Biomolecular Evidence of Early Human Occupation of a High-Altitude Site in Western Central Asia During the Holocene

Abstract

Reconstructions of early human occupation of high-altitude sites in Central Asia and possible migration routes during the Holocene are limited due to restricted archeological sample material. Consequently, there is a growing interest in alternative approaches to investigate past anthropogenic activity in this area. In this study, fecal biomarkers preserved in lake sediments from Lake Chatyr Kol (Tian Shan, Kyrgyzstan) were analyzed to reconstruct the local presence of humans and pastoral animals in this low-human-impact area in the past. Spanning the last ∼11,700 years, this high-altitude site (∼3,500 m above sea level) provides a continuous record of human occupancy in Western Central Asia. An early increase of human presence in the area during the mid-Holocene is marked by a sharp peak of the human fecal sterol coprostanol and its epimer epicoprostanol in the sediments. An associated increase in 5β-stigmastanol, a fecal biomarker deriving from herbivores indicates a human occupancy that most probably largely depended upon livestock. However, sterol profiles show that grazing animals had already occupied the catchment area of Lake Chatyr Kol before and also after a significant presence of humans. The biomarker evidence in this study demonstrates an early presence of humans in a high-altitude site in Central Asia at ∼5,900–4,000 a BP. Dry environmental conditions during this period likely made high altitude regions more accessible. Moreover, our results help to understand human migration in Western Central Asia during the early and mid-Holocene as part of a prehistoric Silk Road territory.

Full text

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ORIGINAL RESEARCH
published: 07 February 2020
doi: 10.3389/feart.2020.00020
Edited by:
Karen L. Bacon,
National University of Ireland Galway,
Ireland
Reviewed by:
Fabien Arnaud,
Centre National de la Recherche
Scientifique (CNRS), France
Nadia Solovieva,
University College London,
United Kingdom
*Correspondence:
Gerd Gleixner
gerd.gleixner@bgc-jena.mpg.de
Specialty section:
This article was submitted to
Quaternary Science, Geomorphology
and Paleoenvironment,
a section of the journal
Frontiers in Earth Science
Received: 09 September 2019
Accepted: 22 January 2020
Published: 07 February 2020
Citation:
Schroeter N, Lauterbach S,
Stebich M, Kalanke J, Mingram J,
Yildiz C, Schouten S and Gleixner G
(2020) Biomolecular Evidence of Early
Human Occupation of a High-Altitude
Site in Western Central Asia During
the Holocene. Front. Earth Sci. 8:20.
doi: 10.3389/feart.2020.00020
Biomolecular Evidence of Early
Human Occupation of a
High-Altitude Site in Western Central
Asia During the Holocene
Natalie Schroeter1, Stefan Lauterbach2, Martina Stebich3, Julia Kalanke4,
Jens Mingram4, Caglar Yildiz5, Stefan Schouten5,6 and Gerd Gleixner1*
1Research Group Molecular Biogeochemistry, Max Planck Institute for Biogeochemistry, Jena, Germany, 2Leibniz
Laboratory for Radiometric Dating and Stable Isotope Research, Kiel University, Kiel, Germany, 3Senckenberg Research
Institute and Natural History Museum Frankfurt – Research Station of Quarternary Palaeontology, Weimar, Germany,
4Section 4.3 – Climate Dynamics and Landscape Evolution, GFZ German Research Centre for Geosciences, Potsdam,
Germany, 5Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research,
Texel, Netherlands, 6Department of Earth Sciences, Faculty of Geosciences, Utrecht University, Utrecht, Netherlands
Reconstructions of early human occupation of high-altitude sites in Central Asia and
possible migration routes during the Holocene are limited due to restricted archeological
sample material. Consequently, there is a growing interest in alternative approaches
to investigate past anthropogenic activity in this area. In this study, fecal biomarkers
preserved in lake sediments from Lake Chatyr Kol (Tian Shan, Kyrgyzstan) were
analyzed to reconstruct the local presence of humans and pastoral animals in this low-
human-impact area in the past. Spanning the last ∼11,700 years, this high-altitude
site (∼3,500 m above sea level) provides a continuous record of human occupancy
in Western Central Asia. An early increase of human presence in the area during the
mid-Holocene is marked by a sharp peak of the human fecal sterol coprostanol and
its epimer epicoprostanol in the sediments. An associated increase in 5β-stigmastanol,
a fecal biomarker deriving from herbivores indicates a human occupancy that most
probably largely depended upon livestock. However, sterol profiles show that grazing
animals had already occupied the catchment area of Lake Chatyr Kol before and
also after a significant presence of humans. The biomarker evidence in this study
demonstrates an early presence of humans in a high-altitude site in Central Asia at
∼5,900–4,000 a BP. Dry environmental conditions during this period likely made high
altitude regions more accessible. Moreover, our results help to understand human
migration in Western Central Asia during the early and mid-Holocene as part of a
prehistoric Silk Road territory.
Keywords: fecal stanols, geochemistry, paleodemography, lake sediments, biomarkers, Silk Road
INTRODUCTION
There is growing evidence that human occupation of high-altitude sites [>3,500 m above sea level
(a.s.l.)] has occurred as early as during the latest Pleistocene and Early Holocene (Brantingham
et al., 2013;Rademaker et al., 2014;Shnaider S. et al., 2018;Zhang et al., 2018). The highest
located and oldest archeological sites have so far been identified on the Tibetan Plateau and in
the southern Peruvian Andes, dating back to at least 30,000 and >11,500 years ago, respectively
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Schroeter et al. Biomolecular Evidence of Human Occupation
(Rademaker et al., 2014;Zhang et al., 2018). Recently, however,
Ossendorf et al. (2019) provided evidence of repeated human
occupation of Fincha Habera (∼3,500 m a.s.l.), located in Africa’s
largest alpine landscape, dating back to 47,000–31,000 years ago,
which makes it the earliest known high-altitude residential site.
These findings indicate that prehistoric human populations were
able to adapt to climatic and environmental extremes at high
altitudes, such as low temperatures, high solar radiation and
low primary productivity, as well as to related physiological
challenges, including hypoxia and cold stress (Rademaker et al.,
2014;Meyer et al., 2017). The prehistoric settlement of high-
altitude regions was likely facilitated by strong immigration
from one resource area to another, and/or by biological
adaptation to a variable climate and environment (Madsen
et al., 2006). In this context, a key period in human history
was the onset of the Holocene since the development of
more favorable climate conditions promoted both the rise and
decline of many prehistoric civilizations (Dong et al., 2012;
Thienemann et al., 2017). Additionally, demographic pressure
on resources potentially opened previously uninhabitable high-
altitude regions, such as the mountain regions of Central Asia,
for settlement and migration.
Owing to its location at the crossroads between East and
West and the related importance for migration and cultural
exchanges (Agatova et al., 2014) for early humans and later as
part of the ancient Silk Road, Central Asia has recently become
a focus region with respect to investigating the occupation of
high-altitude regions by early human civilizations (Bae et al.,
2017;Hessl et al., 2017;Shnaider S. et al., 2018;Yang et al.,
2019a). Due to the harsh environment, featuring dry deserts,
cold mountains and seasonal grasslands, a nomadic pastoral
culture has predominantly prevailed in the mountainous regions
of Arid Central Asia, which is contrary to Monsoon Asia where
sedentary agriculturalists predominated (Hessl et al., 2017;Yang
et al., 2019b). Despite its crucial role in cultural development
and human migration patterns, little is known about the early
history of human occupation of Western Central Asia as part
of the Silk Road territory, especially during the period between
the Paleolithic and the mid-Holocene (Shnaider S. V. et al.,
2018). Western Central Asia encompasses several high-altitude
mountain regions, such as the Tian Shan, the Pamir Mountains
and the Alay Mountains (Shnaider S. et al., 2018). There is
evidence that the Alay Mountains may represent the high-altitude
region in Western Central Asia that has at first been occupied
by humans and was at least temporarily inhabited by hunter-
gatherers since the Paleolithic (Ranov, 1975;Abdykanova, 2014;
Shnaider S. et al., 2018;Taylor et al., 2018). However, the early
colonization of high-altitude Central Asia by humans is still
not well-constrained (Rademaker et al., 2014). Generally, the
success of tracing human presence in such regions largely relies
on finding related archeological sites and respective artifacts.
However, as this is often hindered by a limited number of
settlements and small sample sizes, there has been a growing
interest in alternative approaches to detect early anthropogenic
activity. In this context, the identification of sterols and stanols
as fecal biomarkers is an emergent valuable analytical tool
that can provide evidence for the presence of both humans
and livestock at a certain site. Fecal sterols and stanols are
recalcitrant organic compounds, which can accumulate and
persist in sediments for 1000s of years (Bull et al., 2001,
2003). 5β-Stanols, notably coprostanol (5β-cholestan-3β-ol) and
5β-stigmastanol (24β-ethyl-5β-cholestan-3β-ol), are the products
of anaerobic microbial reduction of sterols, i.e., cholesterol
(Cholest-5-en-3β-ol), in the intestinal tract of mammals (Eyssen
et al., 1973;Macdonald et al., 1983). Owing to their herbivorous
diet, ruminant feces are largely composed of 5β- stigmastanol
and epi-5β-stigmastanol (24β-ethyl-5β-cholestan-3α-ol), which
are derived from β-sitosterol (3β-Stigmast-5-en-3-ol) and
stigmasterol (Stigmasta-5,22-dien-3β-ol), the most common and
the third most common phytosterol (plant sterol), respectively
(Evershed et al., 1997;Bull et al., 2002;Rogge et al., 2006).
Conversely, coprostanol, a cholesterol derivative, accounts for
∼60% of the total sterols in human feces (Leeming et al., 1996;
Bull et al., 2002;Daughton, 2012). Therefore, human fecal input
can be distinguished from those of herbivores (Leeming and
Nichols, 1996;Leeming et al., 1996;Ortiz et al., 2016), enabling
their use in paleoenvironmental and archeological studies (e.g.,
Baeten et al., 2012;D’Anjou et al., 2012;Gea et al., 2017;Engels
et al., 2018;White et al., 2018).
Since sterols and stanols are well-preserved in lacustrine
sediments, the latter provide an ideal natural archive for tracing
the sources of feces in the environment and for assessing
human and livestock occupation of a certain area through
time, both qualitatively and quantitatively (D’Anjou et al., 2012;
White et al., 2018;Kinder et al., 2019). Furthermore, the
vicinity of lakes and rivers provides ideal conditions for natural
settlements as well as important pathways for human migration
(Thienemann et al., 2017).
In this study we analyzed coprostanol, epicoprostanol,
cholesterol, cholestanol and 5β-stigmastanol as fecal biomarkers
in the sediments of Lake Chatyr Kol, in the central Tian Shan
of Kyrgyzstan to elucidate the local presence of humans and
livestock during the Holocene. Investigating the history of human
occupation of this part of the Tian Shan can potentially contribute
to a better understanding of its cultural importance as a
prehistoric trade and migration route along the ancient Silk Road.
Study Area
Lake Chatyr Kol (40◦370N, 75◦180E) is located at 3,535 m a.s.l.
in the southern Tian Shan of Kyrgyzstan, close to the border
to China (Figure 1). The lake occupies the south-western part
of a large intra-montane basin between the At Bashy Range
in the north and the Torugart pass in the south. To the west
of Lake Chatyr Kol lies the Arpa river valley and moraine
landscape. Lake Chatyr Kol is the third largest lake in Kyrgyzstan
(Thorpe et al., 2009) and has a catchment area of approximately
1,084 km2. The lake extends to a maximum width (NW-SE)
of 12 km and a maximum length (SW-NE) of 23 km and has
a surface water area of ∼175 km2(Mosello, 2015). Since Lake
Chatyr Kol is a hydrologically closed alpine lake, the lake water
is slightly brackish (salinity 1.18 g/l in July 2018) and its water
balance is generally controlled by the interplay between snow
meltwater and precipitation input and evaporation. The largest
and only permanent inflow is the Kekagyr River, which enters
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FIGURE 1 | Map of Central Asia showing the study site Lake Chatyr Kol (indicated by a black star) and other sites mentioned in this study. A = Lake Chatyr Kol;
B = Grotto Semetey (Abdykanova, 2014); C = Kok-Aygyr (Ranov and Kydyrov, 1969); D = Terek-1 (Ranov, 1975); E = Aigyrzhal-2 (Motuzaite Matuzeviciute et al.,
2017); F = Alay Valley (Shnaider S. et al., 2018); G = Chegirtke Cave (Taylor et al., 2018); H = Sarazm (Spengler and Willcox, 2013). Figure made with GeoMapApp
(www.geomapapp.org, Ryan et al., 2009).
the lake from the north-east. The present-day regional climate
is temperate continental (Wang et al., 2017) and dominated by
the interaction between the Siberian anticyclonic circulation and
the mid-latitude Westerlies (Aizen et al., 1997;Lauterbach et al.,
2014). Owing to the high mountain ranges of the Tian Shan that
prevent the transport of moisture, rainfall is reduced, especially
in January and February (Aizen et al., 1995, 2001). Mean annual
precipitation consequently amounts to only ∼300 mm/a (Koppes
et al., 2008). The mean annual air temperature (1961–1990)
in Naryn, ∼100 km northeast of Lake Chatyr Kol, is −0.34◦C
(Ilyasov et al., 2013) and the lake is generally ice covered from
October to April. The prevailing dry and cold conditions favor
the preservation of permafrost soils (Shnitnikov et al., 1978) and
thermokarst formations can be frequently observed in this region
(Abuduwaili et al., 2019). Due to the harsh climate conditions,
vegetation is sparse and classified as desert and semi-desert
vegetation. Alpine grasslands dominate this region (Taft et al.,
2011) and there are no trees in the surrounding of the lake.
MATERIALS AND METHODS
Sediment Material and Chronology
In July/August 2012, several vertically overlapping, 2-m-long
sediment cores have been recovered from about 20 m water
depth in the deep south-western part of Lake Chatyr Kol
(40◦36.370N, 75◦14.020E) by using a 60 mm diameter UWITEC
piston corer. Additionally, seven parallel gravity cores have been
withdrawn in 2017 by utilizing a UWITEC gravity corer with
hammer weight (SC17_1–7). The individual sediment cores were
stratigraphically linked using distinct macroscopically visible
correlation layers, allowing the construction of a continuous,
623.5-cm-long composite profile (Kalanke et al., Under review).
With the exception of the upper 63 cm, the sediments are
almost continuously annually laminated (varved). The floating
varve age model (Figure 2), labeled “Chatvd19,” was established
using replicate microscopic varve counts below 63.0 cm depth,
which were performed on petrographic thin sections (Kalanke
et al., Under review) prepared at the GFZ German Research
Centre for Geosciences, Potsdam, Germany. Replicate varve
counts yield a mean deviation of ∼5% which was applied as
an uncertainty for the varved composite profile between 623.5–
63.0 cm depth. The uppermost homogenous 63.0 cm of the
composite profile were chronologically constrained by gamma
spectrometric analysis of 210Pb and 137 Cs performed at the GFZ
German Research Centre for Geosciences, Potsdam, Germany,
on 0.5 cm thick sediment slices of the parallel gravity core
SC17_7. 210Pb activity concentrations were used for age model
constructions based on a constant initial concentration (CIC)
model (cf. Appleby, 2002) and on a constant rate of supply
(CRS) model (Appleby and Oldfield, 1978) in combination
with 137Cs activity concentrations. Both 210 Pb models are in
good accordance with the onset of elevated 137Cs activity
concentrations, representing the onset of global nuclear weapon
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FIGURE 2 | Age model of the Lake Chatyr Kol sediment record (Kalanke
et al., Under review). The age model (black line) is based on varve counting
combined with 210Pb and 137 Cs measurements. Dashed lines indicate the
varve counting error (Kalanke et al., Under review). Black dots with 2 σerror
ranges represent AMS 14C dates of terrestrial plant macro remains.
tests since AD 1945 (Kudo et al., 1998;Wright et al., 1999). The
non-varved interval between the time marker AD 1945 and the
onset of varve deposition at 63.0 cm depth was interpolated by
using a mean sedimentation rate derived from varve thickness
measurements of adjacent varved intervals with an assumed
uncertainty of 10%. The final floating varve chronology had
a basal age of 11619 ±603 a BP and was independently
verified by two AMS 14C ages of terrestrial wood remains at a
composite depth of 380.5 and 528 cm (Poz-63307 and Poz-54302)
(Kalanke et al., Under review). AMS 14C measurements were
conducted at the Poz´
nan Radiocarbon Laboratory in Poland and
the conventional 14C ages were calibrated with OxCal 4.3 (Bronk
Ramsey, 1995) using the IntCal13 calibration curve (Reimer et al.,
2013). All age dates of Lake Chatyr Kol samples reported in this
study refer to the described age model.
Pollen Analyses
Pollen analyses have been carried out on 152 sediment samples.
The samples have been collected volumetrically (ranging between
1–2.6 cm3) at intervals of 4 cm on average from sediment depth
between 0.5 and 623 cm. The preparation involved treatment
with HCl, KOH, HF, hot acetolysis mixture and ultrasonic sieving
(mesh size 6 ×8µm), following the standard methods described
by Berglund and Ralska-Jasiewiczowa (1986). Lycopodium
spores were added to each sample to calculate the pollen
concentrations. Sample residues were stained with safranine,
mounted in glycerine and analyzed using an Olympus BX 40 light
microscope at ×400–1000 magnification. With the exception of
six samples, a minimum of 500 terrestrial pollen grains was
counted. The identification of the palynomorphs was carried
out with the aid of the palynological reference collection of
the Senckenberg Research station of Quaternary Palaeontology,
Weimar, supported by different pollen atlases (Beug, 2004;
Reille, 1995–1999). Pollen percentages were calculated on
the basis of terrestrial pollen, excluding aquatics, spores and
non-pollen palynomorphs.
Fecal Biomarker Analyses
For the present study, bulk sediment samples of 1 cm thickness
were taken from the Lake Chatyr Kol composite profile at
5 cm intervals and subsequently freeze-dried. The freeze-
dried bulk sediment samples were homogenized and lipids
were extracted twice with a dichloromethane/methanol solvent
mixture (9:1, v:v) by using a pressurized solvent speed extractor
(E-916, BÜCHI, Essen, Germany) operated at 100◦C and 120
bar for 15 min. Subsequently, the total lipid extract of each
sample was partitioned into a neutral and an acid fraction by
elution over aminopropyl gel columns (CHROMABONDR
NH2
polypropylene columns, 60 Å, Macherey-Nagel GmbH & Co.,
KG, Düren, Germany) with dichloromethane/isopropanol (3:1,
v:v) and diethyl ether:acetic acid (19:1, v:v), respectively (Richey
and Tierney, 2016). The neutral fraction was further separated
over activated silica gel columns (∼2 g, 0.040–0.063 mm mesh,
Merck, Darmstadt, Germany) into hydrocarbons, ketones and
a polar fraction by elution with hexane, dichloromethane and
methane, respectively. The hydrocarbon fraction, containing
long-chain n-alkanes, was analyzed using a gas chromatograph
(GC) with flame ionization detection (GC-FID, Agilent 7890B
GC) and an Ultra 2 column (50 m length, 0.32 mm ID,
0.52 µm film thickness, Agilent Technologies, Santa Clara, CA,
United States). The GC oven temperature program started at
140◦C (hold for 1 min), heated up to 310◦C at 4◦C/min (hold
for 15 min) and finally increased to 325◦C at 30◦C/min (hold
for 3 min). The PTV injector was operated in splittless mode
and started at 45◦C (hold for 0.1 min) and increased to 300◦C
at 14.5◦C/s (hold for 3 min). The quantification of long-chain
n-alkanes was carried out by peak area comparison with an
external n-alkane standard mixture (nC15–nC33 ).
The polar fraction, containing sterols and stanols, was silylated
with 10 µL N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)
and 10 µL pyridine at 60◦C for 30 min and afterward dissolved
in ethyl acetate. Sterol and stanol concentrations were measured
at the Royal Netherlands Institute for Sea Research (NIOZ),
Texel, Netherlands, as described by de Bar et al. (2019). Gas
chromatographic separation was carried out utilizing an Agilent
7890B GC that was equipped with a fused silica capillary
column (Agilent CP Sil-5, length 25 m, diameter 320 µm, film
thickness 0.12 µm) and coupled to an Agilent 5977A MSD
mass spectrometer (MS). The GC temperature program started
at 70◦C, increased to 130◦C at a rate of 20◦C/min, then heated
to 320◦C at 4◦C/min and was held at 320◦C for 25 min. The
flow rate was 2 mL/min. The MS quadrupole was held at 150◦C
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and the electron impact ionization energy of the MS source
was set to 70 eV. Sterols were identified and quantified via
single ion monitoring (SIM) of the mass-to-charge-ratios m/z
368.3 (cholesterol), 398.3 (stigmastanol) and 370.3 (cholestanol).
Additionally, characteristic mass spectra fragmentation patterns
and relative retention times were compared with the literature
for further identification. Coprostanol and epicoprostanol were
additionally confirmed using reference standards. An external
coprostanol reference standard in five different concentrations
was used for quantification of the sterols.
In order to account for microbial degradation processes, we
applied the ratio established by Bull et al. (1999):
coprostanol +epicoprostanol
coprostanol +epicoprostanol +5α−cholestanol =R1
5α-cholestanol is a product of the degradation of cholesterol by
soil microbial communities (Wakeham, 1989;Bull et al., 2001).
Therefore, considering both 5α-cholestanol and coprostanol
allows to compare input and preservation of stanols in a specific
environment to stanol input from feces (White et al., 2018). In
particular, higher R1 values indicate increased human fecal input,
while lower R1 values reflect low human fecal deposition.
In addition, we utilized the ratio proposed by Evershed
and Bethell (1996) to distinguish between human and higher
mammal feces:
coprostanol
5β−stigmastanol =R2
with R2 values >1.5 suggesting human or porcine fecal matter
(Evershed and Bethell, 1996).
In order to determine distinct temporal intervals in the
Lake Chatyr Kol sediment record, we used Uniform Manifold
Approximation and Projection (UMAP), a novel nonmetric
manifold learning technique for dimension reduction (McInnes
et al., 2018) implemented in R 3.6 as package “umap” 0.2.3.1
(Konopka, 2019;R Core Team, 2019). UMAP was recently
shown to preserve more of the global structure compared
to previous nonmetric techniques such as t-SNE (van der
Maaten and Hinton, 2008;McInnes et al., 2018). The improved
preservation of global structures in UMAP allowed the use of
k-means clustering on the resulting graph. We supplied the
UMAP algorithm with the biomarker profiles as well as their
chronology and interpreted the resulting three clusters as distinct
temporal intervals in the sediment core (Phases I, II, and III)
(Supplementary Figure S1). Data visualizations were performed
in R package “ggplot2” 3.2.1 (Wickham, 2016).
RESULTS AND DISCUSSION
Vegetation Development
The Holocene vegetation development of Lake Chatyr Kol has
been derived from 152 pollen samples collected between 0.5
and 623 cm composite core depth. The sampling intervals yield
a temporal resolution of 77 years on average. Total pollen
concentration is circa 30,000–60,000 grains per cm3through
most of the sequence. Overall, 83 pollen taxa were distinguished,
consisting of 20 arboreal and 63 non-arboreal elements. The
pollen assemblages are characterized by predominance of non-
arboreal taxa contributing between 88.3 and 99.6% to the
terrestrial pollen assemblage (Supplementary Table S1). The
herbaceous flora is mainly composed of Artemisia (34–61%),
Chenopodiaceae (15–29%), Poaceae (7–24%), and Cyperaceae
(0.2–6.9%). Other herbs occur in smaller quantities or only
scattered, each contributing between 0 and 8%. Most abundant
woody taxa are Juniperus (0–6.6%), Betula (0–3.4%), Hippophaë
(0–1.8%) and Picea (0–1.7%), while other tree pollen taxa appear
in trace amounts (Supplementary Table S1).
The dominance of xerophytic herbs and the sparse
representation of trees and shrubs reveal an open landscape
character in the surroundings of the Chatyr Kol Lake throughout
the Holocene (Figure 3). According to studies on modern pollen
assemblages in central and eastern Asia, herbaceous pollen
composition of the Chatyr Kol Lake sediments mainly reflects
semi-arid, alpine meadow, steppe, and lake shore communities
(Beer et al., 2007;Ma et al., 2008;Qin et al., 2015). In terms of
the overall species composition, the pollen signatures show only
gradual changes through the entire record. Therefore, the alpine
landscape of the study region experienced no major vegetation
shifts during the past 11.5 ka.
Sedimentary Fecal Biomarker
Distribution
Several fecal biomarkers were detected in the Lake Chatyr
Kol sediments of which five sterols and stanols, i.e.,
cholesterol, coprostanol, epicoprostanol, 5β-stigmastanol
and 5α-cholestanol, were quantified. Their total sum reveals a
strong increase from relatively low concentrations in the oldest
part of the composite profile [∼146 µg/g dry weight (d.w.)]
to higher amounts (5,342 µg/g d.w.) at a composite depth of
319.5 cm (∼4,900 a BP) (Figure 3). The total concentration of
sterols and stanols ranged between 0.2 and 5,800 µg/g d.w. The
most abundant sterol throughout the record is cholesterol (46%),
followed by 5β-stigmastanol (21%).
The sediment record was divided into three distinct
phases based on three distinct k-means clusters within a
UMAP ordination of the fecal biomarker data (Figure 4
and Supplementary Figure S1). Phase I (∼11,400–8,300 a
BP) shows conditions of prehuman occupation with low
background concentrations of human-specific fecal sterols and
stanols (coprostanol, epicoprostanol, and cholesterol). However,
considerable amounts of 5α-cholestanol and 5β-stigmastanol are
present throughout Phase I, indicating the presence of indigenous
higher mammals. First noticeable human activity is recorded
during Phase II (∼7,600–2,400 a BP), which is characterized
by strong concentration increases in all fecal biomarkers at
three time points, dating to ∼5,900, ∼4,800, and ∼2,400 a
BP, respectively. This phase represents the beginning of human
occupation in the catchment area of Lake Chatyr Kol, possibly
with domesticated livestock, since both human-specific and
higher mammals-specific fecal biomarkers increase significantly
during Phase II. In relation to the total amount of fecal
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FIGURE 3 | Profiles of the sum of long-chain n-alkanes (nC27 +nC29 +nC31), pollen percentages of arboreal pollen (AP, black area) and non-arboreal pollen (NAP,
gray area) with AP + NAP = 100%, sum of fecal biomarkers, R1 (coprostanol + epicoprostanol)/(coprostanol + epicoprostanol + 5α–cholestanol), R2
(coprostanol/5β– stigmastanol) and environmental conditions during the Holocene. Time periods of increased human presence at ∼4,800 and ∼2,500 a BP seem to
coincide with dry phases.
biomarkers, the imprint of human presence was highest at ∼4,800
a BP, where concentrations reach their maxima. Additionally,
there are intervals within Phase II, which show decreased values
of human-specific fecal biomarkers, especially during ∼3,800
–2,700 a BP, whereas concentrations of 5β-stigmastanol are still
elevated. The beginning of Phase III (<2,300 a BP) is marked by
a decline of all biomarkers toward background concentrations
comparable to Phase I. Phase III continues to modern times
and displays the current low level of human occupation of the
catchment area of Lake Chatyr Kol.
Sources of Analyzed Fecal Biomarkers
In order to disentangle the sources of the sterols and stanols,
we applied principal component analysis (PCA). PC1 and PC2
account for 96.2% of the total variance and show a clear
distinction between the fecal biomarkers cholesterol, coprostanol
and epicoprostanol, which are associated to a carnivorous and
omnivorous diet, and the herbivore-derived fecal biomarker 5β-
stigmastanol (Figure 5). We utilized cholesterol, coprostanol
and epicoprostanol as human-specific biomarkers; however it
is possible that carnivorous animals may have contributed to
this biomarker signal. The strong linear correlation between the
concentrations of coprostanol and epicoprostanol (r2= 0.9) and
coprostanol and cholesterol (r2= 0.92) additionally confirms
a combined source of these compounds (Supplementary
Figure S2). This confirms coprostanol, epicoprostanol, and
cholesterol as human biomarkers and 5β-stigmastanol as being
indicative of grazing animals including domesticated livestock.
Since 5β-stanol background concentrations are also detectable
in soils that were not exposed to fecal deposition, diagnostic ratios
of selected sterols that are independent of total concentrations are
advantageous for the interpretation of the total fecal biomarker
spectrum (Grimalt et al., 1990;Bull et al., 1999, 2001). In this
context, several ratios of different sterol and stanol classes have
been proposed to determine the origin of organic matter and to
detect anthropogenic activity (Grimalt et al., 1990;Bull et al.,
2002;Martins et al., 2007). In order to determine the fecal
origin of our sterols and stanols, we applied ratio R1 proposed
by Bull et al. (1999). By including epicoprostanol, R1 corrects
for microbial degradation processes since coprostanol may be
microbially degraded in situ to epicoprostanol (Bull et al., 1999,
2002;Battistel et al., 2015).
R1 values for the sediments of Lake Chatyr Kol range between
0.09 and 0.6 (Figure 3). A R1 value of 0.7 has been proposed
as a threshold with higher R1 values indicating human fecal
deposition and lower values reflecting scarce human presence
(Grimalt et al., 1990). The values in the Lake Chatyr Kol
sediments, however, do not exceed this threshold but as it was
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FIGURE 4 | Fecal sterol and stanol concentrations in the Lake Chatyr Kol sediments. Dashed horizontal lines indicate different time periods discussed in this study.
originally determined from modern-day urban sewage pollution
investigations, it may not be applicable in an archeological
context (Bull et al., 2001;Birk et al., 2011;Baeten et al., 2012).
Nevertheless, elevated R1 values during Phase II, especially at
∼4,800 a BP (R1 = 0.6), demonstrate the fecal nature of the
5β-stanols and most likely indicate the presence of humans
during the mid-Holocene (Figure 3).
To allow for the discrimination between human and herbivore
fecal deposition, the ratio of coprostanol and 5β-stigmastanol
(R2) was established (Evershed and Bethell, 1996), which is
based on the divergent biogenic origin of these 5β-stanols. As a
result of their herbivorous diet and the associated high uptake
of phytosterols, ruminant feces are enriched in 5β-stigmastanol,
whereas coprostanol is the major sterol in human and omnivore
feces. Typically, R2 values between 1.5 and 5.5 are indicative of
humans and pigs, while R2 values in the order of ∼0.25 imply
herbivorous fecal input (Baeten et al., 2012).
R2 values in the Lake Chatyr Kol sediment record fluctuate
between 0.003 and 2.6 (Figure 3). Highest R2 values of 2.5 and 2.6
are observed at ∼11,100 and ∼132 a BP, respectively (Figure 3).
However, these values are controlled by particularly low amounts
of 5β-stigmastanol and may therefore not necessarily reflect
human activity. Conversely, slightly elevated R2 values (>1.5)
during Phase II at ∼5,700 and ∼4,800 a BP confirm a human
origin of the stanols and reinforce the assumption of an early
human occupation of the Lake Chatyr Kol catchment area during
the mid-Holocene. In this context, generally lower R2 values
during Phase I and Phase III indicate predominant herbivore
origin of the stanols during the Early and Late Holocene,
respectively (Figure 3).
Holocene Human and Mammal Presence
in the Lake Chatyr Kol Catchment Area
Phase I (∼11.4k – 8,300 a BP)
The beginning of the Holocene was characterized by a global
temperature rise (Marcott et al., 2013). Such more favorable
climate conditions may have boosted human migration as
climate is generally considered a key factor for cultural and
social development (DeMenocal, 2001;Flohr et al., 2016).
Direct evidence of human occupation of Kyrgyzstan during
this period was observed in the western part of the Alay
Valley in southern Kyrgyzstan at an elevation of ∼2,800 m
a.s.l. (Shnaider S. et al., 2018), where findings of stone tool
assemblages suggest the local presence of prehistoric humans
during the Late Pleistocene or Early Holocene, supposedly mainly
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Schroeter et al. Biomolecular Evidence of Human Occupation
FIGURE 5 | Principal component analysis (PCA) of fecal sterols. The
separation indicates different sources for coprostanol, epicoprostanol,
cholesterol, 5α-cholestanol, and 5β- stigmastanol.
consuming sheep (Shnaider S. et al., 2018;Taylor et al., 2018).
Furthermore, archaeological findings close to Lake Chatyr Kol
indicated the presence of humans in the area around the lake
during the Mesolithic and Neolithic. Ranov and Kydyrov (1969)
described findings of stone tools at two archaeological sites at
Kok-Aygyr, northeast of Lake Chatyr Kol. The relative ages of
these archaeological sites were established by assigning a series
of characteristic artifacts to a local culture dated to 9,530 ±
130 a. Furthermore, Ranov (1975) reported findings from the
archaeological site Terek-1, ∼40 km east of Lake Chatyr Kol,
described as likely Neolithic based on the composition and
craftwork of the excavated assemblages.
The Lake Chatyr Kol fecal sterol record does not indicate
local human presence during the Early Holocene as the amounts
of coprostanol, epicoprostanol, and cholesterol remained near
the background values (median values for Phase I: 0.72, 1.19,
and 1.72 µg/g d.w., respectively) (Figure 4). Yet, elevated
concentrations of 5α-cholestanol and 5β-stigmastanol indicate
an increased presence of indigenous higher mammals in the
catchment area during the Early Holocene. The first biomarker
evidence for an increased occurrence of higher mammals at Lake
Chatyr Kol is found at ∼10,600 and ∼9,500 a BP (Figure 4).
The occupation by mammals over a prolonged period during
the Early Holocene is further supported by generally higher
5β-stigmastanol concentrations between ∼8,300 and ∼8,500 a
BP. More temperate climate conditions in Kyrgyzstan during this
time were inferred from analyzing the sediments of Lake Son
Kul, located ∼125 km north of Lake Chatyr Kol (Huang et al.,
2014;Mathis et al., 2014) and Lake Issyk-Kul, located ∼242 km
northeast of Lake Chatyr Kol (Ricketts et al., 2001).
In order to estimate the flux of vegetation remains within
the catchment area of Lake Chatyr Kol independently from
the fecal biomarkers, we analyzed the concentrations of long-
chain n-alkanes (nC27,nC29,nC31). An increase in the
concentration of long-chain n-alkanes would imply enhanced
organic productivity, since these compounds are commonly
found in terrestrial sources, such as terrestrial plants and grasses
(Eglinton and Hamilton, 1967;Meyers, 2003). Both long-chain
n-alkanes and arboreal pollen increase at the beginning of
the early Holocene (Figure 3). It is therefore likely that more
temperate climate conditions promoted the growth of terrestrial
vegetation, resulting in improved habitability of the Lake Chatyr
Kol catchment area.
Phase II (∼7,600–2,400 a BP)
The earliest biomolecular evidence of human presence in
the Lake Chatyr Kol catchment is provided by significant
increases in the concentrations of coprostanol, epicoprostanol,
and cholesterol at ∼5,900 a BP and particularly at ∼4,800 a
BP (Figure 4). This is further supported by elevated R1 and
R2 values (Figure 3). Since 5β-stigmastanol concentrations also
reveal maximum values at the same time, it is likely that human
presence at that time was associated with domesticated livestock.
Variations in the concentrations of fecal sterols suggest that the
population size of the first human occupancy was low in relation
to the human presence at ∼4,800 a BP as changes in the human
and livestock population would inevitably entail changes in the
concentrations of fecal sterols being transported to the lake (cf.
D’Anjou et al., 2012).
The biomarker evidence for human presence at Lake Chatyr
Kol during Phase II coincides with findings of stone tool
assemblages from Grotto Semetey situated in the adjoining
Arpa river valley dating to ∼5,700 and ∼6,180 cal a BP
(Abdykanova, 2014).
Archeological findings of ceramic fragments and radiocarbon
dating of animal bones (4,240–3,990 cal a BP) at Chegirtke
Cave in southern Kyrgyzstan indicate that humans started
to occupy the mountain foothills of the Alay Valley at least
during the early Bronze Age (Taylor et al., 2018). Since the
excavated bone fragments at this site belong to sheep, goat,
and cattle, it can be concluded that they represent a pastoral
assemblage of domestic animals (Taylor et al., 2018). This
would support our assumption of a livestock-herding human
occupancy around Lake Chatyr Kol at ∼4,800 a BP. Evidence
of human occupancy during the Bronze Age is provided by
archeological excavations at the closely nearby site Aigyrzhal-2
(2,005 m a.s.l.) in central Kyrgyzstan, ∼97 km northeast of Lake
Chatyr Kol (Motuzaite Matuzeviciute et al., 2017). Domestic
animal remains, such as horse and ovicaprines, and remains
of cereals (grains and chaff), indicate that humans in the
Tian Shan mountain valleys developed agricultural interests
during this period (Motuzaite Matuzeviciute et al., 2017). Yet,
at the high-altitude site Lake Chatyr Kol pollen analysis did
not indicate agricultural practice. The oldest directly dated
wheat remains in Central Asia have been found in Tasbas,
eastern Kazakhstan, and have been dated to ∼4,500 cal a
BP (Doumani et al., 2015). Based on archaeobotanical and
carbon isotope data of human bones and dating of crop
remains found in prehistoric sites in Eurasia, Dong et al.
(2017) suggested that western Asian crops spread to eastern
Central Asia and northwestern China between 4,500 and
4,000 a BP. Further evidence of early agricultural and herding
practice in Central Asia stems from the Eneolithic/Early
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Schroeter et al. Biomolecular Evidence of Human Occupation
Bronze Age site of Sarazm in northwestern Tajikistan
(Frachetti, 2012;Spengler and Willcox, 2013). Sarazm was a
sedentary agropastoral settlement, which was occupied from
the fourth to the end of the third millenium BC and provides
evidence for exchange and trade (Spengler and Willcox, 2013).
Paleoenvironmental studies from Kyrgyzstan indicate rather
dry conditions during Phase II. Lauterbach et al. (2014) reported
a pronounced dry interval for Lake Son Kul between 4,950 and
3,900 cal a BP. In accordance, a stalagmite record from Uluu-
2 Cave, ∼250 km west of Lake Chatyr Kol, also suggests dry
conditions between 4,700 and 3,900 cal a BP (Wolff et al., 2017).
These dry conditions likely entailed the necessity of exploiting
new herding grounds and correspondingly affected migration
flows. Human settlement and local herding within the catchment
area of Lake Chatyr Kol were likely favored by an open landscape
and herbaceous vegetation as indicated by pollen data (Figure 3).
Phase II is mainly characterized by a high amount of long-
chain n-alkanes, which is in line with the presence of herbivores
in general (Figure 3; R2). The high amounts of the human specific
fecal biomarkers, however, cover only a short period of time
that is defined by a reduced flux of vegetation remains. Further,
pollen data do not indicate significant increases or compositional
changes of the local vegetation. We therefore suggest the reported
dryness likely influenced the presence of humans at Lake Chatyr
Kol rather than an enhanced supply of vegetation resources.
As there are no pollen data indicative of agricultural practices,
the catchment area of Lake Chatyr Kol was likely inhabited by
pastoralists rather than agro-pastoralists.
Concentrations of human-specific fecal sterols returned
to background values between ∼3,900 a BP and ∼2,400 a
BP (Figure 4) and 5β-stigmastanol concentrations decreased
similarly at ∼3,900 a BP. The decline in the fecal sterol
concentrations was probably triggered by a shift to colder climate
conditions as reconstructed for Lake Karakul in NE Tajikistan
(∼240 km southwest of Lake Chatyr Kol) at ∼3,500 cal a
BP, for Lake Balikun, eastern Tian Shan Mountains, between
4,800 and 3,800 cal a BP (Zhao et al., 2017) and for Lake
Qinghai, located in the northeast corner of the Tibet-Qinghai
Plateau Qinghai, between 5,000 and 3,500 cal a BP (Hou
et al., 2016). 5β-Stigmastanol concentrations however increase
again shortly afterward to relatively high amounts at ∼3,600 a
BP, indicating herbivore activity without a significant presence
of human activity.
The latest short-term increase of human-specific fecal
sterol concentrations occurred between ∼2,500 and ∼2,400 a
BP. As previously observed, this increase is accompanied by
a simultaneous increase of 5β-stigmastanol concentrations,
suggesting livestock farming. Interpreting these elevated
concentrations in the context of local environmental conditions,
they again appear to have contemporaneously occurred to dry
climate conditions. For example, a warm and dry episode has
been identified at Lake Karakuli, ∼240 km south of Lake Chatyr
Kol, between 2,500 and 1,900 cal a BP (Aichner et al., 2015).
Pollen data from the Kashgar oasis at the western margin of
the Tarim Basin show sparse vegetation cover from 2,620 to
1,750 cal a BP, indicating a relatively dry climate (Zhao et al.,
2012). Similarly, Mischke et al. (2010) reported low freshwater
inflow and a low lake level for Lake Karakul between 2,600
and 1,900 cal a BP.
Phase III (∼2,300 a BP – Present)
Phase III is characterized by generally low concentrations of
coprostanol, epicoprostanol and cholesterol, suggesting scarce
human presence within the catchment area of Lake Chatyr Kol.
5β-Stigmastanol concentrations remained on an intermediate
level between ∼1,900 and ∼1,300 a BP but significantly dropped
thereafter (Figure 4).
The economy of Kyrgyzstan largely relies on the agricultural
sector and seminomadic lifestyles continue to exist until present-
day (Rahimon, 2012). A survey of pastoralists in Kyrgyzstan
revealed that in AD 2004 only 12 herding families encamped the
vicinity of Lake Chatyr Kol, owning livestock of not more than
12,000 sheep (Farrington, 2005). At present, the basin of Lake
Chatyr Kol is little used, with the majority of occupancy occurring
during the summer months from June to mid- September
(Farrington, 2005). This could explain the low amounts of
fecal sterols in the youngest part of the Lake Chatyr Kol
sediment record.
Cultural Importance of the Tian Shan
Being located along the ancient Silk Road, the territory of
Kyrgyzstan represented an important corridor for cultural
exchange and trade in the past. The Silk Road was a complex
network of trade and travel routes, which enabled both cultural
interaction and the trading of goods between Central Asia, the
Middle East and the Eastern Mediterranean (Hansen, 2012;Yang
et al., 2019b). Therefore, it functioned as a “Cultural Bridge”
between Asia and Europe (Foltz, 2010;Yang et al., 2019b) and
had a profound influence on societal development. Even though
the relevance of the Silk Road is conventionally constrained to
the second century BC, exchange and migration occurred well
before that (Taylor et al., 2019). Archeological studies elucidated
that ancient trade and travel routes along this corridor already
existed during the Bronze Age (third to second millenium BC)
(Frank and Thompson, 2005;Frachetti et al., 2017;Panyushkina
et al., 2019), although details about pathways and exact timing are
still uncertain.
The progression of mobile pastoralist groups during the
Bronze Age is considered to have contributed to the evolvement
of high-elevation pathways across the Silk Road, but more
research is needed to determine their extent (Frachetti et al.,
2017). Nevertheless, interrelations and early diffusions of
technologies between mobile pastoralist economies across
Eurasia occurred from the third to the second millenium BC
(Mei, 2003;Spengler et al., 2014).
This is in good agreement with our observation of elevated
fecal sterol amounts at ∼4,800 year. BP, indicating anthropogenic
and livestock presence. Indeed, it is suggested that the Tian
Shan was already occupied by pastoralists during the Bronze Age
(Motuzaite Matuzeviciute et al., 2017). Along with the Pamir,
Dzhungar and Altai mountains, the Tian Shan is part of a
proposed “Inner Asian Mountain Corridor” (Frachetti, 2012),
which promoted the exchange of goods, culture flow (Spengler
and Willcox, 2013) and the earliest diffusion of sheep and goat
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Schroeter et al. Biomolecular Evidence of Human Occupation
pastoralism to inner Asia at ∼3,500 BC (Frachetti, 2012). Routes
of nomadic societies near Lake Chatyr Kol have been indicated
by Frachetti et al. (2017), who used flow accumulation modeling
to assess migration along the Silk Road. Specifically, the model
results revealed a path crossing the Torugart Pass in the south,
highlighting the importance of Lake Chatyr Kol as a likely transit
stop along herding routes.
Considering that the human-specific fecal sterols in the Lake
Chatyr Kol sediment record diminished at ∼3,900 year. BP, it is
likely that mobile non-sedentary pastoralists occupied the Lake
Chatyr Kol catchment area around ∼4,800 year. BP but later
left the area again, probably focusing on other, more habitable
regions. The migration from one region to another after the
consumption of resources in one area is commonly observed
along the Silk Road (Yang et al., 2019b). This does not necessarily
entail the collapse of a population, but rather reflects high
adaptability and resilience of social groups (Yang et al., 2019b).
In summary, our study indicates human migration in the area
around Lake Chatyr Kol as early as during the early Bronze
Age, centuries before the establishment of the Silk Road. This
reinforces the influence of small-scale migration patterns on
the evolution of a macro-scale trade and exchange network
(Frachetti et al., 2017).
CONCLUSION
The present study demonstrates the usefulness of fecal biomarker
analyses as a valuable tool to reconstruct temporal anthropogenic
presence in an area as an alternative approach in archeological
studies. Such biomarker analyses applied to the sediments of
Lake Chatyr Kol, Kyrgyzstan reveal a pronounced population
evolvement in the Tian Shan at ∼4,800 a BP. Coinciding dry
environmental conditions likely increased the accessibility of
high-altitude regions and necessitated the exploitation of new
herding grounds. This finding suggests climatic change, inter
alia, as a potential driver for human migration and underlines
the ability of early humans to adapt to variable environmental
conditions. Migration through the high-altitude terrains of the
Tian Shan reveals its cultural importance as an early travel
corridor during the Bronze Age. As one of the rare high-altitude
sites providing evidence of early pastoralists, Lake Chatyr Kol is
of great importance for the understanding of the human history
and migration in Eurasia.
DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to
the corresponding author.
AUTHOR CONTRIBUTIONS
GG conceived the research idea. SL conducted the field
work. MS contributed pollen data. JK and JM generated
the age model. NS conducted laboratory analysis and
data analysis with support from SS and CY. NS prepared
the manuscript. All authors discussed the data and
improved the manuscript.
FUNDING
This study was supported by the BMBF-funded research
projects CADY (Central Asian Climate Dynamics, Grant No.
03G0813) and CAHOL (Central Asian Holocene Climate,
Grant No. 03G0864). NS acknowledges the Max Planck
Society and the International Max Planck Research School for
Global Biogeochemical Cycles (IMPRS-gBGC) for additional
project funding.
ACKNOWLEDGMENTS
The authors thank Michael Köhler, Sylvia Pinkerneil, Roman
Witt, and Robert Schedel for their help during field work and
Stefanie Garz for her help during pollen analysis.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/feart.
2020.00020/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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