Response to Comment by Poinar et al. on “DNA from Pre-Clovis Human Coprolites in Oregon, North America”

Abstract

The arguments of Poinar et al. neither challenge our conclusions nor would contribute to the verification of our data. We counter their questions about
the authenticity of our ancient DNA results and the reliability of the radiocarbon data and stand by the conclusion that our
data provide strong evidence of pre-Clovis Native Americans.

Full text

DOI: 10.1126/science.1168457
, 148-b (2009); 325Science et al.M. Thomas P. Gilbert,
America"
from Pre-Clovis Human Coprolites in Oregon, North
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Response to Comment by Poinar et al.
on “DNA from Pre-Clovis Human
Coprolites in Oregon, North America”
M. Thomas P. Gilbert,
1
Dennis L. Jenkins,
2
Thomas F. G. Higham,
3
Morten Rasmussen,
1
Helena Malmström,
1
Emma M. Svensson,
4
Juan J. Sanchez,
5
Linda Scott Cummings,
6
Robert M. Yohe II,
7
Michael Hofreiter,
8
Anders Götherström,
4
Eske Willerslev
1
*
The arguments of Poinar et al. neither challenge our conclusions nor would contribute to the
verification of our data. We counter their questions about the authenticity of our ancient DNA
results and the reliability of the radiocarbon data and stand by the conclusion that our data
provide strong evidence of pre-Clovis Native Americans.
In our study of the Paisley Cave coprolites (1),
we applied some of the most comprehensive
controls yet applied to ancient DNA. These
controls were stricter than those used by our critics
in related studies on coprolites, ancient humans,
or both (e.g., 2–5). Nevertheless, Poinar et al.(6)
challenge our study on several grounds. Although
ancient human DNA results can rarely be 100%
certain, and studies presenting noteworthy conclu-
sions should be challenged, the arguments presented
in (6) do not undermine our claims.
The initial arguments by Poinar et al.(6)focus
on sample contamination. Contamination has two
contexts with regard to our study. The first is that
most commonly raised in ancient DNA (aDNA)
studies—the recent contamination of ancient
human samples. Given the absence of evidence of
laboratory-derived contamination, we acknowl-
edged (1) that two likely sources exist: unpro-
tected handling at excavation or leaching while
still in the ground. As we stated, the samples
were contaminated through handling, although
not by sources of haplogroup (Hg) A and B
mitochondrial DNA (mtDNA). Poinar et al.(6)
question why we did not resolve who the con-
taminators were. As detailed in (1), the levels of
contamination and DNA degradation made this
impossible. Before we address the issue of leach-
ing, we highlight that there is an additional form
of contamination of equal importance: contami-
nation at time of origin. In a cave inhabited con-
temporaneously by multiple individuals (possibly
with canid companions), speculation based on hu-
man behavior would render it surprising if the
inhabitants did not defecate/urinate in collective
localities. We stated this in our original text,
presenting it as a reason for the presence of canid
DNA in some of the samples. We also note that
this scenario would still require a human presence
andthusisconsistentwithpre-Clovishumanoccu-
pation. As such, the controls in our study were
principally focused on the former (countering
leaching and modern handling). Poinar et al.’s
statement that a central tenet of our hypothesis
requires the presence of only one Native American
sequence in each sample (6) is inaccurate. The
central tenet of our hypothesis is that combined
genetic, nongenetic, and other evidence indicates
that recent contamination with Hg A or B se-
quences is unlikely.
Poinar et al. further note that we neglected to
provide primer sensitivity/optimization data or to
use quantitative polymerase chain reaction (qPCR).
Ignoring the fact that such data are rarely pro-
vided in genetic studies, including (2–4), this
challenge is not problematic. Our initial assay,
multiplex PCR with minisequencing (MPMS), is
a sensitive tool that detects contaminants at a 4%
threshold in aDNA extracts (7). Although our
PCR targets ranged from 50 to 105 base pairs
(bp), copy number of aDNA molecules increases
exponentially as size is decreased (4,8). Thus,
one might expect the smallest amplicons to be
most sensitive. In addition, Poinar et al.failto
observe that many of our results, in particular
both the MPMS and cloned Hg B results, derive
from our longest amplicon. Size notwithstanding,
the relative sensitivity of the different primers
themselves is also irrelevant here, because the
MPMS assay requires all eight primers to coam-
plify; the output is always eight sequenced
single-nucleotide polymorphisms (SNPs), which
simply differ by the state of each SNP. The only
conceivable way that sensitivity could be a prob-
lem in this context would be if the particular
derived versus ancestral SNP allele could suffi-
ciently affect the binding of primers that are po-
sitioned multiple bases away, which is highly
unlikely. With regard to a lack of qPCR, we are
unclear what the grounds for this argument are.
Historically, qPCR has been used to prevent
sequence errors derived from postmortem mis-
coding lesions. However, we adopted a simpler,
and widely accepted, alternative—the reproduc-
ibility of data. Indeed, Poinar and colleagues
have used this in several of their own studies on
coprolite aDNA (2,4).
Perhaps the biggest challenge facing our study
was proving that modern DNA had not leached
into the coprolites. Although the only comprehen-
sive means to conclusively rule out leaching is
screening all the cave soil for contaminants, this is
not realistic. We agree that, on their own, some of
the arguments against leaching are not watertight
(e.g., previous observations on DNA movement
in temperate soils and the diagnostic power of
protein). However, in combination, we believe our
arguments suggest that leaching is unlikely. We do
not feel, furthermore, that the criticisms offer any
additional solution to the problem, and we dis-
agree with several. It is not unusual that only 13 of
28 soil controls tested positive for human DNA,
given that, in contrast to the coprolites, they were
not directly handled but simply sampled into
storage containers. With regard to the wood rat
primer sensitivity, as detailed in (1), they are ef-
fective on ancient wood rat coprolites from the
site, and thus clearly work. Given this, and that
such coprolites constitute up to 80% of the sed-
iments in the cave, we argue that leaching is
unlikely.
Poinar et al.(6) also raise questions about the
Paisley Caves site, assemblages, and
14
C dating.
Direct dating indicates that the pre-Clovis assem-
blage includes a stemmed point, five nondiag-
nostic chipped stone tools, debitage, a hand stone
with horse protein residues, and a butcher-cut
grouse sternum. The assemblage is not Clovis but
is Paleoindian (9). Obsidian pre-Clovis artifacts
were subjected to hydration dating (OH). OH rate
variability is caused by a combination of inherent
characteristics and environmental variables (10).
Controlling for effective hydration temperature
(EHT) and employing mean group OH measure-
ments rather than individual measurements often
greatly improves OH rate accuracy and concor-
dance between
14
CandOHresults(11 –14). EHT
has been calculated for multiple microsettings at
the site by recording temperatures every 45 min
between 2005 and 2008. Although more work
needs to be done, we have observed good con-
cordance between matched OH and
14
C dates.
With regard to sedimentary disturbances, these
were generally traceable in Cave 5 (13). Sedi-
ments dip and thicken differentially toward the
cave center, causing substantial elevational var-
iation among penecontemporaneous specimens.
Site formation processes caused occasional intra-
stratigraphic age reversals. However, the general
TECHNICAL COMMENT
1
Centre for Ancient Genetics, University of Copenhagen,
Universitetsparken 15, 2100 Copenhagen, Denmark.
2
Museum
of Natural and Cultural History, 1224 University of Oregon,
Eugene, OR 97403–1224, USA.
3
Research Laboratory for
Archaeology and the History of Art, Dyson Perrins Building,
South Parks Road, Oxford OX1 3QY, UK.
4
Department of
Evolutionary Biology, Uppsala University, Norbyvagten 18D,
74236 Uppsala, Sweden.
5
National Institute of Toxicology and
Forensic Science, Canary Islands Delegation, 38320 Tenerife,
Spain.
6
Paleo Research Institute, 2675 Youngfield Street,
Golden, CO 80401, USA.
7
Department of Sociology and
Anthropology, California State University, 9001 Stockdale
Highway, Bakersfield, CA 93311, USA.
8
Max Planck Institute
for Evolutionary Anthropology, Deutscher Platz 6, 04103
Leipzig, Germany.
*To whom correspondence should be addressed. E-mail:
ewillerslev@bio.ku.dk
10 JULY 2009 VOL 325 SCIENCE www.sciencemag.org148-b
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integrity of deposits is well supported by the ma-
jority of stratigraphically correct dates obtained
on artifacts, bone collagen, and human coprolite
dates.
Our dated samples from Paisley Caves con-
sisted only of identifiable fibrous plant matter
carefully extracted from the human coprolites, as
stated in (1) [see the materials and methods and
figure S2 in the supporting online material for
(1)]. In their previous published work [e.g., (4)],
on the other hand, Poinar and colleagues homog-
enized coprolite remains before accelerator mass
spectrometry (AMS) dating. We suspect this to
be the source of their misinterpretation and con-
fusion over our results. The d
13
C values we pub-
lished do not relate to bulk coprolitic carbon, and
it is, therefore, impossible to infer general char-
acteristics of human diet from them and to then
link this with possible lacustrine reservoir effects.
The C3 plant d
13
C values do not provide evidence
that the humans are herbivores, as Poinar et al.(6)
imply. We selected only plant matter from the
coprolites for dating to avoid potentially proble-
matic bulked samples, which could conceivably
include carbon from soil or sediment of different
age. The variation in the two d
13
C values for
specimen 1374-PC-5/5D-31-2 may well reflect
small amounts from two different types of plant
matter being extracted and included in the analy-
sis, for example C3 (d
13
C ranging from ~−20 to
−35 per mil) and C4 plants (~−11 to −15 per mil).
Further material from 1294-PC-5/6B-40 is cur-
rently being AMS dated to confirm which of the
two ages thus far obtained (Beta-2134231 and
OxA-16376) is more reliable. We consider a fresh-
water reservoir effect to be unlikely in explaining
this difference, or to have a major effect on the
other results, but we will report new dating results
for this specimen in due course. We contend that
the radiocarbon results are (i) reproducible be-
tween two independent laboratories (with the ex-
ception of the 1294-PC-5/6B-40 sample noted
previously); (ii) not subject to a reservoir effect
because they yield d
13
C values, which are pre-
dominantly indicative of terrestrial C3 plant re-
mains; and (iii) of fibrous plant material which
ought to give reliable AMS determinations when
effectively pretreated.
In summary, although we accept that the
sample contamination makes our data set far
from perfect, we feel that the arguments pre-
sented in (6) would neither help resolve the
data nor seriously challenge our conclusions.
Ultimately, perhaps the only resolution may
come from new, sterile excavations at this
unique site.
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5 December 2008; accepted 22 April 2009
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