Journal of Archaeological Science (2002) 29, 301–306doi:10.1006/jasc.2001.0693, available online at http://www.idealibrary.com on

Re-examination of Ancient DNA in Texas Rock Paintings

E. J. Mawk, M. Hyman and M. W. Rowe

Department of Chemistry, Texas A&M University, College Station, TX 77843, U.S.A.

(Received 20 February 2001, revised manuscript accepted 14 May 2001)

An unsuccessful attempt to study ancient DNA from Pecos River genre rock paintings located in shelter 41VV75 in theLower Pecos River region of southwest Texas is described. We were unable to extract any ancient DNA from thesePecos River genre rock paintings, casting doubt on a previous study that reported extraction and characterization ofancient DNA in paint samples of the same genre at the same site. � 2002 Elsevier Science Ltd. All rights reserved.

Keywords: MITOCHONDRIAL DNA, CYTOCHROME C OXIDASE SUBUNIT II GENE, DNASEQUENCING, POLYMERASE CHAIN REACTION, PECOS RIVER GENRE ROCK PAINTINGS.

Introduction

T he Lower Pecos River region of southwestTexas, inhabited from approximately 11,000radiocarbon years ago up to the time of Spanish

contact (Hester, 1988; Turpin, 1990), is an area rich inprehistoric rock paintings. Thousands of the moreimpressive rock paintings in North America are on thewalls of the many rock shelters in the area. Five distinctgenres of rock paintings in the Lower Pecos Riverregion have been classified: Historic, Red Linear, RedMonochrome, Bold Line Geometric and Pecos River(Kirkland & Newcomb, 1967). Pecos River is the oldestgenre, with 19 samples having published radiocarbonages ranging from 2750 to 4200 years (Hyman &Rowe, 1997; Pace et al., 2000; and references therein).The images range from approximately one-third meterto five meters and are yellow, red and black in colour.

No ethnographic information is available on thereligious/social significance of the rock paintings in theLower Pecos River region. Only by studying the rockpaintings will researchers ascertain when, how andpossibly why, they were created (Turpin, 1994; Boyd,1998). The mineral compositions of the paint pigmentsare well determined: Fe oxides and hydroxides foryellow to reds to brown, and Mn oxide, and hydrox-ides, for black (Hyman, Turpin & Zolensky, 1996).Pictographs sampled were covered with mineral accre-tion layers that normally contain calcium carbonate(calcite), calcium oxalate (whewellite and weddellite)and occasionally gypsum (CaSO4·2H2O) (Hyman,Turpin & Zolensky, 1996; Russ et al., 1996; Mawk &Rowe, 1998; Russ et al., 1999). The paints appear to bewithin a biologically produced matrix (Russ et al.,1999).

The nature of any organic binder/vehicle(s), how-ever, are undetermined. Edwards, Drummond & Russ

3010305–4403/02/$-see front matter

(1998) used Fourier transform Raman spectroscopyand found organic matter with aliphatic �CH vibra-tions in black paint samples from two sites in theLower Pecos River region, but none in red pictographsthere. However, organic material in red paints fromthe same region have been found to contain organicmaterial of undetermined origin (see Hyman & Rowe,1997 for example).

To remedy this later situation, Reese et al. (1996)investigated the organic binder/vehicle using DNA-based methods. Ancient DNA was first extracted andcharacterized by Higuchi et al. (1984) from a museumspecimen of the extinct horse species Equus quagga.Ancient DNA is expected to degrade into fragments of250 bases or smaller (Lindahl, 1993). Many of thenucleotides making up ancient DNA are also chemi-cally altered, making amplification with PCR moredifficult (Gelenberg, Bickel & Weihs, 1996). Low copynumber, small fragment size and altered nucleotides inancient DNA makes modern DNA contamination aproblem. Relatively few molecules of modern DNAhave the potential to mask any ancient DNA duringPCR amplification. According to Austin et al. (1997)some amberized insect and plant ancient DNA reports(Cano et al., 1993; Cano & Borucki, 1995; DeSalleet al., 1992; Poinar, Cano & Poinar, 1993) were theresult of modern DNA contamination. Woodward,Weynard & Bunnell (1994) reported extracting andamplifying ancient DNA from dinosaur bone fragments,but other researchers (Hedges & Schweitzer, 1995;Henikoff, 1995; Zischler et al., 1995; Allard, Young& Huyen, 1995) showed that the Cytochrome b genesequence obtained by Woodward, Weynard & Bunnellwas not ancient dinosaur DNA, but the result of eithermodern or ancient mammal DNA contamination.

Reese et al. (1996) performed DNA analysis on twoPecos River genre rock paintings using polymerase

� 2002 Elsevier Science Ltd. All rights reserved.

302 E. J. Mawk et al.

chain reaction (PCR) and phylogenetic analysis in anattempt to determine the organic binder/vehicle source.Using primers for a fragment of the Histone 4 gene, ahighly conserved gene found in the nuclear genome ofevery plant and animal, Reese et al. determined that amember of the Order Artiodactyla was utilized in thepreparation of the paints for the sampled rock paint-ings. Artiodactyls are hoofed, even-toed mammalssuch as bison and deer. A more specific determinationwas not possible due to the conservative nature of theHistone 4 gene. The research presented here attemptedto replicate the work of Reese et al. (1996) and toextend the determination of the organic binder/vehiclesource to the species level. However, we were unableto confirm ancient DNA in the rock paintings andreluctantly conclude that the results of Reese et al. wereprobably contamination.

Experimental Method

Sample collection

We collected Pecos River genre rock art from site41VV75, Seminole Canyon State Historic Park, Texas.To minimize damage to the art, we took samplesof rock paintings that appeared about to spall. Weminimized modern DNA contamination from casualcontact by sampling rock paintings located high on therock shelter walls. Unpainted rock adjacent to sampledrock painting samples was collected to allow monitor-ing of background DNA contamination. All sampleswere levered off the rock wall with a chisel. The chiselwas cleaned with bleach followed by ethanol andacetone rinses, allowed to air dry, and wrapped intreated Al foil. All Al foil was treated first with anacetone rinse, followed with an ethanol rinse, andallowed to air dry. All samples were wrapped in treatedAl foil and placed in previously unused plastic bags fortransport to the laboratory.

Gene and primer selectionThe previous work of Reese et al. (1996) failed toachieve a species level determination due to a lack ofsufficient base pair sequence variation in the Histone 4gene. To achieve our goal of a species level determi-nation for the organic binder/vehicle source material ofPecos River genre rock art, we chose the mitochondrialgene Cytochrome C Oxidase Subunit II (COII). TheCOII gene was chosen for two main reasons: (1) TheCOII gene is located in the genome of the mitochon-dria, an organelle found in the cells of eukaryotes.Each eukaryotic cell contains hundreds to thousandsof mitochondria, which increases the odds of findingenough ancient DNA molecules to amplify. Being aeukaryotic gene, COII excluded bacterial DNA, whichwe would expect to find growing on the pictographsurfaces, as a source of contamination. (2) Mitochon-drial genes, COII specifically, are known to be highly

variable in their base pair sequences, even betweenclosely related species (Vawter & Brown, 1986). Janecket al. (1996) used COII to study the population geneticsof the artiodactyl subfamily bovinae and Honeycuttet al. (1995) used COII and Cytochrome b gene tostudy mammalian mitochondrial DNA evolution. Thisdegree of variability should allow for a species leveldetermination.

The COII gene is 684 bases in length, about 400bases more than the largest expected fragment ofDNA, �250 bases (Lindahl, 1993). We needed toselect primers, small pieces of DNA 10–20 bases inlength, for a region of the COII gene under 250 bases.Our selection task was to locate/identify a highlyvariable base pair region bracketed on each side byhighly conserved base pair regions. The conservedregions would define the universal primer sites and thehighly variable region would give us the required basepair sequence variability to obtain a species determi-nation. To identify the conserved region, we collectedCOII sequence information for past and presentspecies common to the Lower Pecos River region.The following species were used to design the COIIFragment primers: cattle (Bos taurus), white-taildeer (Odocoileus virginianus), mule deer (Odocoileushemionus), human (Homo sapiens), pronghorn antelope(Antilocapra americana), javalina (Tayasu tajacu),rabbit (Oryctolagus cuniculis) and goat (Capra hircus).We chose goat, cattle, javalina and human as possibleDNA contamination sources. All COII sequences arefrom GenBank (an online database of DNA sequencedata maintained by the National Center for Biotech-nology Information, http://www.ncbi.nlm.nih.gov/Genbank/index.html), with the exception of mule deer,rabbit and javalina. The later were determined in ourlaboratory with previously established COII primers(Janeck et al., 1996). From the COII sequence data, theCOII Fragment forward (F) and reverse (R) primerswere designed. COII Fragment F has the sequence5�-TA GAT GCC CAA GAR GTR GAR AC-3� andCOII Fragment R has the sequence 5�-ATA GTC TGTGTA TTC RTA RCT TCA-3�. R indicates the equalprobability of a G or a C in that location. The COIIFragment primers amplify PCR product 127 bases inlength and were tested with DNA from all species ofinterest. In addition elk/red deer (Cervus elephus),sheep (Ovis aries) and American bison (Bison bison)DNA’s were included to verify that the primers workeduniversally as designed. The COII Fragment primerssuccessfully amplified all DNA. We determined anoptimal annealing temperature of 53�C for the COIIFragment primer set. The COII Fragment primerswere synthesized by Genosys Biotechnologies, Inc.(1442 Lake Front Circle, Suite 185, The Woodlands,TX 77380–3600). Initial work with these primers failedto produce any PCR product with extracts from PecosRiver genre rock paintings. We believed that theancient DNA extracts did not contain enough templateto produce enough product to be seen with agarose gel

Re-examination of Ancient DNA in Texas Rock Paintings 303

electrophoresis. To work around the lack of COIIFragment product, we designed and employednested PCR primers to reamplify COII Fragment PCRproducts.

DNA extractionPrecautions (Handt et al., 1994) to control modernDNA contamination were used throughout this work.Two laboratories that had never been used foreukaryotic DNA work were utilized, an advantage thisresearch has over the earlier work of Reese et al. Allequipment and laboratory surfaces were cleaned withsodium hypochlorite solution and/or ethanol. Aerosolresistant pipette tips were used with all pipetters toprevent cross contamination (Intermountain ScientificCorporation, 420 North Kays Drive, Kaysville, UT84037). Extraction blanks, extractions performedwithout a sample, were used throughout this researchto monitor background DNA contamination in thelaboratory. We included an extraction blank with everyancient DNA extraction attempt. The Pecos Rivergenre paint samples and unpainted rock backgroundsamples were treated to multiple extraction attemptswith the three extraction protocols.

Extraction protocol one. In our first extraction proto-col, the surface layer of each sample was removed. Thepainted and unpainted rock samples were placed inseparate sterile 100 m by 20 m disposable Petridishes. The surfaces of each sample were wetted with500 �l of phosphate buffered saline (PBS, 8·0 g NaCl,0·2 g KCl, 1·44 g Na2HPO4, 0·24 g KH2PO4 in 800 mlof distilled H2O, pH adjusted to pH 7·4 with HCl,diluted to 1 l and autoclaved to sterilize). Using a newsterile razor blade (sterilized with an ethanol rinse andair-dried), we removed surface material from eachsample. Removed surface material was transferred toan Eppendorf tube for DNA extraction with the‘‘Dried Blood, CSF, and Bone Marrow on Hemato-logical Slides’’ protocol, included with the QIAampTissue Extraction Kit (Cat No. 29306, QIAGEN Inc.,28159 Avenue Stanford, Santa Clarita, CA 91355).Extraction blanks were prepared by pipetting 500 �lof PBS into a clean and sterile petri dish, swirling thePBS around the inside of the petri dish, and transfer-ring the PBS to an Eppendorf tube extraction. Weextracted two red pictograph samples twice with thisprotocol.

Extraction protocol two. After failure to extract ancientDNA with protocol one, we attempted to extract DNAwith the extraction protocol described by Reese (1994).The extraction technique of Reese (1994) dependedupon extracting ancient DNA directly from the rockart sample, without removing the pigment from therock fragment. After one extraction attempt with thisprotocol, we switched to using a pH 7·5 Tris buffer(60·25 g of Tris in 400 ml of DIUF water with the pH

adjusted to 7·5 with 6 molar HCl) instead of the pH 4·1citrate buffer as described by Reese. Duplicate extrac-tion attempts were made on four red pictographsamples.

Extraction protocol three. The third extraction protocolcombined elements of extraction protocols one andtwo. There were three variants. (1) We initially usedpieces of painted and unpainted rock samples withoutscraping off any material. We selected rock piecessmall enough to fit in a 15 ml Falcon tube and intro-duced enough pH 4·1 extraction buffer solution (Reese,1994) to just cover the samples. An extraction blank of5 ml pH 4·1 extraction buffer was prepared. After 7days we removed 500 �l of the extraction buffer, cen-trifuged the aliquot for 1 min and transferred the clearsupernatant to a 1·7 ml Eppendorf tube. The ‘‘DriedBlood, CSF, and Bone Marrow on HematologicalSlides’’ protocol was used to concentrate and purifyany extracted DNA from the supernatant. We madeone extraction attempt on a red pictograph sampleusing this protocol.

(2) We removed surface material from samples witha sterile razor blade, as described above. The removedmaterial was transferred to an Eppendorf tube, centri-fuged to pelletize the rocky material, and the excessPBS was removed. Five hundred �l of pH 4·1 extrac-tion buffer was added, the tubes capped and eachsample allowed to soak for 9 days. The Eppendorftubes were then centrifuged and 400 �l of supernatantremoved for treatment with the ‘‘Dried Blood, CSF,and Bone Marrow on Hematological Slides’’ protocol.We made one extraction attempt re-using the redpictograph sample from the first variant with thisprotocol.

(3) Again we removed the surface material with arazor blade, after wetting the surface of each samplewith 1 ml of d2aH2O. Removed surface material wastransferred to an Eppendorf tube, and the remainingsample rinsed with 500 �l of d2aH2O. The samplerinse was collected and transferred to the sample tube.The extraction blank consisted of 1·5 ml of d2aH2O.We centrifuged each sample to congregate therocky material, and then removed the excess d2aH2O.Then 1 ml of pH 7·5 extraction buffer (describedabove) was added to each sample. The Eppendorftubes were secured inside a sterile petri dish, which wassealed with parafilm, and secured to the bed of aForma Scientific Orbital Shaker, model 4520 (FormaScientific, PO Box 649, Millcreek Road, Marietta,OH 45750). The samples were shaken at 225 rpm for7 days, after which they were removed, centrifuged topellet the rocky material and a 1 ml aliquot wasremoved for DNA extraction with a QIAquick PCRPurification Kit (Cat No. 28104, QIAGEN, Inc., 28159Avenue Stanford, Santa Clarita, CA 91355). Wemade one extraction attempt, using four new samples,three red and one mixed red and black, using thistechnique.

304 E. J. Mawk et al.

PCR amplificationEach ancient DNA extract was amplified with theCOII Fragment primers and the COII Nest primer sets.We prepared PCR with 25, 50 and 100 �l reactionvolumes. The 100 �l reactions were prepared by mixing10 �l 15 m MgCl2, 10 �l Taq buffer, 1 �l 10 pmole/�ldNTP, 1 �l forward primer (50 pmole/�l), 1 �l reverseprimer (50 pmole/�l) and 0·1 �l Taq polymerase (0·5units of enzyme). Typical ancient DNA template vol-umes were 1, 2, 5 and 20 �l. The reactants and ancientDNA sample were diluted with enough d2aH2O toachieve 100 �l. We prepared the 25 and 50 �l PCRvolumes by scaling down the 100 �l reaction volumesappropriately. A volume of 0·1 �l Taq polymerase wasused regardless of reaction volume. Each reaction wascovered with a layer of Nujol Mineral Oil (PE AppliedBiosystems, Foster City, CA 94404) if needed to pre-vent evaporation. All reagents were purchased fromPromega (Promega Corporation, 2800 Woods HollowRoad, Madison, WI 53711–5399). The Taq polymeraseand the Taq buffer B were used as received. We dilutedthe MgCl2 from 25 m to 15 m. The dNTP’s(100 m) were aliquoted and diluted to 10 m in eachdNTP (cATP, cCTP, cGTP, cTTP). All solutions wereprepared with d2aH2O and autoclaved before use.Positive (1 �l goat DNA) and negative (no DNAadded) controls were used with all PCR’s.

We used three DNA thermocycler models for PCR:(1) A Perkin Elmer Model 2400 DNA Thermocycler;(2) a GenAmp� PCR System 9700; and (3) a PerkinElmer Cetus DNA Thermal Cycler (PE Applied Bio-systems, Foster City, CA 94404). The reaction volumesused for each model were 25, 50 and 100 �l respect-ively. Mineral oil was only used with the Perkin ElmerCetus DNA Thermal Cycler. The following tempera-ture program was used across all models. The DNAwas denatured at 94�C for 30 s, the reaction tempera-ture was lowered to the primer annealing temperature(45�C to 55�C) and maintained for 1 min, and the cyclefinished with a 4 min temperature ramp to 72�C, main-tained for 1 min. After 35 cycles were performed, thereaction temperature was maintained at 72�C for10 min and then stored indefinitely at 4�C.

Gel electrophoresis and DNA sequencingAll PCR products were separated on a 1–4% (wt/v)agarose gel. A 1% gel is composed of 0·5 g agarose in50 ml of 1� TEA buffer (1:20 dilution of 20� TEA,96·8 g 2-amino-2-hydroxymethyl-1,3-propanediol and14·9 g Na2EDTA·H2O dissolved in 1·5 l d2aH2O, pHadjusted to 8·0 with acetic acid, diluted to 2 l andsterilized by autoclaving). A 10 �l aliquot of PCRproduct is placed in a sample well and electrophoresedwith 100 milliamps for 20–30 min in 200 ml of 1�TEA buffer. Bromcresol blue was the tracking dye. Amolecular size standard of enzyme digested P BlueScript or � DNA was used to gauge PCR product size.Ethidium bromide was used to visualize the gel.

For an ancient DNA extraction PCR that generateda product in a reaction containing ancient DNA anddid not amplify a product in the negative, the ancientDNA extract PCR mixture was purified with aQIAquick PCR Purification Kit (QIAGEN, Inc.,28159 Avenue Stanford, Santa Clarita, CA 91355). TheQIAquick PCR Purification Kit prepares the PCRproduct for sequencing by removing excess primersand reaction ingredients. Five �l of the purified prod-uct was electrophoresed on a 1% agarose gel against5 �l of PGEM, a DNA concentration standard(0·2 g/l). PGEM was included with the Applied Biosys-tems Incorporated ABI/Prism DNA Sequencing Kit,designed to be used with the ABI Prism 377 DNASequencer we used (Perkin-Elmer, Applied Biosystems,850 Lincoln Centre Drive, Foster City, CA 94404).Comparison of band intensities between the purifiedPCR product and PGEM indicated the quantity ofpurified PCR product to use in a sequencing reaction(5–10 �l). Each sequencing reaction had a volume of20 �l, 8 �l sequencing premix, 1 �l primer, 5–10 �l PCRproduct and the balance d2aH2O. For each PCRproduct, we prepared two sequencing reactions, onewith the forward primer and one with the reverseprimer. The sequencing reactions were thermocycledfor 24 cycles with the following temperature program:96�C for 30 s, 50�C for 15 s, and then 60�C for 4 min.

After cycle sequencing was complete, we purified thesequencing products using Centri-Sep spin columns(Princeton Separations, P.O. Box 300, Adelphia,NJ 07710). The purified sequencing products werethen concentrated and dried using a Savant SpeedVac Concentrator SVC100H connected to a SavantRefrigerated Vapor Trap RVT400 (Savant Instru-ments, Inc. 100 Colin Drive Holbrook, NY 11741–4306). The dried products were submitted forsequencing with an ABI Prism 377 DNA Sequencer(Perkin-Elmer, Applied Biosystems, 850 LincolnCentre Drive, Foster City, CA 94404).

After sequencing, the information was imported intoMacVector (Version 4.5.3, Kodak, Scientific ImagingSystems, 4 Science Park, New Haven, CT 06511) andcompared to known COII sequence information. If thesequence information compared favourably, it wasimported into PAUP 3.11 (Swofford, 1993) where theforward and reverse sequences were aligned. Thealigned sequences were combined into an overallsequence and added to the database of COII Nest PCRproducts for later analysis.

CloningFor PCR products too short to be sequenced cleanly,the product must be cloned. The ABI 377 Automatedsequencer cannot accurately determine the first 10–20bases of the PCR product it is sequencing. One way toget around this shortcoming is to sequence the PCRproduct with the forward and reverse primers. The last10–20 bases of the reverse sequence are substituted for

Re-examination of Ancient DNA in Texas Rock Paintings 305

the first 10–20 bases of the forward sequence when thereverse sequence is reversed and complimented.Another method is cloning of the COII Nest PCRproducts with the TA Cloning Kit (InvitrogenCorporation, 1600 Faraday Avenue, Carlsbad,CA 92008). We ligated the COII Nest PCR productinto the pCR�2.1 plasmid vector, and transformed theligated product into One Shot� competent cells withheat shock. Fifty and 200 �l aliquots of transformedcompetent cells were plated on 15 m�150 m sterileL-broth petri dishes. L-Broth contains 100 �g/ml IPTG(isopropyl-beta-d-thiogalactopyranoside), 20 �g/mlampicillin and 40 �g/ml X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). We incubated theplates overnight at 37�C, afterward placing the plates ina 4�C cold room to allow blue/white colony develop-ment. Ten white colonies were picked and replated. Wesequenced any remaining replated white colonies withPuc primers. Puc primers are designed to anneal topCR 2.1 vector and amplify across the PCR productinsert. After sequencing, the plasmid DNA and primersequences were identified and removed and the COIINest sequences aligned.

Results and DiscussionUsing the three extraction techniques described earlier,a total of nine extractions were performed. Eachextraction included an extraction blank (no rocksample), a pictograph sample, and an unpainted rocksample. All these extractions failed to amplify anyDNA using only the COII Paint primers. At that point,the COII Nest primers were employed and some COIIPaint PCR products were amplified. However, PAUP3.11 analysis and BLAST (Altschul et al., 1997)searches of the collected DNA sequences indicatedmodern DNA contamination. The collected COII datafailed to group together during the PAUP analysis aswas expected. The lack of grouping indicated an incon-sistent source of DNA. Additional DNA sequenceinformation was subjected to a BLAST search. BLASTsearches of multiple sequences from the samePecos River genre sample returned inconsistentdeterminations for the source material.

ConclusionsBased on the lack of positive results, we are forced toconclude that the work of Reese et al. (1996) was inerror. How the Reese et al. study may have erred is notclear. We cannot definitely prove that ancient DNAdoes not exist in Pecos River genre rock art, only thatwe did not detect it.

AcknowledgementsThe researchers would like to acknowledge the helpof Trina Guerra, Charlie Young, Dr Cathy Lehn,

Dr Susan Tanksley, Melanie Wike, Dr Sara Davis andDr Scott Davis. We would also like to thankDr Baldwin and his laboratory for the use of theirfacilities. Support for this work was provided by anInterdisciplinary Grant, Texas A&M University. Wereceived permission for sampling from the TexasHistorical Commission on the Texas Department ofParks and Wildlife and Fisheries permit.

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