is the rst such direct occurrence on stone tools in the Midwest. Given the osteological evidence from other sites, however, these results are not unexpected and at the same time provide another important point of reference for the use of this species. Additional support for the importance of caribou comes by way of the south-to-north chronocline in the location of large Paleoindian aggregation sites in the Great Lakes area spanning the Gainey and subsequent Parkhill phases (Seeman et al., 1994: 83). As a category, protein residues testing positive for bear were the second largest taxon of large mammals (N 5) represented. Ethnographically, bear hunting in eastern North America was often a cold-season activity geared to take advantage of hibernation (Hallowell, 1926: 3136, 5354; Smith, 1975: 118; Rogers 1962: C44). This is consistent with the extensively heat-damaged lithic materials at Nobles Pond and other early Paleoindian sites, which have likewise been interpreted as evidence of cold season occupancy (Seeman et al., 1994: 81). Interpretations of the protein residue evidence for bovine (bison) occurrences at Nobles Pond are more problematic than for deer, caribou, or bear. To date, no direct association of this taxon with an early Paleoindian site has been documented in the region, although bison were probably present in Ohio at that time (McDonald, 1994: 26, 30; see also Webb et al., 1984). At least two other eastern Paleoindian sites have yielded positive tests for bovine protein residue that have been interpreted as pertaining to bison (Newman and Julig, 1989: 129; McAvoy and McAvoy, 1997; see also Loy and Dixon, 1998: 38; Newman et al., 1996: 681; Gramly, 1991: 7). The two uted points and a hafted end scraper testing positive for bovine protein residues at Nobles Pond were in close proximity to one another in Block C and could arguably represent a single kill. Ancient protein residues are preserved on stone tools at Nobles Pond, and results indicate that uted points are the most likely tool class on which they will occur. This makes sense in light of their designed purpose as the business-end of projectile weaponry. The decreased incidence on end scrapers, conversely, is consistent with available microwear data from the site, and lithic studies elsewhere suggesting a broader range of functionality for end scrapers than simply the processing of animal hides. Interpretations as to the meaning of these occurrences must be tempered by an appreciation of site formational issues and the range of means by which animal proteins can come in contact with stone tools. Manufacturing and maintenance activities, particularly in the use of protein adhesives or mastics, may have had a greater bearing on which protein residues are present on stone tools than previously has been recognized. The consistent targeting of particular areas on stone tools for protein residue analysis rather than simply immersing the whole

2748

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750

33

deer

32

32

bear

deer bear deer bovine deer deer caribou deer bear deer deer bear caribou bovine & bear deer & elk bovine & deer deer
327

50

100 m.

Fig. 2. Spatial distribution of provenienced tools tested for blood protein residue, Nobles Pond (33ST357) (positive, solid circle; negative, open circle). All positives from mammals of body weight of 50 kg or greater are labeled. Mapped cases 127 or 98% of total sample.

tool would seem one methodological renement that would aid in disentangling the effects of manufacture, use, and rehafting in this regard. In sum, the overall pattern of protein residues identied at Nobles Pond cohere with expectations based on currently available background knowledge, as they do at a number of other sites. Mammoth, bison, sheep, bear, caribou, and musk ox from Paleoindian sites in Alaska (Loy and Dixon, 1998:42); bovine positives from a bison jump on the northern Plains (Newman et al., 1996: 681); only human protein residue from a Civil War surgeons kit (Newman et al., 1998: 556; pennipeds, cervids, bear, and smaller animals from a late Paleoindian site at the mouth of the Saint Laurence (Dumais, 2000: 106); horse protein residue on Clovis points near bone-bearing deposits of this same species in Alberta (Kooyman et al., 2001: 688); bovine, sheep, bear, and human protein residues from an Archaic site in Wyoming where sheep and bison dominate the faunal assemblage (Shanks et al., 1999: 1189);

human protein from the walls of ceramic vessels in an expected case of cannibalism (Malar et al., 2000); clam and trout protein residues on ground stone tools appraised on other evidence to be used in the processing of sh and shellsh (Newman, 1993); protein residues in these and other archaeological contexts meet expectations based on other, independent lines of reasoning and support the conclusion that researchers should continue to collect and analyze preserved ancient protein residues. Comparative data from other Gainey phase sites in the Midwest would be particularly welcome. Certainly the Nobles Pond results indicate that the method holds particular promise for interpreting spatial patterning in a variety of situations. The present study makes use of a large sample, but even larger and better controlled samples are called for. Results of the present study suggest that these samples should be targeted towards hafted tools and that moderate cleaning and curation should not preclude the testing of such materials, a nding consistent with recent actualistic studies.

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750 Table 3 Positive protein reactivity by site area, Nobles Pond (33ST357) Positive reactivity for: Deer Caribou Elka Bovineb Bear Large animal subtotal Rabbit Dog Guinea pig Mouse Chicken Small animal subtotal Cat Total Lithic concentrations (south) (%) 10/29 2/29 1/29 3/29 4/29 20/29 4/29 1/29 1/29 1/29 1/29 8/29 (34) (7) (3) (10) (14) (69) (14) (3) (3) (3) (3) (28) Away from lithic concentrations (north) (%) 2/14(14) 0/14(0) 0/14(0) 0/14(0) 1/14(7) 3/14(21) 7/14(50) 2/14(14) 0/14(0) 0/14(0) 0/14(0) 9/14(64) 2/14(14) 14/14(100)

2749

1/29 (3) 29/29 (100)

a A total of two tools tested positive for elk. However, one was an unprovenienced surface nd and is not tallied in this table. b One of these three positives came from the backdirt of a backhoe trench, but can be condently associated with the eastern margin of Block C.

Acknowledgements We acknowledge and thank the numerous individuals and groups that contributed donations to fund protein residue analysis at Nobles Pond. Included and notable here are: the Sugarcreek, Johnny Appleseed, Chippewa Valley, and Cuyahoga Valley chapters of the Archaeological Society of Ohio, the Manseld Gem and Mineral Society, and numerous friends and volunteers that participated in our Adopt-an-Artifact program. The Nobles Pond excavation was funded in part by grants from the Timken Foundation and the National Science Foundation (BNS88-14809). References
Balikci, A., 1970. The Netsilik Eskimo. Natural History Press, Garden City, NY. Barnard, H., Shoemaker, L., Rider, M., Craig, O.E., Parr, R.E., Sutton, M.Q., Yohe II, R.M., 2007a. Introduction to the analysis of protein residues in archaeological ceramics. In: Barnard, H., Eerkens, J. (Eds.), Theory and Practice of Archaeological Residue Analysis. BAR International Series 1650. Archaeopress, Oxford, pp. 216 231. Barnard, H., Ambrose, S.H., Beehr, D.E., Forster, M.D., Lanehart, R.E., Malainey, M.E., Parr, R.E., Rider, M., Solazzo, C., Yohe II, R.M., 2007b. Mixed results of seven methods of organic residue analysis applied to one vessel with the residue of a known foodstuff. Journal of Archaeological Science 34, 2837. Birket-Smith, K., 1929. The Caribou Eskimos: Material and Social Life and Their Cultural Position. II Analytical Part. Gyldendalske Boghandel. Nordisk Forlag, Copenhagen. Cannon, K.P., 1995. Blood residue analysis of ancient stone tools reveal clues to prehistoric subsistence patterns in Yellowstone. CRM 18 (2), 1416. Cannon, M.D., Meltzer, D.J., 2004. Early Paleoindian foraging: examining the faunal evidence for large mammal specialization and regional variability in prey choice. Quaternary Science Reviews 23, 19551987. Deller, D.B., Ellis, C.J., 1992. Thedford II: A Paleoindian Site in the Ausable River Watershed of Southwestern Ontario, Memoir No. 24. Museum of Anthropology. University of Michigan, Ann Arbor, MI. Dorrill, M., Whitehead, P.H., 1979. The species identication of very old human bloodstains. Forensic Science International 13, 111116. Downs, E.F., Lowenstein, J.M., 1995. Identication of archaeological blood proteins: a cautionary note. Journal of Archaeological Science 22, 1116. Dumais, P., 2000. The La Martre and Mitis Late Paleoindian sites: a reection on the peopling of southeastern Quebec. Archaeology of Eastern North America 28,1112. Frison, G.C., 1987. The tool assemblage, unnished bifaces, and stone aking material sources for the Horner site. In: Frison, G., Todd, L. (Eds.), The Horner Site: The Type Site of the Cody Cultural Complex. Academic Press, Orlando, FL, pp. 233278. Gaensslen, R.E., 1983. Sourcebook in Forensic Serology, Immunology, and Biochemistry. U. S. Department of Justice, Washington, D.C. Grayson, D.K., Cole, S.C., 1998. Stone tool assemblage richness during the Middle and Early Upper Paleolithic in France. Journal of Archaeological Science 25, 927938. Gerlach, S.C., Newman, M.E., Knell, E.J., Hall Jr., E.S., 1996. Blood protein residues on lithic artifacts from two archaeological sites in the De Long Mountains, Northwestern Alaska. Arctic 49, 110.

Gramly, R.M., 1988. Palaeo-Indian sites south of Lake Ontario, western and central New York State. In: Laub, R., Miller, N., Steadman, D. (Eds.), Late Pleistocene and Early Holocene Paleoecology and Archeology of the Eastern Great Lakes Region, vol. 33. Buffalo Society of Natural Sciences, Buffalo, NY, pp. 265280. Gramly, R.M., 1991. Blood residues upon tools from the East Wenatchee Clovis site, Douglas County, Washington. Ohio Archaeologist 41(4), 4-9. Grimes, J.R., Eldridge, W., Grimes, B.G., Vaccaro, A., Vaccaro, F., Vaccaro, J., Vaccaro, N., Orsini, A., 1984. Bull Brook II. Archaeology of Eastern North America 12, 159183. Grinnell, G.B., 1923. The Cheyenne Indians: Their History and Ways of Life. Yale University Press, New Haven, CT (reprinted: Cooper Square Publishers, New York, 1962; also, University of Nebraska Press, Lincoln, NE). Hallowell, A.I., 1926. Bear ceremonialism in the northern hemisphere. American Anthropologist 28 (1), 1175. ` , P., 2002. Red blood cell haemotaphonomy of experimental human bloodHortola stains on techno-prehistoric lithic raw materials. Journal of Archaeological Science 29, 733739. Jackson, L.J., 1998. The Sandy Ridge and Halstead Paleoindian Sites: Unifacial Tool Use and Gainey Phase Denition in South-Central Ontario, Memoirs 32. Museum of Anthropology. University of Michigan, Ann Arbor, MI. Jillson, W.R., 1936. Big Bone Lick: An Outline of Its History, Geology, and Paleontology. Standard Printing Company, Louisville. Kay, M., 1996. Microwear analysis of some Clovis and experimental chipped stone tools. In: Odell, G. (Ed.), Stone Tools: Theoretical Insights into Human Prehistory. Plenum Press, New York, pp. 315344. Kooyman, B.P., Newman, M.E., Ceri, H., 1992. Verifying the reliability of blood residue analysis on archaeological tools. Journal of Archaeological Science 19, 265 269. Kooyman, B.P., Newman, M.E., Cluney, C., Lobb, M., Tolman, S., McNeil, P., Hills, L.V., 2001. Identication of horse exploitation by Clovis hunters based on protein analysis. American Antiquity 66, 686691. Lambert, P.M., Banks, L.L., Billman, B.R., Marlar, R.A., Newman, M.E., Reinhard, K.J., 2000. Response to the claim of cannibalism at Cowboy Wash. American Antiquity 65, 397406. Lowie, R.H., 1935. The Crow Indians. Holt. Rinehart and Winston, New York. Loy, T.H., 1983. Prehistoric blood residues: detection on tool surfaces and identication of species of origin. Science 220, 12691271. Loy, T.H., Dixon, E.J., 1998. Blood residues on uted points from eastern Beringia. American Antiquity 63, 2146. Luedtke, B.E., 1992. An Archaeologists Guide to Chert and Flint. University of California, Los Angeles, Institute of Archaeology, Archaeological Research Tools 7. Los Angeles, CA. McAvoy, J.M., McAvoy, L.D., 1997. Archaeological Investigations of Site 44SX202, Cactus Hill, Sussex County, Virginia, Research Report Series No. 8. Virginia Department of Historic Resources, Richmond, VA. McDonald, H.G., 1994. The Late Pleistocene vertebrate fauna in Ohio: coinhabitants with Ohios Paleoindians. In: Dancey, W. (Ed.), The First Discovery of America: Archaeological Evidence of the Early Inhabitants of the Ohio Area. The Ohio Archaeological Council, Columbus, OH, pp. 2341. McPherson, J., 2006. How to make a primitive arrow. Primitive Archer 14 (4), 3948. Mails, T.E., 1972. The Mystic Warriors of the Plains. Doubleday, Garden City, NY. Malar, R.A., Leonard, B.L., Billman, B.R., Lambert, P.M., Malar, J.E., 2000. Biochemical evidence of cannibalism at a prehistoric site in southwestern Colorado. Nature 407, 7478. Meltzer, D.J., 1989. Was stone exchanged among eastern North American Paleoindians? In: Ellis, C., Lothrop, J. (Eds.), Eastern Paleoindian Lithic Resource Use. Westview Press, Boulder, CO, pp. 1139. Newman, M.E., 1993. Appendix 1: immunological analysis of artifacts from site CASLO-762, Cambria, San Luis Obispo County, California. In: Breschini, G., Haversat, T. (Eds.), Pitted Stones from CA-SLO-697 and CA-SLO-762. Cambria, San Luis Obispo County, California. http://www.californiaprehistory.com/reports01/reo0015.html. Newman, M.E., Julig, P., 1989. The identication of protein residues on lithic artifacts from a stratied boreal forest site. Canadian Journal of Archaeology 13, 119132. Newman, M.E., Ceri, H., Kooyman, B.P., 1996. The use of immunological techniques in the analysis of archaeological materials a response to Eisele; with report of studies at Head-Smashed-In Buffalo jump. Antiquity 70 (269), 677682. Newman, M.E., Byrne, G., Ceri, H., Dimik, L.S., Bridge, P.J., 1998. Immunological and DNA analysis of blood residues from a surgeons kit used in the American Civil War. Journal of Archaeological Science 25, 553557. Nova Scotia Museum, 1996. Archaeology in Nova Scotia-Debert Paleoindian site. http://museum.gov.ns.ca/arch/sites/debert/debert.htm. Petraglia, M., Knepper, D., Glumac, P., Newman, M., Sussman, C., 1996. Immunological and microwear analysis of chipped-stone artifacts from Piedmont contexts. American Antiquity 61, 127135. Rogers, E.S., 1962. The Round Lake Ojibwa. Royal Ontario Museum, Art and Archaeology Division, Occasional Paper 5. Ontario Department of Lands and Forests for the Royal Ontario Museum. Rots, V., Williamson, B.S., 2004. Microwear and residue analyses in perspective: the contribution of ethnoarchaeological evidence. Journal of Archaeological Science 31, 12871299. Seeman, M.F., 1994. Intercluster lithic patterning at Nobles Pond: a case for disembedded procurement among early Paleoindian societies. American Antiquity 59, 273288. Seeman, M.F., Summers, G., Dowd, E., Morris, L., 1994. Fluted point characteristics at three large sites: the implications of modeling early Paleoindian settlement patterns in Ohio. In: Dancey, W. (Ed.), The First Discovery of America:

2750

M.F. Seeman et al. / Journal of Archaeological Science 35 (2008) 27422750 Great Lakes Region, Volume 33. Buffalo Society of Natural Sciences, Buffalo, NY, pp. 95113. Storck, P.L., 1997. The Fisher Site: Archaeological, Geological and Paleobotanical Studies at an Early Paleo-Indian Site in Southern Ontario, Canada, Memoir 30. Museum of Anthropology. University of Michigan, Ann Arbor, MI. Storck, P.L., Spiess, A.E., 1994. The signicance of new faunal identications attributed to an early Paleoindian (Gainey Complex) occupation at the Udora site, Ontario, Canada. American Antiquity 59, 121142. Tankersley, K.B., 1989. Late Pleistocene Lithic Exploitation and Human Settlement in the Midwestern United States. PhD dissertation, Department of Anthropology, Indiana University, Bloomington, IN. Tankersley, K.B., 1994a. Was Clovis a colonizing population in eastern North America? Journal of Archaeological Science 21, 117124. Tankersley, K.B., 1994b. Clovis mastic and its hafting implications. In: Dancey, W. (Ed.), The First Discovery of America: Archaeological Evidence of the Early Inhabitants of the Ohio Area. The Ohio Archaeological Council, Columbus, OH, pp. 95116. Tuross, N., Barnes, I., Potts, R., 1996. Protein identication of blood residues on experimental stone tools. Journal of Archaeological Science 23, 289296. Webb, S.D., Milanich, J.T., Alexon, R., Dunbar, J.S., 1984. A Bison antiquus kill site, Wacissa River, Jefferson County, Florida. American Antiquity 49, 384392. Witthoft, J., 1952. A Paleo-Indian site in eastern Pennsylvania: an early hunting culture. Proceedings of the American Philosophical Society 96, 464495.

Archaeological Evidence of the Early Inhabitants of the Ohio Area. The Ohio Archaeological Council, Columbus, OH, pp. 7793. Shanks, O.C., Kornfeld, M., Hawk, D., 1999. Protein analysis of Bugas-Holding tools: new trends in immunological studies. Journal of Archaeological Science 26, 11831191. Shanks, O.C., Bonnichsen, R., Vella, A., Ream, W., 2001. Recovery of protein and DNA trapped in stone tool microcracks. Journal of Archaeological Science 28, 965 972. Shanks, O.C., Kornfeld, M., Ream, W., 2004. DNA and protein recovery from washed experimental stone tools. Archaeometry 46, 663672. Shortt, M.W., 2002. The Osprey Beach locality: a Cody Complex occupation on the south shore of West Thumb. In: Anderson, R., Harmon, D. (Eds.), Yellowstone Lake: Hotbed of Chaos or Reservoir of Resilience? 6th Biennial Scientic Conference on the Greater Yellowstone Ecosystem. National Park Service, US Department of the Interior, pp. 232241. Shott, M.J., 1993. The Leavitt Site: A Parkhill Phase Paleo-Indian Occupation in Central Michigan, No. 25. Museum of Anthropology. University of Michigan, Ann Arbor, MI. Smith, B.D., 1975. Middle Mississippi Exploitation of Animal Populations, Anthropological Paper No. 57. University of Michigan, Ann Arbor, MI. Steadman, D.W., 1988. Vertebrates from the Late Quaternary Hiscock site, Genesee County, New York. In: Laub, R., Miller, N., Steadman, D. (Eds.), Late Pleistocene and Early Holocene Paleoecology and Archaeology of the Eastern