Appendix A

PROTEIN RESIDUE ANALSIS


PROTEIN RESIDUE ANALYSIS OF A FLUTED POINT FROM THE BOG SITE, CA-SDI-2506, SOUTHERN CALIFORNIA

By

Kathryn Puseman
and
Jaime Dexter

 

Paleo Research Institute, Golden, Colorado

 

Paleo Research Institute Technical Report 06-55

 

Prepared For

George Kline
San Diego State University
San Diego, California

August 2008

Introduction

A single obsidian fluted point and an associated soil control from the Bog Site, CA-SDI-2506, in the Lost Valley area in northeastern San Diego County, California, were tested for possible protein residues.  Although the site has been culturally affiliated with a seasonal camp of the late prehistoric and protohistoric Cupeño People and possibly the Mountain Cahuilla, the fluted point is believed to represent the early Holocene Clovis Culture approximately 10,000 to 12,000 years ago.  Obsidian sourcing analysis indicates that the source of the lithic material was Lookout Mountain in the Casa Diablo Complex, about 540 kilometers north of the site.  Protein residue analysis was undertaken to determine if proteins were present on the artifact surface that would provide information concerning animals that were hunted with this tool.

Methods

The artifact submitted for protein residue analysis was tested using an immunologically-based technique referred to as cross over immunoelectrophoresis (CIEP or COE).  The method for CIEP is based on forensic work by Culliford with changes made by Newman following the procedure used by the Royal Canadian Mounted Police Serology Laboratory in Toronto, Canada.  Further changes were made at Paleo Research Institute following the advice of Dr. Richard Marlar at the Thrombosis Research Laboratory in the Denver VA Medical Center and the University of Colorado Health Sciences Center.

The artifact was washed using 1-2ml of a 0.02M Tris hydrochloride, 0.5M sodium chloride, and 0.5% Triton X-100 solution.  The artifact was placed in an ultrasonic bath for 30 minutes, on a rotating mixer for 30 minutes, then in the ultrasonic bath for an additional 30 minutes.  Because soils contain compounds such as bacteria and animal feces that can cause false positive results for artifacts buried in the soil, a control sample also was tested.  One gram of soil was added to 1 ml of the Tris/NaCl/Triton solution, then refrigerated for several days prior to testing.

The residues extracted from the artifact and the soil control first were tested against pre-immune goat serum (serum from a non-immunized animal) to detect non-specific binding of proteins.  Samples testing negative against pre-immune serum then are tested against prepared animal antisera obtained from ICN Pharmaceuticals, Inc. and Sigma Chemical Company, and against antisera raised under the direction of Robert Sargeant in Lompoc, California, and Dr. Richard Marlar.  Appropriate positive and negative controls were run for each antiserum.  A positive control consists of the blood of an animal for which the antiserum is know to test positively, and a negative control consists of the serum/blood of the animal in which the antiserum was raised, either rabbit or goat.

CIEP is performed using agarose gel as the medium.  Two holes are punched in the gel about 5 mm apart.  The protein extract from the artifact was placed in the cathodic well and the antiserum is placed in the anodic well.  The sample was electrophoresed in Barbital buffer (pH 8.6) for 45 minutes at a voltage of 130v to drive the antigens and antibodies towards each other.  Positive reactions appear as a line of precipitation between the two wells.  Gels are stained with coomassie blue to make the precipitate line easier to see.  Positive reactions were re-tested with dilute antisera to determine between true and false positives.  Antisera are diluted to increase specificity of reactions, usually 1:10 or 1:20.  Positive reactions obtained after this step are reported.

Identification of animals represented by positive results is usually made to the family level.  All mammalian species have serum protein antigenic determinations in common; therefore, some cross reactions will occur between closely and sometimes distantly related animals .  For example, bovine antiserum will react with bison blood, and deer antiserum will react with other members of the Cervidae (deer) family, such as elk and moose.

Discussion

CA-SDI-2506 is located near Shingle Spring in the Lost Valley, California, at an elevation of 4600 to 4800 feet.  Shingle Spring represents the headwaters of the San Luis Rey Watershed.  Vegetation in the area consists of oak (Quercus) and pine (Pinus) woodlands flanked by mountain chaparral and a riparian environment near the spring.  A meadow containing grasses (Poaceae) and sedges (Cyperaceae) is located about one-half kilometer west-southwest of the site.  This meadow may have been a Pleistocene lake that has since silted up (George Kline, personal communication, May 28, 2006).  Shallow lake and marsh conditions prevailed in southern and western California from about 11,000 to 8,000 B.P.  These areas contained varied and abundant plant and animal resources, which in turn would have attracted early hunter-gatherers to the area .

The site originally was described as a late prehistoric Cupeño seasonal camp.  A variety of artifacts were found in the Late Prehistoric blackened midden deposits at depths of up to 50 cm below the present ground surface.  Sample 4408 consists of an obsidian fluted point recovered at a depth of 100 cm in an otherwise sterile deposit (Table 1).  The accompanying soil control (sample 4395) was collected approximately 20 cm west of the point at a depth of 103 cm below the surface.  The fluted point is believed to represent the Clovis Culture of the late Pleistocene/early Holocene, about 12,000 to 10,000 years ago.  This time period is noted to have experienced warming temperatures, glacial retreat, rising seas, evaporation of pluvial lakes, and major vegetation shifts, and the extinction of the Rancholabrean fauna .          

The fluted point and the soil control were tested against the various antisera listed in Table 2. Sample 4408 tested positive to deer antiserum (Table 3), suggesting use of the point to hunt a member of the Cervidae (deer family).  Soil control sample 4395 yielded a negative result to deer antiserum, indicating that the positive result for the artifact is not due to soil contamination.  Members of the Cervidae in North America include deer (Odocoileus sp.), elk (Cervus elaphus/canadensis), moose (alces), and caribou (Rangifer sp.).  Moose and caribou are found mainly in Canada, although moose can be found in the northeastern United States and southwest through the Rocky Mountains to northeastern Utah and northwestern Colorado. 

Elk are found primarily in the Rocky Mountain region and along the Pacific Northwest coast, with great numbers found in Colorado, Wyoming, Montana, and Washington.  Tule elk were once found in the California Central Valley Lower Sonoran zone, and Canadian elk can be found in the California Transition zone .  Elk are reported to have once ranged through most of the United States and Canada.  Prior to European settlement, elk are noted to have been among the most common and widely distributed wild ungulates in North America, but their numbers decreased due to hunting and reduction in habitat from settlement and farming.  Mule deer (Odocoileus hemionus) are found primarily in the western portion of North America, from southern Yukon and Mackenzie south through the western UnitedStates, extending east to Wisconsin and west Texas.  Prehistoric ranges may have extended further east.  White-tailed deer (Odocoileus virginianu) currently is the most widespread and numerous member of the Cervidae.  White-tailed deer are found throughout most of the United States in a variety of habitats, except for northern Arizona, southwest Colorado, northwest New Mexico, and most of California, Nevada and Utah, as well as in southern Canada, through Central America, and into northern South America.

Summary and Conclusions

Protein residue analysis was conducted on a fluted projectile point found at site CA-SDI-2506 in Lost Valley, San Diego County, California.  This point is believed to be associated with the Clovis Culture.  The point yielded a positive result to deer antiserum, suggesting that it was used to hunt a member of the deer family.  A negative result to deer antiserum for the soil control indicates that the positive result was not due to soil contamination.

TABLE 1
PROVENIENCE DATA FOR SAMPLES FROM SITE CA-SDI-2506

Sample
No.

Feature No.

Depth
(cmbs)

Provenience/Description

Analysis

4408

 

100

20S 6W; Obsidian fluted point

Protein residue

4395

 

103

20S 6W; Soil control from the west wall of unit, 20 cm west of fluted point

Protein residue

 

TABLE 2
LIST OF ANTISERA USED IN TESTING ARTIFACT AND SOIL CONTROL FROM SITE CA-SDI-2506

ANTISERA

SOURCE

POSSIBLE RESULTS

ANIMALS:

 

 

  Bear

ICN Pharmaceuticals, Inc.

Black bear, Brown bear, Grizzly, Polar bear

  Bison

Prepared under the direction of Dr. Richard Marlar at the University of Colorado Health Sciences Center

Bison, Domestic bovids

  Bovine

Sigma Chemical Company

Domestic bovids, Bison

  Camel

ICN Pharmaceuticals, Inc.

Camel, Llama, Prehistoric camelids

  Cat

Sigma Chemical Company

Domestic cat, Mountain lion, Bobcat, Lynx, other wild cat species

  Chicken

Sigma Chemical Company

Domestic chicken, Partridge, Quail, Grouse, Ptarmigan, Pheasant

  Deer

ICN Pharmaceuticals, Inc.

White tail deer, Mule deer, Elk, Moose, Caribou

  Dog

Sigma Chemical Company

Domestic dog, Coyote, Wolf, Fox

  Elephant

Robert Sargeant

Elephant, Mammoth

  Goat

Sigma Chemical Company

Pronghorn, Mountain goat, Domestic goat

  Horse

ICN Pharmaceuticals, Inc.

Horse, Donkey, Zebra, Extinct species of wild horse

  Human

ICN Pharmaceuticals, Inc.

Human

  Rabbit

Sigma Chemical Company

Rabbit, Jackrabbit (hare)

  Sheep

ICN Pharmaceuticals, Inc.

Domestic sheep, Bighorn sheep

  Turkey

Sigma Chemical Company

Domestic turkey, Wild turkey, Ducks

FISH:

 

 

  Striped bass

Robert Sargeant

Perciformes order (Spiny-rayed or percoid fish)

 

TABLE 3
POSITIVE PROTEIN RESIDUE RESULTS FOR SAMPLES
FROM SITE CA-SDI-2506

Sample
No.

 

Description

Positive Result
(Antiserum Type)

Possible Animal(s)
Represented

4408

Obsidian fluted point

Deer                       

Cervidae (Deer family)

References Cited

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Culliford, Brian J.
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Culliford, Brian J.
1971 The Examination and Typing of Bloodstains in the Crime Laboratory. United States Department of Justice, U.S. Government Printing Office, Stock 2700-0083, Washington D.C.

Gaensslen, R. E.
1983 Sourcebook in Forensic Serology, Immunology, and Biochemistry. United States Department of Justice, Washington D.C.

Moratto, Michael J.
1984 California Archaeology. Academic Press, Inc., Orlando, Florida.

Newman, M. and P. Julig
1989 The Identification of Protein Residues on Lithic Artifacts from a Stratified Boreal Forest Site. Canadian Journal of Archaeology 13:119-132.

Rickel, Bryce
2005 Chapter 2: Large Native Ungulates. In Assessment of Grassland Ecosystem Conditions in the Southwestern United States: Wildlife and Fish  Volume 2, edited by D. M. Finch, pp. 13-33. USDA Department of Agriculture, Forest Service, Rocky Mountain Research Station, General Technical Report RMRS-GTR-135.

Teer, J. G.
1996 The White-Tailed Deer: Natural History and Management. In Rangeland Wildlife, edited by P. R. Krausmann, pp. 193-209. The Society of Range Management, Denver, Colorado.

Warren, C. N., and M. G. Pavesic
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Weide, M. L.
1968 Cultural Ecology of Lakeside Adaptation in the Western Great Basin. Ph.D. dissertation, Department of Anthropology, University of California, Los Angeles.

Whitaker, John O., Jr.
1980 The Audubon Society Field Guide to North American Mammals. Audubon Field Guides. Alfred A. Knopf, Inc., New York, New York.

Wisdom, M. J., and J. W. Thomas
1996 Elk. In Rangeland Wildlife, edited by P. R. Krausmann, pp. 157-181. The Society of Range Management, Denver, Colorado.