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Ancient Mammalian and Plant DNA from Late Quaternary Stalagmite Layers at Solkota Cave, Georgia

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Metagenomic analysis is a highly promising technique in paleogenetic research that allows analysis of the complete genomic make-up of a sample. This technique has successfully been employed to archaeological sediments, but possible leaching of DNA through the sequence limits interpretation. We applied this technique to the analysis of ancient DNA (aDNA) from Late Quaternary stalagmites from two caves in Western Georgia, Melouri Cave and Solkota. Stalagmites form closed systems, limiting the effect of leaching, and can be securely dated with U-series. The analyses of the sequence data from the Melouri Cave stalagmite revealed potential contamination and low preservation of DNA. However, the two Solkota stalagmites preserved ancient DNA molecules of mammals (bear, roe deer, bats) and plants (chestnut, hazelnut, flax). The aDNA bearing layers from one of the two Solkota stalagmites were dated to between ~84 ka and ~56 ka BP by U-series. The second Solkota stalagmite contained excessive detrital clay obstructing U-series dating, but it also contained bear bones with a minimum age of ~50 BP uncalibrated years and ancient DNA molecules. The preservation of authentic ancient DNA molecules in Late Quaternary speleothems opens up a new paleogenetic archive for archaeological, paleontological and paleoenvironmental research.
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Ancient Mammalian and Plant DNA
from Late Quaternary Stalagmite
Layers at Solkota Cave, Georgia
M. C. Stahlschmidt1,2, T. C. Collin3, D. M. Fernandes4,5, G. Bar-Oz6, A. Belfer-Cohen7, Z. Gao8,
N. Jakeli9, Z. Matskevich10, T. Meshveliani9, J. K. Pritchard8,11,12, F. McDermott13 & R. Pinhasi
4
Metagenomic analysis is a highly promising technique in paleogenetic research that allows analysis
of the complete genomic make-up of a sample. This technique has successfully been employed to
archaeological sediments, but possible leaching of DNA through the sequence limits interpretation. We
applied this technique to the analysis of ancient DNA (aDNA) from Late Quaternary stalagmites from
two caves in Western Georgia, Melouri Cave and Solkota. Stalagmites form closed systems, limiting the
eect of leaching, and can be securely dated with U-series. The analyses of the sequence data from the
Melouri Cave stalagmite revealed potential contamination and low preservation of DNA. However, the
two Solkota stalagmites preserved ancient DNA molecules of mammals (bear, roe deer, bats) and plants
(chestnut, hazelnut, ax). The aDNA bearing layers from one of the two Solkota stalagmites were
dated to between ~84 ka and ~56 ka BP by U-series. The second Solkota stalagmite contained excessive
detrital clay obstructing U-series dating, but it also contained bear bones with a minimum age of ~50 BP
uncalibrated years and ancient DNA molecules. The preservation of authentic ancient DNA molecules in
Late Quaternary speleothems opens up a new paleogenetic archive for archaeological, paleontological
and paleoenvironmental research.
Ancient DNA (aDNA) genomics is a valuable information source on past biological diversity and evolutionary
trajectories of species13. A particular focus has been on the analysis of human bones yielding high coverage
genomes of archaic humans46 and enabling novel insights into human dispersals and migrations79. Additionally,
several studies employed a metagenomic approach to the study of DNA sequence data retrieved from soils and
sediments from various environments, including caves10, lakes11, arid12 and arctic environments13,14. Slon et al.15
using a shotgun sequencing approach and analysing the deamination pattern for identication of authentic
ancient DNA16, reported on the recovery of archaic human aDNA as well as other mammalian aDNA from
archaeological deposits at several sites. is metagenomic research shows that not only bones but many other
components of the archaeological and paleontological record, such as deposits themselves, may serve as a preser-
vation medium for ancient DNA.
e retrieval of authentic aDNA strands from deposits is made possible by the binding of DNA to various
sediment and soil components, including clays1719, silica20,21, humic acids22 and calcite23. However, soil chem-
istry, e.g. pH20, and soil transformation processes, such as the dissolution and precipitation of minerals, greatly
impacts preservation. Furthermore, post-depositional movement of sediment components through turbation,
such as bioturbation, as well as other soil translocation processes, such as clay illuviation, may negatively impact
the integrity and complicate the interpretation of aDNA found in sediments and soils24,25.
1Department of Human Evolution, Max-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany. 2School
of Archaeology, University College Dublin, Dublin, Ireland. 3School of Medicine, University College Dublin, Dublin,
Ireland. 4Department of Evolutionary Anthropology, University of Vienna, Vienna, Austria. 5CIAS, Department of Life
Sciences, University of Coimbra, Coimbra, Portugal. 6Zinman Institute of Archaeology, University of Haifa, Haifa,
Israel. 7Institute of Archaeology, The Hebrew University of Jerusalem, Jerusalem, Israel. 8Department of Genetics,
Stanford University, Stanford, USA. 9Department of Prehistory, Georgian State Museum, Tbilisi, Georgia. 10Israel
Antiquities Authority, Jerusalem, Israel. 11Departments of Biology, Stanford University, Stanford, USA. 12Howard
Hughes Medical Institute, Stanford University, Stanford, USA. 13School of Earth Sciences, University College
Dublin, Dublin, Ireland. Correspondence and requests for materials should be addressed to M.C.S. (email: mareike_
stahlschmidt@eva.mpg.de) or R.P. (email: ron.pinhasi@univie.ac.at)
Received: 17 January 2019
Accepted: 15 April 2019
Published: xx xx xxxx
OPEN
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Speleothems are another potential source for aDNA and have long been explored as paleoenvironmental
archives using other methods, mainly stable isotopes studies and U-series dating26. Paleoenvironmental studies
most commonly use stalagmites, which form on the cave oor below a drip and in which calcite precipitates in
distinctive and continuous layers. Preservation conditions for DNA are ideal inside the stalagmites and especially
for those located deeper inside caves, where low temperatures limit the production of reactive oxygen species27,
there is little exposure to UV light28 and a stable pH as well as very low permeability inside the stalagmite and
hence a low risk of DNA migration between consecutive stalagmite layers. Few DNA studies have been conducted
on speleothems and they are mainly restricted to the surface of speleothems2931 with the exception of a study by
Zepeda Mendoza et al.32, who analysed two samples from the inside of popcorn calcite from a dolerite granite
gneiss cave. However, while they reported that aDNA was preserved inside the speleothems, they concluded that
this type of speleothem is unsuitable as a biological paleoarchive32. ‘Popcorn’ calcite exhibits rather irregular
and complex multi-dimensional growth patterns compared with the relatively simple sequential deposition of
consecutive layers in stalagmites, making the latter a geometrically simpler and therefore more reliable archive.
We here present a rst metagenomic study exploring aDNA metagenomics combined with U-series dates of
stalagmites from two caves from Western Georgia, Solkota and Melouri Cave, as archives on species that inter-
acted with or inhabited these cave systems. In 2016, we surveyed six caves in the Imereti region of Georgia (Fig.1):
three archaeological cave sites - Satsurblia Cave33, Dzudzuana34, Kotias Klde35 - and three non-archaeological
cave sites - Melouri Cave, Datvi Cave, Solkota. e latter three caves contained cave bear bones, but were not
archaeologically explored, and only these sites had favourable speleothems for the aim of this study. Each of the
three archaeological cave sites had a large entrance, permitting light and air to enter into the cave, which typically
makes them less suitable for quantitative paleoenvironmental reconstructions based on stable isotope studies26.
We therefore chose to proceed in our analysis with one stalagmite from Melouri Cave (MEL) and with two stalag-
mites from Solkota (SKK) (Fig.2). Ancient DNA was detected in several locations inside the two Solkota stalag-
mites (SKK 16 3 and 5). However, analysis of the stalagmite from Melouri Cave revealed potential contamination
and low preservation of DNA (see results below, SI Text 1 and Fig.1) and we focus here on the Solkota samples.
Solkota cave lies near the village of Kumistavi above the river Semi. Solkota is part of the same karst system as
Satsurblia Cave, Melouri Cave and Datvi Cave, the Tskaltubo karst system in the Sataplia-Tskaltubo Limestone
Massif36. e cave entrance of Solkota is located in a sinkhole with a very steep slope and little light penetrat-
ing into the cave entrance. e rear of the cave consists of a steep, muddy slope leading upwards with bedrock
exposed at the top of the slope. Another, former entrance may have been present here. Next to limestone boul-
ders the cave contains clay-rich mud and water concentrated in ponds and rills. e cave is rich in speleothems
(stalagmites, owstone, stalactite, curtains, straws) as well as in bone and we also found three lithic akes. Bones
are oen exposed in rill beds and we collected 40 bones. One bone was identied as capra, two as canids and the
remaining 37 as cave bear (Ursus spelaeus or Ursus deningeri). We also observed several bear hibernation dens in
the inner parts of the cave. We collected one large stalagmite, which had been growing on top of three cave bear
long bones (SKK 16 5) from the secondary context of a rill bed (Fig.2B,C). e bones comprised of right and le
distal humerus and a distal sha of a tibia. Carnivore gnaw marks were observed on the surface of the bones. We
collected a second stalagmite (SKK 16 3) from the top of the slope at the rear of the cave, close to its potential root
(Fig.2A).
Results
U-series. Uranium concentrations in speleothem SKK 16 3 are relatively low, typically in the range 35–70
ppb (Table1). ree samples from SKK 16 3 (3/10, 3/6.5, 3/5) (Fig.3) have high 232 contents (c. 42–77 ppb)
resulting in low (230/232) values (between 2.1 and 3.2), and therefore unacceptably large age uncertainties
aer corrections for detrital thorium have been applied (SI Table1). Similarly, the 232 contents for all samples
from SKK 16 5 (Fig.4) are too high to calculate ages for this speleothem (SI Table1). However, six samples from
Figure 1. Location of the study sites. e studied cave sites are located in Western Georgia (map created with
ASTER GDEM59): (1) Location of Satsurblia, Solkota, Melouri and Datvi Cave; (2) Location of Dzudzuana and
Kotias Klde.
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SKK 16 3, corresponding to d (depth from top) values of 17.5, 13.2, 6, 5.5, 4 and 1.7 cms, have low 232 contents
yield moderately high (230/232) ratios in the range 14.7–148 (Table1), permitting the calculation of precise
U-series ages following correction for the detrital clay component. As discussed in the Methods section, clay-
rich samples from the cave were measured separately using a total dissolution approach to constrain the actual
(230/232) value of the detrital component in the speleothems, considerably reducing the uncertainties in the
corrected U-series ages compared to the standard approach of simply assuming a (230/232) value for the detri-
tal component. Overall, detrital corrected U-series dates for the key stalagmite SKK 16 3 from Solkota range from
83.79 ± 0.64 ka at a depth from top (d) of 13.2 cm to 50.02 ± 0.68 ka at a d of 1.7 cm (Fig.3). In detail however,
considerable complexity in the speleothem’s growth history is evident.
e date from the sample closest to the base of the speleothem (17.5 cm d) yields an age of 80.26 ± 1.87 ka,
out of stratigraphic order, and just outside the error limits of the next three dates above (83.79 ± 0.64, 84.57 ± 0.76,
83.32 ± 1.48 at ds of 13.2, 6 and 5.5 cms respectively, Fig.3). is may indicate some minor post-depositional
migration of uranium in the lower section of the speleothem. Regardless, the similarity of the next three dates (all
three within their 2σ errors) indicates an interval of very rapid speleothem growth around 84 ka and no detectable
post-depositional uranium migration. Warm, wet intervals favour high speleothem growth rates and we note that
this time interval coincides with climatic amelioration during Greenland Interstadial 21.1e (GI-21.1e)37 during
Marine Isotope Stage (MIS) 5a.
Figure 2. Sampling locations of the Solkota Cave stalagmites SKK 16 3 and 5 (photos taken by MCS). (A) e
nd spot of SKK 16 3 (red circle) next to its possible root (white arrow). Note the scarcity of sediment here.
(B) Discovery location of SKK 16 5 (red circle) in a rill bed next to multiple bone remains (blue dots). (C)
Stalagmite SKK 16 5 with cave bear bones at its base.
Sample 238U ppb (230/238U) (234U/238U) (230/232) 232 ppb Age ka
uncorrected Age ka
corrected
SKK16 3/17.5 37.083 ± 0.003 0.6317 ± 0.0020 1.1221 ± 0.0009 14.70 ± 0.04 4.8733 ± 0.0043 88.63 ± 0.54 80.26 ± 1.87 1.84
SKK16 3/13.2 35.263 ± 0.004 0.6138 ± 0.0019 1.1245 ± 0.0014 148.10 ± 0.41 0.4466 ± 0.0030 84.59 ± 0.55 83.79 ± 0.64 0.63
SKK16 3/6 35.34 ± 0.02 0.6140 ± 0.0017 1.1087 ± 0.0012 59.414 ± 0.153 1.1162 ± 0.0003 86.64 ± 0.50 84.57 ± 0.76 0.75
SKK16 3/5.5 64.113 ± 0.005 0.6338 ± 0.0011 1.1156 ± 0.0010 19.086 ± 0.031 6.5056 ± 0.0012 89.95 ± 0.36 83.32 ± 1.48 1.46
SKK 16 3/4 53.907 ± 0.003 0.5001 ± 0.0008 1.1546 ± 0.0007 17.157 ± 0.026 4.8015 ± 0.0012 61.07 ± 0.18 56.69 ± 0.95 0.94
SKK16 3/1.7 38.183 ± 0.005 0.4510 ± 0.0009 1.1658 ± 0.0018 23.266 ± 0.047 2.2618 ± 0.0004 52.72 ± 0.24 50.02 ± 0.68 0.67
Table 1. U-series data for speleothem SKK 16 3. Parentheses denote activity ratios. Dates reported in this table
are considered reliable aer detrital corrections have been applied (see SI Table 1 for dates strongly aected
by detrital correction and with no reliable age calculation). e following decay constants were used: 230:
9.1577E-6, 232: 4.9475E-11, 234U: 2.826E-6, 238UE 1.551E-10. e nal column on the right hand side shows
the ages calculated aer correction for detrital thorium using a measured (230/232) value of 0.95 ± 0.1 for the
detrital end-member.
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Examination of the cut surface of stalagmite SKK16 3 reveals the presence of three distinctly visible deposi-
tional hiatuses at ds of 4.5 (hiatus 1), 4.8 (hiatus 2) and 5.6 cms (hiatus 3) (Fig.3). ese provide clear evidence
that the speleothem growth was discontinuous above the interval dated at 83.32 ± 1.48 ka. e DNA sample SKK1
is located between hiatus 3 and the combined hiatus 1 and 2. Consequently, the reliable bracketing ages for SKK 1
are 84.57 ± 0.76 ka at 6 cms d (older layer), 83.32 ± 1.48 ka at 5.5 cms d (same layer) and 56.7 ± 0.95 ka at 4 cms
d (younger layer) (Table1, Fig.3). e latter date corresponds to a warm MIS3 interval in the N. Hemisphere
(GI-16.1). e DNA sample SKK3 was taken from the same spot as the u-series sample at 17.5 cm d (Fig.3) with
an age of around 80.26 ± 1.87 ka and is capped by the u-series age 84.57 ± 0.76 ka at 6 cms d (Table1).
Radiocarbon. A fragment of bone from the bottom of stalagmite SKK 16 5 was sent for AMS radiocarbon
dating at the Research Laboratory for Archaeology and the History of Art, University of Oxford. e age of the
bone is beyond the range of radiocarbon, giving it a minimum age of 50.200 BP uncalibrated (OxA-36539).
Ancient DNA. All samples were aligned to the human reference genome (GRCh37/hg19) and damage pat-
terns were assessed. Alignments to the human genome were either too short, <35 bp (base pairs), and aligned
uniquely to the human genome or they were longer, >75 bp, and showed a low deamination rate, indicative of a
high likelihood of contaminant modern human DNA (SI Fig.2). As such, all primate sequences were excluded
from further study due to potential for human contaminant DNA.
Analysis of the Melouri cave samples (MEL1–4) showed that the majority of aligned reads fell within the
ranges of <35 bp, prone to misalignments, and >75 bp, with low deamination indicating potential contamination
and low preservation of DNA of ancient origin (SI Table2). e Melouri samples were therefore excluded from
Figure 3. e cut stalagmite SKK 16 3 (photos taken by MCS). (A) SKK 16 3 before sampling. e stalagmite
was partially cut open with a rock saw and then broken open (broken surface is to the right of the dashed
blue line) to reduce contamination by the saw blade. ree dark lines stemming from hiatuses in speleothem
formation can be observed at ds of 4.5 (h1), 4.8 (h2) and 5.6 (h3) cms (black arrows). Note that hiatuses
h1 and h2 combine to the right (h1, 2). (B) SKK 16 3 aer sampling for DNA analysis and U-series dating.
U-series samples (red, dotted line if unsuccessful analysis) and samples for DNA analysis (green, dotted line
if unsuccessful analysis) were oen taken in close association. Reliable U-series ages are reported next to their
sampling location. Note however, that the age of 80.26 ± 1.87 ka in the same sampling locality as SKK3 is less
reliable as it is out if stratigraphic order. DNA sample SKK1 was taken in the same layer as U-series sample
SKK 16 3/5.5, between hiatuses h3 and the combined hiatus h1 and h2 and dating to 83.32 ± 1.48 ka. Its age
is capped by U-series ages from layers above (56.7 ± 0.95 ka) and below (84.57 ± 0.76). SKK1 may contain dust
particles from the hiatus events.
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further analysis. In contrast, almost all Solkota cave samples (SKK1, 3–12) showed preservation of aDNA, most
with multiple genera identications (Fig.5). In the case of SKK 2, screening prior to sequencing showed no dis-
cernible presence of DNA and this sample was therefore excluded from further analysis.
An initial global alignment to the Blastn database with MGmapper revealed 16 commonly occurring genera
in the Solkota samples (SI Table2). e reassessment for false positives (following the approach by Slon et al.15
and see method section below), positively identied 6 genera: Capreolus (roe deer), Rhinolophus (bat), Ursus
(bear), Castanea (chestnut), Corylus (hazelnut), and Linum (ax) (Fig.5 and SI Table3). e combined number
of uniquely aligned reads to the reference species of these genera (SI Tables3 and 4) per speleothem section varied
between 4541 (SKK12) and 72056 (SKK3), and the per-species damage patterns between 0 and 54% (Fig.5, SI
Figure 4. e cut stalagmite SKK 16 5 (photo taken by MCS). SKK 16 was sampled for U-series dating (red dotted
line, unsuccessful analyses) and DNA analysis (green, dotted line if unsuccessful analysis), which include samples
from the stalagmites as well as the incorporated bones (SKK 7 and 12 from cortical bone and SKK 10 from trabecular
bone). Similar to SKK 16 3, stalagmite SKK 16 5 was also partially cut open with a rock saw and then broken open
(le of the blue dashed line) and both contexts were sampled. Note the brown colour of the speleothem, indicating the
presence of detrital clay, which impeded the U-series dating. However, each sample gave aDNA reads.
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Table3). e genus most frequently found was Capreolus (roe deer) which was positively identied in 7 of the 11
samples, followed by Rhinolophus (bat) in six, Castanea (chestnut) in ve, Ursus (bear) and Corylus (hazelnut)
in four, and Linum (ax) in only two (Fig.5, SI Table3). In SKK 10 we conrmed the presence of six ancient
genera, the highest number among all Solkota samples. Interestingly, the samples from the bear bone embedded
within the speleothems (SKK7, 10 and 12) also contained exogenous aDNA, including aDNA of other mammalia
genera and plantae with damage patterns ranging from 11.87 to 27.50% (Fig.5). e samples from the bear bone
embedded in the speleothem matrix provided high numbers of aligned reads to Ursus (8465 for SKK7, but only
126/125 for SKK 10/12) and display a strong deamination pattern above 50% for SKK 7 and nearly 30% for SKK
10 and 12 (Fig.5, SI Table3). Together, the aligned reads for bear, the clear damage pattern, the minimum age of
the bone and the zooarchaeological observations indicate that the speleothem embedded bone originates from
cave bear. Sample SKK 1 also displayed a strong deamination pattern for bear reads, nearly 50%, here, however, no
bear bone was present. Negative control analysis identied no ancient molecules aligned to any of the mentioned
genomes (SI Table3), indicating no cross-contamination between samples.
Discussion and Conclusion
Growth phases of stalagmite SKK 16 3 can be linked to global climatic records. e speleothem’s rapid but inter-
mittent growth around 84 ka coincides with climatic amelioration during Marine Isotope Stage (MIS) 5a, the
Greenland Interstadial 21.1e (GI-21.1e)37. e resumption of growth at 56.7 ± 0.95 coincides with a warm interval
in MIS3, Interstadial GS 16.137. For both time periods, interstadial GI-21e and GI 16.1, no dates for human occu-
pation in the region have been reported. However, this may be the result of limited dating of human occupation
deposits beyond the range of radiocarbon in this region, many Middle Paleolithic sites still lack absolute dating
(Bronze Cave, Sakaja and Ortvala38, Koudaro I, Undo39, Djruchula and Tsona40). Speleothem growth at Solkota
Cave suggests episodic favourable climatic condition in the region during parts of MIS5 and MIS3, which could
also have supported human occupation. However, climatic interpretations need to be further investigated with
stable isotope data and can now also be coupled with environmental aDNA from the same stalagmite.
Our rst metagenomic analyses presented here allowed the documentation of aDNA from inside the stalag-
mites with characteristic deamination damage to the DNA. We were able to identify the aDNA inside the stalag-
mites down to genera and to show the preservation of aDNA from mammals (bear, roe deer, horseshoe bat) and
plants (chestnut, hazelnut, ax) from various layers inside the speleothem as well as from the incorporated bone
(Fig.6). e identied plants and large mammals indicate a generally forested environment. Similar landscape is
also reconstructed from later Paleolithic sites of the area, such as Kotias Klde41,42 and Satsurblia33,42. Apart from
sample SKK 2, all samples from stalagmites SKK 16 3 and 5 contained aDNA from one or more genera. SKK 1 and
3 from stalagmite SKK 16 3 gave each one genera conrmation, bear and roe deer respectively. For stalagmite SKK
16 5, the number of detected genera range from 1 to 6 per sample. Bone samples from this stalagmite (SKK7, 10,
12) exhibit a higher number of conrmed genera (4–6 per sample) than pure speleothem samples (SKK4, 5, 6, 8,
9, 11) (1–3 per sample). e preservation of aDNA with characteristic deamination damage in most of the studied
samples show that both stalagmite and bone embedded in stalagmites are a promising medium for aDNA preser-
vation. However, only for the aDNA from stalagmite SKK 16 3 absolutes ages could be inferred. SKK 3, containing
roe deer, can be condently assigned to be older than ~84 ka. e same layer that preserved the ancient bear DNA
in SKK1, between hiatus 3 and the combined hiatus 1 and 2, was dated to 83.32 ± 1.48 ka with U-series. However,
this layer is rather thin and it is possible that the aDNA sample contained dust from either hiatus event and the
bracketing the U-series dates, 84.57 ± 0.76 ka (older layer) and 56.7 ± 0.95 ka (younger layer), provide a more
reliable chronological frame. For stalagmite SKK 16 5 only a minimum age for the bone, >50.200 uncalibrated,
could be deduced and the age of the stalagmite, which formed aer the deposition of the bone, remains open.
The diverse occurrence of the aDNA with variability inside the stalagmites, between sampling contexts
(pure speleothem versus bone embedded in the speleothem), between stalagmites from the same cave and from
Figure 5. Deamination frequencies in the Solkota samples (graph made by DMF). e graph presents the
average deamination frequencies at the 5 and 3 bases for the terminals ends. Only genera exceeding a 10%
deamination threshold were accepted as ancient and are presented here. Solid bars represent mammalian
genera, patterned bars represent plantae.
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dierent caves of the same cave system, Solkota and Melouri Cave, open up the question of the formation history
of the aDNA in this context. is formation history includes the DNA source, DNA adsorption, transport (agent),
deposition and preservation of DNA inside the stalagmites. Zepeda Mendoza et al.32 noted in their analysis,
that aDNA inside the studied popcorn speleothem contained aDNA originating from outside the cave as well as
from dierent parts inside the cave. Similarly, in our study aDNA from cave dwelling genera (bear, bat) as well as
non-cave dwelling genera (roe deer, hazelnut, chestnut and ax) are present. is mixture of allochthonous and
autochthonous sources suggests also a mixture of depositional processes. A number of possible processes can be
imagined. First, water is one possible transport agent, which inltrates through soils above the cave through the
epi-karst system into the cave, transporting plant, animal, bacterial, insect and fungal DNA. Another possible
biogenic process is the direct contact of the organism, from which the DNA derived, with the speleothem, e.g.
bears rubbing on speleothems, food remains (roe deer, nuts) adhering to the bear and being transported into
the cave, bats and bear defecating and urinating. A nal possible process is the gravitational transport of DNA
adhering to sediment particles into the cave, as can easily be imagined with the steep entrance slope at Solkota
cave. Clays, ne organic matter, silica grains and other minerals can all occur inside speleothems32,43,44. Aer
deposition and adsorption of the DNA and formation of the speleothem, DNA preservation and integrity is pro-
moted by the closed system of the stalagmite, making them a probably more reliable archive than sedimentary
deposits and soils. In addition, precise dating of layers containing aDNA is possible. However, possible minor
post-depositional migration of uranium in the lower part of speleothem SKK 16 5 (the lowermost age is out of
stratigraphic order) opens up the question of the integrity of speleothem to post-depositional migration of aDNA.
is being said, if DNA fragments are strongly adsorbed to sediment components, e.g. clay minerals1719 depos-
ited during the hiatus events within the speleothem, post-depositional migration by drip waters that percolate
through the speleothem structure is less likely for the DNA than for water-soluble uranium.
e preservation of aDNA inside speleothems entails diverse prospects for archaeological and paleoenviron-
mental research. Paleoenvironmental speleothem records from cave sites are associated with contemporaneous
archaeological, paleontological and paleobotanical records via correlating dates. e detection of mammalian and
plant aDNA inside speleothems reveals a potentially direct link between these records. Furthermore, stalagmites
can serve as an additional archive for old excavation, where all sediments, archaeological and paleontological
remains have already been removed, or for sites where bone and overall organic preservation is poor.
Materials and Methods
For sample extraction and to limit/control for sample contamination, speleothems were sawed only partially open
with a rock saw, using deionized water for cooling, and were then broken open to reveal surfaces for sampling.
Aer stratigraphic interpretation of the speleothems, samples were taken with a micro drill using layer-parallel
elliptical sampling pits and including samples from the sawed and broken area (Figs3 and 4). U-series samples
Figure 6. Genera identied by the aDNA analyses in stalagmites SKK 16 3 and 5 (photos taken by MCS). ©
MPI for Evolutionary Anthropology.
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were taken to constrain the age of calcite layers that contain aDNA and some were taken in tandem with the DNA
samples, from adjacent/overlapping sampling locations. Sample size was 100–200 mg for U-series and 25–60 mg
for DNA analysis. Samples for the latter were taken in a clean laboratory, using bleach to clean the surface of the
speleothems and about 2 mm of the exposed inner speleothems surface was removed with the micro drill before
sampling to limited contamination. Samples for DNA sequencing include samples from the speleothem as well as
samples from the bone inside speleothem SKK 16 5, cortical and trabecular bone (Fig.4).
U-series. Methods for U-series dating methods were similar to those described by Fankhauser et al.44.
Briey, sample powders were weighed and spiked with a mixed 233U/236U/229 tracer. Following dissolution and
spike equilibration, separation of uranium and thorium was completed by anion exchange chromatography. All
measurements were carried out using a ermoFisher Neptune® high-resolution inductively coupled plasma
mass spectrometer with an Aridus® desolvation nebuliser at the School of Earth Sciences, University College
Dublin. 238U/236U and 233U/236U ratios were measured using three Faraday collectors, while the 234U ion beam
was measured in a secondary electron multiplier (SEM). Calibration of the SEM relative to the Faraday detectors
was achieved by sample-standard bracketing, using the certied 235U/238U ratio of the IRMM-3184 standard.
Mass-fractionation corrections for uranium were applied based on the certied 233U/236U ratio of the mixed spike.
e minor isotopes of thorium (230 and 229) were measured using the SEM, whilst two Faraday collectors
were used to simultaneously measure the much larger 232 ion beam. A standard-sample bracketing method
using the IRMM-318444 standard a uranium standard was applied for the thorium mass fractionation correction
and for the SEM/Faraday yield calibration.
As discussed in the results, several sub-samples from the speleothem contained significant amounts of
non-carbonate ‘detrital’ thorium as evidenced by high 232 concentrations and low 230/232 ratios (Table1).
is necessitated corrections for inherited non-radiogenic 230. In the literature this correction is oen achieved
by simply assuming that the 230/232 ratio of the inherited (non-carbonate) fraction is equivalent to a typical
upper crustal 238U/232 activity ratio of 0.8 ± 0.445. In this study, in order to reduce the dating uncertainties asso-
ciated with the detrital correction we measured the U-series isotope ratios in clay-rich samples from the cave to
constrain the actual non-carbonate (silicate clay mineral) 230/232 ratio.
Ancient DNA. DNA samples were extracted and prepared within a clean room environment at a dedicated
ancient DNA laboratory at University College Dublin (UCD), Ireland. Unilateral air-ow hoods, tyvek suits,
hair nets, face masks and non-powdered gloves were used to limit contamination. Upon amplication further
steps were performed in a modern laboratory environment. DNA extraction was undertaken according to the
method outlined by Collin et al.46,47 (Collin manuscript in preparation). is protocol, developed for the extrac-
tion of aDNA from anthropogenic sediments, reduces the action of potentially damaging geopolymers on DNA
by chemical inhibition and increases the range as well as quantity of isolated DNA fragments thereby reducing
dependency on DNA capture techniques for exploratory samples. e extraction protocol consisted of samples
being placed into Matrix E lysing tubes (MP-BIO-116914050) and submerging them in 1 mL of extraction buer
up to a nal concentration of 0.45 M EDTA, 0.02 M TrisHCL (pH 8.0), 0.025% SDS, 0.5 mg/mL Proteinase K and
dH2O. Samples were incubated at 39 °C overnight using an Eppendorf ermomixer® C with a rotational speed
of 1600rpm to ensure maximal bead movement. Supernatant was collected and cleaned following Dabney et al.48
and DNA libraries were prepared following Meyer and Kircher49. Negative controls were included at all stages and
pooled to investigate the presence of damaged reads indicative of cross-contamination during DNA extraction
and library preparation.
DNA samples were amplied using a universal Illumina primer and Polymerase chain reaction (PCR) follow-
ing Gamba et al.50 and were repeated 15 times following Collin et al.46,47. Assessment of PCR reaction concen-
trations were performed on an Agilent 2100 Bioanalyser following instructions of the manufacturer. Based on
these concentrations samples were pooled into a 4 nM working solution and sequenced on an Illumina NextSeq.
500/550 using the high output v2 (75 cycle) reagent kit at UCD Conway Institute of Biomolecular and Biomedical
Research. Genera were initially identied by cross referencing raw sequencing data with the National Centre for
Biotechnology (NCBI) genomic database using Basic Local Alignment Search Tool (BLAST)5153 at an evalue of
1e-05 and MGmapper54 with a 0.8 fraction of matches + mismatches and minimum alignment score of 2547. In
order to reduce the chances of false positive identications of taxa at the family or genus level we followed a sim-
ilar approach to that of Slon et al.15. Aer the initial alignment using Blastn, an oine nucleotide BLAST + data-
base was generated with the genomic sequences of the 16 main eukaryote species detected (12 animals and 4
plants, SI Table2) with “makeblastdb” (genome versions for each species presented in SI Table4). Each sample’s
trimmed reads were aligned to this database using default “blastn” parameters and the resulting output data
was imported into MEGAN Community Edition v.6.2.1355. For the last common ancestor (LCA) parameters
we used a minimum bitscore of 35 within the top 10% of the best alignments, minimum support count of 2, and
the default “naive” LCA algorithm15. A minimum 1% of the total assigned reads was necessary to accept a taxa
to be present following Slon et al.15 and were then used for downstream analysis (SI Table3). e sets of reads
assigned to each species were extracted into independent les and then aligned to the correspondent genome
for authentication. We used BWA v.0.7.5a-r405 “aln”56 with permissive parameters (o 2 n 0.01) and disabled
seed (-l 1000), and then the aligned reads were ltered for a minimum quality of 25, sorted, duplicates removed,
and indexed using samtools v.1.3.157. Using mapDamage257 we investigated and quantied the presence of C to
T substitutions on the 5’ end and G to A on the 3’ end of the sequences, and used a minimum value of 10% on
both sides for a taxon to be identied as ancient6,16. Average read lengths were calculated using Genome Analysis
Toolkit’s “ReadLengthDistribution” (see SI Fig.3 for an averaged deamination length plot)58.
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Acknowledgements
e research presented here was supported by a New Interdisciplinary Initiatives Fund grant (SF1362) to MCS
by the University College Dublin. Further support came from a Government of Ireland Postdoctoral Fellowship
to MCS (GOIPD/2015/775), a Medical Trainee PhD Scholarship, Anatomy, School of Medicine, University
College Dublin awarded to TCC and from the Moshe and Bina Stekelis Foundation and Moshe Stekelis Chair in
Prehistoric Archaeology for excavation at Satsurblia Cave in 2016 to ABC, hosting our eld expedition. We are
grateful to the excavation team of Satsurblia, the people of Kumistavi and the Georgian State Museum. We further
would like to thank Mick Murphy from the UCD School of Earth Sciences for assistance with the U-series lab
work and Susanna Sawyer from Department of Evolutionary Anthropology, University of Vienna, for assistance
with sequencing data interpretation. Figure1 was created with ASTER GDEM, a product of METI and NASA,
and with help from R. C. Power, Max-Planck Institute of Evolutionary Anthropology. We are grateful to S. Tüpke
and the Max-Planck Institute of Evolutionary Anthropology for making the silhouettes in Fig.6.
Author Contributions
M.C.S. and R.P. conceived and supervised the project with contributions from T.C.C. and F.M.D. M.C.S. wrote
the paper with contributions from R.P., T.C.C., F.M.D., G.B.O. and D.M.F. M.C.S. and F.M.D. performed the
u-series dating, T.C.C., D.M.F., Z.G., J.P., R.P. the DNA analysis. A.B.C. and G.B.O. identied lithics and bones
respectively. M.C.S., F.M.D., R.P., A.B.C., G.B.O., N.J., Z.M. and T.M. conducted eld work related to the project.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-43147-0.
Competing Interests: e authors declare no competing interests.
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... Those sequences that align to multiple taxa within a grouping are binned into a higher taxonomic level until multiple assignments are no longer occurring. To reduce the chances of false positive identifications of taxa at the family or genus level a minimum of 1% of the total assigned reads was necessary to accept a taxon as present and for use in downstream analyses 3,21 . The resulting taxa identifications are used as the main taxon labels from this point forward, and they inform the user on which reference sequences to download for in-depth sequence alignment using BWA, the second authentication. ...
... Mapping quality (MAPQ) refers to the degree of confidence that a sequence is correctly mapped to reference genome coordinates. In aDNA research a MAPQ of between 25-30 is typically used to extract aligned reads from poorly and non-aligned reads 21,43,45,46 . To test the percentage difference in authentic ancient reads passing into subsequent steps using a MAPQ of 25 and 30, sequences were processed and taxonomic assignments were identified using MEGAN. ...
... BLAST results were input into MEGAN v6.2.13 1 using a bit-score of 40 within the top 10% of best alignments, and the default "naïve" LCA algorithm. A minimum of 1% of the total assigned reads was necessary to accept a taxon as present and use for downstream analyses 3,21 . Passing sequences were aligned to their corresponding genome using the original cut fasta file and BWA v07.5a.r405 29 aln function with a disabled seed (-l 1000). ...
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Bioinformatic pipelines optimised for the processing and assessment of metagenomic ancient DNA (aDNA) are needed for studies that do not make use of high yielding DNA capture techniques. These bioinformatic pipelines are traditionally optimised for broad aDNA purposes, are contingent on selection biases and are associated with high costs. Here we present a bioinformatic pipeline optimised for the identification and assessment of ancient metagenomic DNA without the use of expensive DNA capture techniques. Our pipeline actively conserves aDNA reads, allowing the application of a bioinformatic approach by identifying the shortest reads possible for analysis (22-28bp). The time required for processing is drastically reduced through the use of a 10% segmented non-redundant sequence file (229 hours to 53). Processing speed is improved through the optimisation of BLAST parameters (53 hours to 48). Additionally, the use of multi-alignment authentication in the identification of taxa increases overall confidence of metagenomic results. DNA yields are further increased through the use of an optimal MAPQ setting (MAPQ 25) and the optimisation of the duplicate removal process using multiple sequence identifiers (a 4.35-6.88% better retention). Moreover, characteristic aDNA damage patterns are used to bioinformatically assess ancient vs. modern DNA origin throughout pipeline development. Of additional value, this pipeline uses open-source technologies, which increases its accessibility to the scientific community.
... (Опалко & Опалко, 2021). Археоботанічні свідчення (Sheng et al., 2019), і зокрема метагеномний аналіз стародавньої ДНК (aDNA) пізньочетвертинних печерних сталагмітів (Stahlschmidt et al., 2019), дають підстави припускати, що прадавня людина часів неоліту вже використовувала горіхи фундука. Унаслідок підсвідомого добору в процесі доместикації рослин зазвичай істотно змінювалися їхні генотипи, що сприяло розширенню культигенних ареалів й поширенню меж первинних осередків на інші території. ...
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Aim. Value of cultivated Corylus L. as a fruit, ornamental and oilseed crop with prospects for use in the food industry, feed production and pharmacy are grown under the hazelnut name, makes it necessary to improve the methods of conservation and reproduction of Corylus spp., which can be valuable sources of initial material for breeding. Involvement in a hybridization of the well-known cultivars of hazelnuts with Chinese hazel (C.chinensis Franch.) contributed to the creation of several new cultivars, in particular ‘Sofiyivsky 1’ (‘Ukraine-50’×C. chinensis), Sofiyivsky 2’ (‘Dar Pavlenka’×C. chinensis), and ’Sofiyivsky 15’ (‘Garibaldi’×C. chinensis). However, in the process of studying the morphological features of C. chinensis from the collection of NDP “Sofiyivka” and analysis of the effectiveness of its interspecific interbreeding with other Corylus revealed their differences from the data given in the literature sources, which initiated our research. Materials and methods. Study of species-specific features of C. chinensis, hybridization, progeny analysis, clonal selection, propagation of selected seedlings, and generalization of the observations were performed using commonly used methods. Results and discussion. Comparison of morphological features of the C. chinensis imported from the Berlin Botanical Garden (Botanischer Garten Berlin-Dahlem) and its vegetative descendants with descriptions and photos given in the online database founded by the Royal Botanic Gardens Kew (Great Britain), showed the similarity of features of leaves, bark, and trunk with incomplete similarity of the infructescence, its shape, and downiness. It may indicate a hybrid origin of the introduced plant (Corylus…, 2017). The obtained data related to the value of C. сhinensis in hybridization with hazelnut cultivars using its male parent contradict the literature data that report on successful hybridization in direct combinations of C. chinensis×C. avellana and the incompatibility of these species in reciprocal crossing. Conclusions. It was found that the studied C. chinensis plants of generative age generally correspond to the descriptions of the species given in scientific sources and the electronic databases “Plants of the world Online” and “World Flora Online” in their morphological characteristics. However, the identified certain discrepancies indicate the need to continue their study, and the study of the others obtained from native sources of C. chinensis representatives, cultivars, and numerous interspecific hybrids using molecular and genetic DNA analysis methods.
... D'autres analyses, comme l'étude des enregistrements de pollens dans les spéléothèmes (Bastin, 1978 ;Renault-Miskovsky et Texier, 1980 ;McGarry et Caseldine, 2004) sont moins courantes. Plus récemment, c'est l'ADN ancien enregistré et préservé dans les spéléothèmes qui fait l'objet de recherches (Stahlschmidt et al., 2019). ...
Article
Speleothems (carbonated cave deposits) are natural archives that are characterized by their ability to record past environments as well as by their high temporal resolution, especially when laminated annually. Their potential for study is not limited to research on palaeo-climatic reconstructions. For example, speleothems can trap anthropogenic particles such as soot, and these fuliginous speleothems have a high informative potential in archaeology. In this paper, we will present the potential archaeological applications of fuliginochronological analysis in several cases, in different temporal contexts. The most recent cases (Belgium, France and Slovenia), for which we sometimes have historical and textual information allowing us to check the recorded archaeological facts, will make it possible to validate the micro-chronological archival potential of speleothems in the context of a fuliginochronological study. Then, we will extend the use of this method to a French Palaeolithic site in order to highlight the informative potential of this approach in Prehistory.
... Next-generation sequencing allows detailed metagenomic analysis of a wide range of ancient samples. Studies have attempted to recreate biological communities from material including coprolites (Bon et al., 2012;Appelt et al., 2014), dental calculus (Warinner et al., 2015;Weyrich et al., 2017), ice cores (Willerslev et al., 2007), sediment (Birks and Birks Hilary, 2015;Smith et al., 2015), stalagmites (Stahlschmidt et al., 2019), rodent middens (Kuch et al., 2002) and mollusc shells (Der Sarkissian et al., 2016). Our understanding of contamination and best laboratory practice has made good progress (Gilbert et al., 2005;Shapiro et al., 2019) and methods for authenticating ancient DNA sequences are developing (Key et al., 2017;Renaud et al., 2019). ...
Article
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Assigning metagenomic reads to taxa presents significant challenges. Existing approaches address some issues, but are mostly limited to metabarcoding or optimized for microbial data. We present PIA (Phylogenetic Intersection Analysis): a taxonomic binner that works from standard BLAST output while mitigating key effects of incomplete databases. Benchmarking against MEGAN using sedaDNA suggests that, while PIA is less sensitive, it can be more accurate. We use known sequences to estimate the accuracy of PIA at up to 96% when the real organism is not represented in the database. For ancient DNA, where taxa of interest are frequently over-represented domesticates or absent, poorly-known organisms, more accurate assignment is critical, even at the expense of sensitivity. PIA offers an approach to objectively filter out false positive hits without the need to manually remove taxa and so make presuppositions about past environments and their palaeoecologies.
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Morphometrics are fundamental for the analysis of size and shape in fossils, particularly because soft parts or DNA are rarely preserved and hard parts such as shells are commonly the only source of information. Geometric morphometrics, that is, landmark analysis, is well established for the description of shape but it exhibits a couple of shortcomings resulting from subjective choices during landmarking (number and position of landmarks) and from difficulties in resolving shape at the level of micro‐sculpture. With the aid of high‐resolution 3D scanning technology and analyses of fractal dimensions, we test whether such shortcomings of linear and landmark morphometrics can be overcome. As a model group, we selected a clade of modern viviparid gastropods from Lake Lugu, with shells that show a high degree of sculptural variation. Linear and landmark analyses were applied to the same shells in order to establish the fractal dimensions. The genetic diversity of the gastropod clade was assessed. The genetic results suggest that the gastropod clade represents a single species. The results of all morphometric methods applied are in line with the genetic results, which is that no specific morphotype could be delimited. Apart from this overall agreement, landmark and fractal dimension analyses do not correspond to each other but represent data sets with different information. Generally, the fractal dimension values quantify the roughness of the shell surface, the resolution of the 3D scans determining the level. In our approach, we captured the micro‐sculpture but not the first‐order sculptural elements, which explains that fractal dimension and landmark data are not in phase. We can show that analyzing fractal dimensions of gastropod shells opens a window to more detailed information that can be considered in evolutionary and ecological contexts. We propose that using low‐resolution 3D scans may successfully substitute landmark analyses because it overcomes the subjective landmarking. Analyses of 3D scans with higher resolution than used in this study will provide surface roughness information at the mineralogical level. We suggest that fractal dimension analyses of a combination of differently resolved 3D models will significantly improve the quality of shell morphometrics. In our study, we compare established morphological and morphometrical methods to the new approach of fractal geometry with respect to a group of freshwater gastropods from Lake Lugu (China) as a model group. Our results show that fractal geometry can help to overcome shortcomings of these established methods, since it might help to objectivize shell morphologies.
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Recent studies have demonstrated the potential to recover ancient human mitochondrial DNA and nuclear DNA from cave sediments. However, the source of such sedimentary ancient DNA is still under discussion. Here we report the case of a Bronze Age human skeleton, found in a limestone cave, which was covered with layers of calcite stone deposits. By analyzing samples representing bones and stone deposits from this cave, we were able to: i) reconstruct the full human mitochondrial genome from the bones and the stones (same haplotype); ii) determine the sex of the individual; iii) reconstruct six ancient bacterial and archaeal genomes; and finally iv) demonstrate better ancient DNA preservation in the stones than in the bones. Thereby, we demonstrate the direct diffusion of human DNA from bones into the surrounding environment and show the potential to reconstruct ancient microbial genomes from such cave deposits, which represent an additional paleoarcheological archive resource.
Preprint
Environmental DNA (eDNA) metabarcoding is a common tool for measuring and cataloguing biodiversity, yet standard methodological approaches to generate metabarcoding data sets have yet to emerge, in part due to challenges understanding the biological and technical biases that affect eDNA profiles. Here, we explore how two experimental choices – depth of sequencing of PCR amplicon libraries and the number of PCR replicates – influence estimates of α and β diversity. We extracted DNA from six soil samples from three ecologically distinct locations, performed 24 PCR replicates from each using two common metabarcodes, and sequenced each to an average depth of 83,898 reads. We found PCR replicates are consistent in composition and relative abundance of abundant taxa, allowing differentiation of samples and sites. However, rare taxa were unique to one or a few replicates, suggesting that even large numbers of experimental replicates may be insufficient to catalogue biodiversity fully. We recommend that to differentiate sites, separately sequencing only a minimum of two PCR replicates to a depth that allows 1,000 reads identified to taxa, is sufficient to differentiate sites. We also conclude that metabarcoding is impractical for exhaustive taxonomic inventory and, because rare taxa are not amplified consistently, taxonomic tallies that rely on consensus among replicates artificially lower richness estimates. These findings provide new considerations for eDNA experimental design and data interpretation.
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The concept of geoheritage is related to places of geological interest, generally of aesthetic, cultural, socioeconomic and/ or scientific value. Many geosites are of karstic nature, because of their intrinsic beauty, their singularity and high geodi-versity. Caves are among the most visited and economically exploited geological landforms. They constitute geosites as a whole, with their scenic landscapes, hydrogeological importance and the presence of bewildering natural rock and mineral formations including stalactites, stalagmites, flowstones and many other bizarre speleothem shapes. In some cases, a single speleothem, and the palaeoclimate record it contains, can be on its own of extraordinary importance to science. Once studied , these samples are often stored in research institution collections, rarely accessible to the wide public. In this paper, we report on the museumization of a stalagmite that has delivered a unique and exceptionally long glacial climate record from southern Italy, shedding light on the causes that led to the Neanderthal contraction and Modern Human expansion in this mild Mediterranean climate between 45 and 42 thousands years ago. The proposed museumization aims to demonstrate the potential of speleothems, after scientific application, in terms of educational and tourist resources. This approach allows to highlight the scientific importance of karst and cave geosites to the wide public, promoting their conservation and the valorisation of the studied cave-material.
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Analysis of ancient environmentalDNA(eDNA) has revolutionized our ability to describe biological communities in space and time, by allowing for parallel sequencing of DNA from all trophic levels. However, because environmental samples contain sparse and fragmented data from multiple individuals, and often contain closely related species, the field of ancient eDNA has so far been limited to organellar genomes in its contribution to population and phylogenetic studies. This is in contrast to data from fossils, where full-genome studies are routine, despite these being rare and their destruction for sequencing undesirable. Here, we report the retrieval of three low-coverage (0.033) environmental genomes from American black bear (Ursus americanus) and a 0.043 environmental genome of the extinct giant short-faced bear (Arctodus simus) from cave sediment samples from northern Mexico dated to 16–14 thousand calibrated years before present (cal kyr BP), which we contextualize with a new high-coverage (263) and two lower-coverage giant short-faced bear genomes obtained from fossils recovered from Yukon Territory, Canada, which date to �22–50 cal kyr BP. We show that the Late Pleistocene black bear population in Mexico is ancestrally related to the presentday Eastern American black bear population, and that the extinct giant short-faced bears present in Mexico were deeply divergent from the earlier Beringian population. Our findings demonstrate the ability to separately analyze genomic-scale DNA sequences of closely related species co-preserved in environmental samples, which brings the use of ancient eDNA into the era of population genomics and phylogenetics.
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Although many large mammal species went extinct at the end of the Pleistocene epoch, their DNA may persist due to past episodes of interspecies admixture. However, direct empirical evidence of the persistence of ancient alleles remains scarce. Here, we present multifold coverage genomic data from four Late Pleistocene cave bears (Ursus spelaeus complex) and show that cave bears hybridized with brown bears (Ursus arctos) during the Pleistocene. We develop an approach to assess both the directionality and relative timing of gene flow. We find that segments of cave bear DNA still persist in the genomes of living brown bears, with cave bears contributing 0.9 to 2.4% of the genomes of all brown bears investigated. Our results show that even though extinction is typically considered as absolute, following admixture, fragments of the gene pool of extinct species can survive for tens of thousands of years in the genomes of extant recipient species.
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Archaeogenetic studies have described the formation of Eurasian 'steppe ancestry' as a mixture of Eastern and Caucasus hunter-gatherers. However, it remains unclear when and where this ancestry arose and whether it was related to a horizon of cultural innovations in the 4th millennium BCE that subsequently facilitated the advance of pastoral societies likely linked to the dispersal of Indo-European languages. To address this, we generated genome-wide SNP data from 45 prehistoric individuals along a 3000-year temporal transect in the North Caucasus. We observe a genetic separation between the groups of the Caucasus and those of the adjacent steppe. The Caucasus groups are genetically similar to contemporaneous populations south of it, suggesting that - unlike today - the Caucasus acted as a bridge rather than an insurmountable barrier to human movement. The steppe groups from Yamnaya and subsequent pastoralist cultures show evidence for previously undetected Anatolian farmer-related ancestry from different contact zones, while Steppe Maykop individuals harbour additional Upper Palaeolithic Siberian and Native American related ancestry.
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The extent and nature of symbolic behavior among Neandertals are obscure. Although evidence for Neandertal body ornamentation has been proposed, all cave painting has been attributed to modern humans. Here we present dating results for three sites in Spain that show that cave art emerged in Iberia substantially earlier than previously thought. Uranium-thorium (U-Th) dates on carbonate crusts overlying paintings provide minimum ages for a red linear motif in La Pasiega (Cantabria), a hand stencil in Maltravieso (Extremadura), and red-painted speleothems in Ardales (Andalucía). Collectively, these results show that cave art in Iberia is older than 64.8 thousand years (ka). This cave art is the earliest dated so far and predates, by at least 20 ka, the arrival of modern humans in Europe, which implies Neandertal authorship.
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An increasing amount of species and gene identification studies rely on the use of next generation sequence analysis of either single isolate or metagenomics samples. Several methods are available to perform taxonomic annotations and a previous metagenomics benchmark study has shown that a vast number of false positive species annotations are a problem unless thresholds or post-processing are applied to differentiate between correct and false annotations. MGmapper is a package to process raw next generation sequence data and perform reference based sequence assignment, followed by a post-processing analysis to produce reliable taxonomy annotation at species and strain level resolution. An in-vitro bacterial mock community sample comprised of 8 genuses, 11 species and 12 strains was previously used to benchmark metagenomics classification methods. After applying a post-processing filter, we obtained 100% correct taxonomy assignments at species and genus level. A sensitivity and precision at 75% was obtained for strain level annotations. A comparison between MGmapper and Kraken at species level, shows MGmapper assigns taxonomy at species level using 84.8% of the sequence reads, compared to 70.5% for Kraken and both methods identified all species with no false positives. Extensive read count statistics are provided in plain text and excel sheets for both rejected and accepted taxonomy annotations. The use of custom databases is possible for the command-line version of MGmapper, and the complete pipeline is freely available as a bitbucked package (https://bitbucket.org/genomicepidemiology/mgmapper). A web-version (https://cge.cbs.dtu.dk/services/MGmapper) provides the basic functionality for analysis of small fastq datasets.
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Contents I. II. III. IV. V. VI. VII. VIII. IX. X. XI. References SUMMARY: Recent advances in sequencing technologies now permit the analyses of plant DNA from fossil samples (ancient plant DNA, plant aDNA), and thus enable the molecular reconstruction of palaeofloras. Hitherto, ancient frozen soils have proved excellent in preserving DNA molecules, and have thus been the most commonly used source of plant aDNA. However, DNA from soil mainly represents taxa growing a few metres from the sampling point. Lakes have larger catchment areas and recent studies have suggested that plant aDNA from lake sediments is a more powerful tool for palaeofloristic reconstruction. Furthermore, lakes can be found globally in nearly all environments, and are therefore not limited to perennially frozen areas. Here, we review the latest approaches and methods for the study of plant aDNA from lake sediments and discuss the progress made up to the present. We argue that aDNA analyses add new and additional perspectives for the study of ancient plant populations and, in time, will provide higher taxonomic resolution and more precise estimation of abundance. Despite this, key questions and challenges remain for such plant aDNA studies. Finally, we provide guidelines on technical issues, including lake selection, and we suggest directions for future research on plant aDNA studies in lake sediments.
Poster
Traditional approaches to assess anthropogenic sediments (AS) focus on flotation and wet sieving for macro- and micro-organisms. While informative, they offer limited breadth of the wide range of organisms available. Metagenomic Next Generation Sequencing (NGS) shotgun methods provide a means of analysing AS by comprehensively listing genes within a sample. Current methodologies, however are optimised for contemporary sediments, increasing the margin of error for ancient samples. This research aims to develop an optimised protocol for aDNA isolation from AS. Key steps in DNA isolation were identified and split into three stages of experimentation to determine the 1) impact of dispersal and separation (DS) techniques, 2) sample size and 3) water saturation on aDNA quality and overall yields. Bulk samples from well-documented Irish Early Medieval (1100 BP) sites were subdivided into sizes ranging 1g to 10g and subjected to a range of DS methods. aDNA was isolated using an optimised method based on published techniques. Samples were cleaned and prepared according to Dabney’s method and sequenced using an Illumia MiSeqTM. Preliminary results indicate that the method of DS greatly impacts overall aDNA quality and yields, with yields increasing regardless of the aDNA isolation method ( p<0.05). Larger sample sizes introduced complications with aDNA catchment and aDNA yields increased depending on water saturation ( p<0.05). In conclusion, the DS technique before introduction of extraction buffer plays a direct role on quantity and quality of aDNA. An optimised protocol for aDNA isolation would allow for new analyses of anthropogenic activity through previously untapped ancient sediment.
Conference Paper
Ancient DNA (aDNA) studies traditionally focus on skeletal remains, a limitation in archaeological, environmental and forensic contexts with poor preservation. Here we present and discuss a novel extraction protocol which aims to detect metagenomic biomarkers within anthropogenic sediment and its application to the metagenomics of sediment samples from two archaeological sites: Drumclay Crannog, Ireland, and Satsurblia Cave, Western Georgia. Bulk samples were secured from Georgian Upper Paleolithic, 31,000-15,000 BP (Satsurblia) and Early Irish Medieval period, 1600-1100 BP (Drumclay). Additionally, extracts from an Upper Palaeolithic bone tool (Satsurblia) were analysed. Samples were isolated using the novel extraction buffer and aDNA libraries were prepared using a double stranded DNA protocol (Meyer and Kircher 2010). Shotgun sequencing was undertaken using the NextSeq platform. Data was analysed for deamination frequency, aDNA strand length and misincorporation events through C>T substitution data, mapDamage2.0 and Geneious bioinformatics software. The extracts yielded aDNA from each site and a number of biomarkers were identified: Drumclay Crannog; Mycobacterium bovis (Bovine TB, 0.14% mean mapped to total metagenomic reads), with a high prevalence of Bos taurus (Cattle, 0.49%) in early habitation contexts compared to later (0.11%). Later contexts shift towards Salmo salar (Salmon, 0.22%) and Gadus morhua (Cod, 0.19%). Oryza sativa (Asian rice; 0.39%), and Xanthomonas oryzae (blight-causing bacteria, 0.12%) were isolated from early contexts. Satsurblia Cave; Multiple adjacent hearth contexts are positive for Homo sapiens (26%), Sus scrofa (Boar, 0.29%), Capreolus capreolus (European Roe Deer, 0.45%), Capra aegagrus (Goat, 1.1%), Canis lupus (Wolf, 0.92%) Bos taurus (0.44%) and Fragaria vesca (Strawberry, 0.22%). Mycobacterium bovis (0.28%), Capreolus capreolus (0.55%) and Capra aegagrus (0.47%) were identified from the bone tool. Prior to exposure to tuberculosis, Drumclay was largely dependent on meat products; upon exposure is a shift to fish, possibly indicating awareness towards public health and limitation of disease. Presence of Asian rice and blight-causing bacteria suggests trade; conceivably the earliest instance of rice within North-Western Europe. Possible presence of tuberculosis at Satsurblia places this 7000 years earlier than previously identified. Animal and wild-plant biomarkers indicate hunter-gatherer subsistence while wolf/dog remains could suggest their occupation of the cave during seasons/periods in which it was unoccupied by humans.
Article
Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using targeted enrichment of mitochondrial DNA we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples we detect Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility to detect the presence of hominin groups at sites and in areas where no skeletal remains are found.