Ancient DNA analysis of marmot tooth remains from the Shamanka II and Lokomotiv-Raisovet cemeteries...

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Quaternary International xxx (2015) 1e7

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Ancient DNA analysis of marmot tooth remains from the ShamankaII and Lokomotiv-Raisovet cemeteries near Lake Baikal: Speciesidentification and genealogical characteristics

Ryuichi Masuda a, *, Robert J. Losey b, Vladimir I. Bazaliiskii c, Bair Badmaev d

a Department of Natural History Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japanb Department of Anthropology, 13-15 Tory Building, University of Alberta, Edmonton T6G 2H4, AB, Canadac Laboratory of Archaeology and Paleoecology, Irkutsk State University, Karl Marx 1, 664003 Irkutsk, Russiad Institute of General and Experimental Biology, Siberian Division, Russian Academy of Sciences, 670047 Ulan-Ude, Russia

a r t i c l e i n f o

Article history:Available online xxx

Keywords:Ancient DNABaikalHoloceneMarmotMarmota sibiricaMitochondrial DNA

* Corresponding author.E-mail address: [email protected] (

http://dx.doi.org/10.1016/j.quaint.2015.03.0501040-6182/© 2015 Elsevier Ltd and INQUA. All rights

Please cite this article in press as: Masuda,Raisovet cemeteries near Lake Baikal: Spdx.doi.org/10.1016/j.quaint.2015.03.050

a b s t r a c t

We analyzed mitochondrial DNA (mtDNA) from ancient marmot teeth (~7550e6800 cal. BP), which wererecovered during archaeological excavations of two contemporary cemeteries near Lake Baikal, Russia:one archaeological site is the Shamanka II cemetery located on the southwest shoreline of Lake Baikal,and the other is the Lokomotiv-Raisovet cemetery located about 77 km to northwest and within themodern city of Irkutsk. Although the teeth had not been identified to species based on their morphology,our ancient DNA analysis revealed that all incisors from ten individuals were of the tarbagan marmot(Marmota sibirica), which is currently not distributed around those archaeological sites. In contrast, theblack-capped marmot (Marmota camtschatica), which also has a dominant distribution in Eastern Siberiaand whose incisors are morphologically similar to M. sibirica, was not identified from our ancient toothsamples. In addition, the mtDNA sequence variation showed that the genealogy of marmots in theShamanka II cemetery could have been different from that of the Lokomotiv-Raisovet cemetery. Thesedata indicate that the ancient people at the Shamanka II cemetery could have used M. sibirica fromdifferent regions than those utilized at the Lokomotiv-Raisovet site. This suggests non-overlappingmarmot hunting ranges for the people buried at the two Middle Holocene cemeteries.

© 2015 Elsevier Ltd and INQUA. All rights reserved.

1. Introduction

Forager cemeteries in the Lake Baikal region of Siberia oftencontain abundant faunal remains, and these items have significantinterpretive value in understanding various aspects of past humansocieties, including food procurement ranges and various forms ofsocial interaction. Animals are widely known to be focal points forhunteregatherer interaction. For example, seasonally availablefaunal resources such as spawning fish andmigrating ungulates candraw people from distant regions to specific places on the land-scape. At such places, various social relationships can occur, rangingfrom the sharing of food to the exchange of information and evenmarriage partners. These human engagements, as well as thoseoccurring between people and animals at these places, potentially

R. Masuda).

reserved.

R., et al., Ancient DNA analyecies identification and gen

have material consequences, and these can be investigated inmortuary sites, including through the technical study of buriedfaunal remains.

In this paper, we study tarbagan marmot (Marmota sibirica)teeth from two Middle Holocene cemeteries near Lake Baikal(Russian Federation) to assess if some people buried at the sites hadoverlapping marmot hunting ranges. The tarbagan marmot is ahibernating steppe and forest-steppe animal and historically is acommon food source. The abundance of their remains at the twosites suggests they could have been an important shared food itemfor the two cemetery groups that was procured from a singlelocation. Additionally, stable isotope studies of human remainspreviously indicated some similarity in the general structure ofdiets at the two cemetery sites, which are within a few dayswalking distance of each other. This might also indicate some de-gree of interaction between the two groups. However, given theambiguous nature of this isotope data, it is not yet possible to usethem to determine how closely people from the two sites

sis of marmot tooth remains from the Shamanka II and Lokomotiv-ealogical characteristics, Quaternary International (2015), http://

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R. Masuda et al. / Quaternary International xxx (2015) 1e72

interacted. For example, did these two groups trade, intermarry, orsimply live nearby and share isotopically similar foods, with nodirect sharing of food resources? Here we analyze mitochondrialDNA from a sample of the marmot teeth from the two cemeteries toassess if these animals were procured from the same populationand region, and thus were a faunal focal point for humaninteraction.

2. Background

The marmot teeth analyzed in this study were recovered duringarchaeological excavations of two contemporary cemeteries nearLake Baikal, Russia (Fig. 1). The first, the Shamanka II cemetery, islocated on the southwest shoreline of Lake Baikal, while the second,the Lokomotiv-Raisovet cemetery, is about 77 km to northwest andwithin the modern city of Irkutsk. This latter cemetery overlooksthe confluence of the Irkut and Angara rivers. Excavations at theShamanka cemetery have resulted in the discovery of 96 gravescontaining the remains of a minimum of 154 individuals dating tothe Early Neolithic period, or ~7550 e 6800 cal. BP, and an addi-tional 11 later-dating graves also have been uncovered (Bazaliiskii,2003; Bazaliiskii and Weber, 2005, 2006, 2008; Bazaliiskii et al.,2006; Weber et al., 2006; Weber et al., in press). The Lokomotiv-Raisovet cemetery has proven to contain at least 87 graves, butdetailed information is only available for 71 of these. The docu-mented graves held the remains of at least 124 human individuals,with the vast majority dating to the Early Neolithic period(Mamonova and Bazaliiskii, 1991; Bazaliiskiy and Savelyev, 2003,2008; Weber et al., 2006; Weber et al., in press).

Fig. 1. Geographical locations of the Shamanka II and Lokomotiv-Raisovet cemeteriesnear Lake Baikal, Russia. The upper map shows the location of the Baikal area ineastern Eurasia.

Please cite this article in press as: Masuda, R., et al., Ancient DNA analyRaisovet cemeteries near Lake Baikal: Species identification and gendx.doi.org/10.1016/j.quaint.2015.03.050

Faunal remains from the two cemeteries were identified by Dr.R. Losey in 2008e2010. Incisors and their fragments identified asgenusMarmota (order Rodentia, family Sciuridae) numbered 643 atthe Lokomotiv-Raisovet cemetery and 1521 at the Shamanka IIcemetery (Fig. 2). The incisors could not be identified to specieslevel due to the similarities in size and shape of such teeth in thetwo marmot species currently occurring in Eastern Siberia, namelythe tarbagan marmot (Marmota sibirica) and the black-cappedmarmot (M. camtschatica). The Shamanka II cemetery also con-tained four mandibles that were morphologically identified asMarmota sibirica; all had their incisors removed. All Marmota re-mains at both cemeteries were found in graves dating to the EarlyNeolithic period, ~7550 e 6800 cal. BP (Bazaliiskiy and Savelyev,2003; Weber et al., 2006; Weber et al., in press), and appear tohave been parts of the garment or ornamentsworn by the deceased,with the exception of the four mandibles. Suchmarmot remains arenearly entirely absent (one specimen was identified at a single site,Sagan-Zaba II) from the Holocene habitation sites along the AngaraRiver and the shores of Lake Baikal (c.f., Medvedev, 1969; Ermolova,1978; Savel'ev et al., 2001; Klement'ev et al., 2005; Goriunova et al.,2007; Losey et al., 2008, 2014; Nomokonova et al., 2010, 2011),suggesting these animals were procured elsewhere.

The tarbagan marmot is a social species that lives in coloniesfound in dessert, steppe, and forest-steppe habitats, and historicallywas present in Northwestern China, Mongolia, and parts of Siberia(Russia; Zimina, 1978). These marmots were present in the upperportion of the Tunka Valley west of the Shamanka II cemetery asrecently as the 1970s (Zimina, 1978). The nearest other remaining

Fig. 2. Marmot incisors excavated together with a human skeleton from the ShamankaII cemetery. Some Early Neolithic people living near Lake Baikal likely used the marmotincisors for clothing or jewelry.


R. Masuda et al. / Quaternary International xxx (2015) 1e7 3

populations are found in southern Buriatia, about 100 km south ofShamanka, and along the Selenga River south of Ulan-Ude (Zimina,1978; Erbajeva and Alexeeva, 2009; Townsend, 2009). Adult tar-bagan marmots weigh from six to eight kilograms, and in thenorthern portions of their range (southern Trans-Baikal) hibernatefromearly September throughMarch, but generally do not exit theirburrows until April (Zimina, 1978; Murdoch et al., 2009). Intensivefeeding and weight gain occurs throughout summer, and by Augustthe animals are at their fattest. The subcutaneous fat layers in themarmots act as energy stores for the hibernation period, and adultfemales can have up to 1 kg of fat in their bodies by the end ofsummer. Marmots are a highly valued food item in parts ofMongolia and Siberia, have high-quality furs, and are used in a suiteof traditional medicines (Zimina, 1978; Belikov, 1994; Pegg, 2001;Kolesnikov et al., 2009). Historically, tarbagan marmots were hun-ted during the non-hibernation period, particularly in late summerand early fall when they were at their fattest (Zimina, 1978).

The black-capped marmot is typically found in alpine and arctictundra, with the populations closest to the study area currentlybeing located in Barguzin Mountains along the northeast coast ofLake Baikal (Zimina, 1978). The adults in the Barguzin Mountainpopulation are about half the size of tarbagan marmots, weighingon average around three kilograms. This marmot also inhabitscolonies, and hibernates from September/October toMay. Far less isknown about its traditional uses, perhaps due to the fact that it livesin habitats not often visited by people, namely high altitude areasabove the tree line.

Table 1Profiles of marmot samples examined in the present study

Sample ID Tissue Taxonomicspecies

Sampling localit

#16-37 Shamanka Ancient incisor Marmota spp. Shamanka II#39-1 Shamanka Ancient incisor Marmota spp. Shamanka II#56-807 Shamanka Ancient incisor Marmota spp. Shamanka II#59-161 Shamanka Ancient incisor Marmota spp. Shamanka II#104-71 Shamanka Ancient incisor Marmota spp. Shamanka II#3-62 Raisovet Ancient incisor Marmota spp. Lokomotiv-Raiso#11-7 Raisovet Ancient incisor Marmota spp. Lokomotiv-Raiso#22-36 Lokomotiv Ancient incisor Marmota spp. Lokomotiv-Raiso#24-45 Lokomotiv Ancient incisor Marmota spp. Lokomotiv-Raiso#38-10 Lokomotiv Ancient incisor Marmota spp. Lokomotiv-Raiso#S1 Modern incisor Marmota sibirica Dzhidinskii raio

#S2 Modern incisor Marmota sibirica Dzhidinskii raio

#S21 Modern incisor Marmota sibirica Selenginskii raio

#ads17 Modern incisor Marmota sibirica Tarbagataiskii ra

#ads22 Modern incisor Marmota sibirica Ivolginskii raion

#ads31 Modern incisor Marmota sibirica Tarbagataiskii ra

#Mar1 Modern muscle Marmota sibirica Tarbagataiskii ra

#Mar2 Modern muscle Marmota sibirica Selenginskii raio

#Mar3 Modern muscle Marmota sibirica Selenginskii raio

#C13 Modern incisor Marmota camtschatica Severobaikalskii




Sample IDs of archaeological remains are shown with “grave no.efind no.”.Asterisks show the 288-bp sequences, which were identical with the corresponding regiowithout asterisks indicate the sequences newly determined in the present study.


Notably, neither Shamanka II nor Lokomotiv-Raisovet is pres-ently located in steppe or forest-steppe areas suitable for tarbaganmarmots, but such ecozones are located in the Angara Valleydownstream of the Lokomotiv-Raisovet site, the Kuda Valley justnortheast of Irkutsk, and about 40 km west of the Shamanka IIcemetery in the Tunka Valley. The nearest potentially suitablehabitat for black-capped marmots is probably the alpine zones ofthe Eastern Sayan Mountains to the west-southwest of the twocemeteries.

Although the Shamanka II and Lokomotiv-Raisovet cemeteriesare 77 km apart and in quite different settings, stable carbon andnitrogen isotope values of human remains from the two sitesoverlap to some extent (Weber et al., 2011). This might suggestoverlapping foraging ranges for some individuals from the twosites, including overlapping marmot hunting areas, but thecomplexity of the isotope ecology of the region makes such in-terpretations difficult to substantiate. For example, the stableisotope values of most terrestrial prey species are essentiallyidentical across the entire region, and thus human stable isotopevalues provide no clear indication of where such resources wereprocured. Further, the isotope values of some riverine and lake fishalso may overlap (prehistoric values are not well-established), andsome fish also move between the lake and the Angara River (seeLosey et al., 2012). The overlap in isotope values at the two ceme-teries could simply be the result of some individuals consumingisotopically similar foods, and these foods could have been pro-cured from non-overlapping areas of the landscape.

ies Supplier mtDNAhaplotype

Accession no.

Irkutsk State University S02 LC014961Irkutsk State University S01 LC014960Irkutsk State University S03 Same as AF143937*Irkutsk State University S02 LC014961Irkutsk State University S01 LC014960

vet Irkutsk State University S03 Same as AF143937*vet Irkutsk State University S03 Same as AF143937*vet Irkutsk State University S03 Same as AF143937*vet Irkutsk State University S04 LC014962vet Irkutsk State University S05 LC014963n Buryatia Institute of General and

Experimental BiologyS03 Same as AF143937*

n Buryatia Institute of General andExperimental Biology

S03 Same as AF143937*



ion Buryatia Institute of General andExperimental Biology


Buryatia Institute of General andExperimental Biology










raion Buryatia Institute of General andExperimental Biology

C01 LC014964


C02 same as AF143922*


C02 Same as AF143922*


C02 Same as AF143922*

n in the registered sequences with those accession numbers. The accession numbers



3. Materials and methods

3.1. Samples and DNA extraction

The incisors of the marmots excavated from the Shamanka II(five individuals) and Lokomotiv-Raisovet (five individuals) cem-eteries were curated in the Laboratory of Archaeology and Paleo-ecology, Irkutsk State University until use (Table 1). Themorphological characteristics of the teeth allowed the incisors ofindividual marmots to be identified (upper versus lower, and leftversus right), but it was difficult to identify the specimens tospecies, as several marmot species are naturally distributed withinthe broader region (Steppan et al., 1999). For modern Marmotasibirica, upper incisors of six individuals and muscle tissues ofthree individuals were used as controls of species identification. Inaddition, upper incisors of four modern M. camtschatica wereanalyzed. These modern specimens, all with information oncollection locations, were stored in the Institute of General andExperimental Biology, Russian Academy of Sciences (Ulan-Ude),and used for comparison with the ancient marmot tooth samples(Table 1).

For DNA extraction, about 0.1e0.2 g of powder were obtainedfrom the root of each incisor using electric drills on a clean bench.Total DNA was extracted from the incisor powder, and the extractwas concentrated to 100e200 ml of TE buffer according to themethod of Masuda et al. (2001, 2006). In addition, total DNA frommuscle tissues from modern individuals of M. sibirica for compar-ison was extracted using the DNA Blood&Tissue kit (Qiagen) ac-cording to the manufacturer's instruction.

3.2. Amplification by polymerase chain reaction and directsequencing

First, the whole cytochrome b gene in the mitochondrial DNA(mtDNA) was amplified from muscle tissue DNA of M. sibiricausing polymerase chain reaction (PCR) with a primer pair, Cb-M1and Cb-MR2, reported by Matsuhashi et al. (1999) and Tamadaet al. (2005). The PCR products were sequenced using themethod described below. Then, based on the obtained nucleotidesequences, two primers MSB-F1 (50-CCG GAT TAT TTC TAG CAATAC AC-30), MSB-R1 (50-AAC TGA TGA GAA GGC TGT TG-30) andMSB-R2 (50-TAC ACT CCT CGG CCC ACA TG-30), were newlydesigned for PCR amplification of shorter fragments from ancientDNA. A primer pair of Cb-M1 and MSB-R1 was used for ampli-fication of fragment A, and another pair of MSB-F1 and MSB-R2was used for that of fragment B. Both fragments A and B of thecytochrome b gene were directly sequenced by the followingmethod. Some part of both fragments overlapped each other, andconsequently those sequences yielded a total of 334 base-pairs(bp).

The PCR for incisor DNA was performed in a total volume of20 ml containing 10 ml of 2 X Multiplex PCR Master Mix (Qiagen),0.2 ml of each primer (25 pmol/ml), 0.4 ml of bovine serum albu-min (20 mg/ml) for inactivation of any enzyme-inhibitors, and1e2 ml of concentrated DNA extract, adjusted by double-distilledwater; cycling conditions were 95 �C for 15 min; 40 cycles of94 �C for 30 s, 50 �C for 3 min (or 56 �C for 1.5 min), and 72 �C for1 min (or 72 �C for 1.5 min); and 72 �C for 10 min. To check thePCR amplification, an aliquot (10 ml) from the PCR product wasrun on a 2% agarose gel, stained by ethidium bromide, and thenvisualized under an ultraviolet illuminator. The PCR productswere purified with the QIAquick PCR Purification Kit (Qiagen)and directly applied to the BigDye Terminator v1.1 CycleSequencing Kit for an ABI 3130 or 3730 DNA automatedsequencer (Applied Biosystems). Sequencing was done using


each of forward and reverse PCR primers as a sequencing primerfor confirmation of complementary sequences in both strands ofPCR products.

In order to secure the authenticity of the nucleotide sequencedata, the extract with no sample obtained by the same method asused on the incisor samples was used as a negative controlwhenever sample extractionwas performed in an air-filtrated cleanbench after the ultraviolet exposure and the treatment of DNAAWAY (Molecular BioProducts). Most experiments were donewhilewearing disposable gloves and were made on a clean bench toprevent any external DNA contamination. Whenever PCRs usingDNA extracts were done, it was confirmed that no PCR productswere generated from the negative control with the same reactioncondition.

3.3. Data analysis

Nucleotide sequences obtained in the present study werealigned using MEGA 5.0 software (Tamura et al., 2011). Minimumspanning networks among mtDNA haploytpes were constructedusing TCS 1.21 software (Clement et al., 2000). For identification ofspecies and the phylogenetic analysis, the partial nucleotide se-quences (288 bp) of the 5‘ end of the whole cytochrome b gene(1140 bp) were cited for the three marmot species that aredistributed in northeast Eurasia and are phylogenetically closelyrelated to one another (Steppan et al.,1999). The accession numberscited from the DNA data bank of Japan (DDBJ) were as follows:AF143937 and AF143938 for M. sibirica (Steppan et al., 1999);AF143921, AF143922 (Steppan et al., 1999) and AF100715(Kruckenhauser et al., 1999) for M. camtschatica; and AF142928 forthe Himalayan marmot (M. himalayana) (Steppan et al., 1999). The288-bp region of the M. sibirica cytochrome b was identical be-tween AF142937 and AF143938.

4. Results and discussion

4.1. Determination and variation of marmot cytochrome bsequences

We determined 334-bp partial sequences of the mtDNA cyto-chrome b gene for all the nine modern Marmota sibirica and thefour modern M. camtschatica samples (Table 1; Supplementaryfigure S1). The homology search (BLAST search in the DDBJ)showed that the 334-bp sequences consisted of 46-bp sequences oftRNA genes and the 50 end of 288 bp in the marmot mtDNA cyto-chrome b gene. Because at least the latter 288 bp are available in thedatabase for sequence comparison, this part was used for thesubsequent analysis.

For the modern M. sibirica samples, all seven individuals sharedone 288-bp sequence (named haplotype S03), which is identicalwith that from accession no. AF143937 from M. sibirica previouslyreported by Steppan et al. (1999). For the modern M. camtschaticasamples, one individual had a new sequence (named haplotypeC01) and the other three shared another new sequence (namedC02). The 288-bp part from AF100715 reported by Kruckenhauseret al. (1999) was named C03 in the present study as the thirdsequence ofM. camtschatica. Because no samples forM. himalayanawere available in the present study, the 288-bp part from AF142928reported by Steppan et al. (1999) was cited and named haplotypeH01 for analysis.

The 288-bp sequences of cytochrome b determined in the pre-sent study (Table 1) were deposited to the DDBJ/EMBL/GenBanknucleotide sequence databases with accession numbersLC014960eLC014965.



4.2. Species identification of ancient marmot incisors based onmolecular phylogeny

We successfully determined 334-bp sequences of the cyto-chrome b gene for all of the ten ancient marmot incisors from theShamanka II and Lokomotiv-Raisovet cemeteries, and found thatthey were separated into five haplotypes (Table 1; Supplementaryfigure S1). Then, a homology search showed that three ancientmarmot samples from the cemeteries shared one commonsequence, which is identical with haplotype S03 (Tables 1 and 2). Inaddition, the homology search result indicated that the four hap-lotypes from the other six ancient marmots had the highest simi-larities (0.7e1.4% differences; 2e4 bp/288 bp) with haplotype S03,and that all of the five haplotypes have higher similarities (0.3e2.1%differences; 1e6 bp/288 bp) with one another. Therefore, the fourremaining haplotypes were thus named S01, S02, S04 and S05(Tables 1 and 2).

Table 2Nucleotide number differences (above diagonal) and percentage differences (belowdiagonal) between marmot mtDNA haplotypes. Haplotypes S01 e S05 for Marmotasibirica; C01 e C04 for M. camtschatica; and H01 for M. himalayana.

S01 S02 S03 S04 S05 C01 C02 C03 C04 H01

S01 1 4 6 6 15 16 16 17 18S02 0.3 3 5 5 14 15 15 16 17S03 1.4 1.0 2 4 13 14 14 15 18S04 2.1 1.7 0.7 2 13 12 14 15 20S05 2.1 1.7 1.4 0.7 11 10 12 13 18C01 5.2 4.9 4.5 4.5 3.8 1 1 2 16C02 5.6 5.2 4.9 4.2 3.5 0.3 2 3 17C03 5.6 5.2 4.9 4.9 4.2 0.3 0.7 1 17C04 5.9 5.6 5.2 5.2 4.5 0.7 1.0 0.3 18H01 6.3 5.9 6.3 6.9 6.3 5.6 5.9 5.9 6.3

Table 3Frequency of mtDNA haplotypes of Marmota sibirica from the Shamanka II andLokomotiv cemeteries and modern Buryatia

Locality Haplotypes

S01 S02 S03 S04 S05

Ancient Shamanka II 2 2 1 0 0Lokomotiv 0 0 3 1 1

Modern Buryatia 0 0 7 0 0

A further sequence comparison of the five haplotypes of ancientmarmots with modern marmot sequences (S03, C01, C02, C03, C04and H01) (Table 2) showed that haplotypes S01eS05 of ancientmarmots had 3.5e5.9% sequence differences (10e17 bp/288 bp)from C01eC04 of M. camtschatica and 5.9e6.9% sequence differ-ences (17e20 bp/288 bp) from H01 of M. himalayana. Meanwhile,the sequence differences between C01eC04 of M. camtschatica andH01 of M. himalayana were 5.6e6.3% (17e20 bp/288 bp) (Table 2).

In addition, the minimum spanning network (Fig. 3) showedthat haplotypes S01eS05 and the known S03 belonging toM. sibirica are clustered and closely related with one another. The“S01eS05” cluster had 10 steps separating it from cluster

Fig. 3. Minimum spanning network among mtDNA haplotypes, which were identified fromtissues of the three taxonomic marmot species, Marmota sibirica, M. camtschatica and M. himrelated with S01, S02, S04 and S05, all of which were found in ancient incisors. By contrast, hawith those of M. sibirica. Small closed circles show presumed haplotypes. One bar betweeancient incisors are of M. sibirica, and occurrence of some sequence variations within ancie


“C01eC04” of M. camtschatica, which had 16 steps between it and“H01” of M. himalayana.

These results of the sequence similarities and phylogenetic re-lationships strongly show that haplotypes S01eS05 all belong toM.sibirica. Therefore, we concluded that the analyzed incisors fromthe Shamanka and Lokomotiv-Raisovet cemeteries were of M.sibirica.

4.3. Genealogy and frequency differences in mtDNA haplotypes ofancient incisors from two archaeological sites

Table 3 shows the frequency of mtDNA haplotypes found in theanalyzed archaeological samples. In the Shamanka II samples,haplotype S01 was found in two individuals, S02 in two individuals,and S03 in only one. By contrast, in the Lokomotiv-Raisovet sam-ples, haplotype S03 was present in three individuals, S04 was inone individual, and S05 in one. This frequency difference indicatesthat the marmots found in the Shamanka II cemetery likely origi-nated from different regional population(s) than those found in theLokomotiv-Raisovet cemetery. However, we have not examined themtDNA phylogeography of modern M. sibirica populations aroundLake Baikal because of the lack of availability of samples from thisregion. Only a single small remnant population ofM. sibirica existedin the region historically (as was present as recently as the 1970s),and was found at the far western end of the Tunka Valley (Zimina,1978). Because haplotype S03 was shared by nine specimens (fromBuryatia) of M. sibirica used in the present study and two in-dividuals (from Chitinsk and Buryatia) reported by Steppan et al.(1999), this haplotype could be distributed widely in Trans-Baikal(Eastern Siberia). Therefore, it may be natural that S03 was foundboth in the Shamanka II and Lokomotiv-Raisovet cemeteries. Thenatural distribution of the other haplotypes S01, S02, S04, and S05in the Middle Holocene could be resolved in the future by studyingnot only modern, but also paleontological and archaeologicalsamples from across Eastern Siberia.

the marmot incisors of the Shamanka II and Lokomotiv cemeteries as well as from thealayana. As shown in Table 1, haplotype S03 was identified from M. sibirica, and closelyplotypes C01 e C04 forM. camtschatica and H01 forM. himalayana are remotely relatedn two haplotypes means one nucleotide substitution. This network indicates that allnt M. sibirica.



4.4. Reconstruction of Early Neolithic hunting ranges from marmotlineages

As mentioned earlier, marmot remains are nearly entirely ab-sent from the Holocene habitation sites along the Angara Riverand the shores of Lake Baikal, suggesting that these animals wereprocured in an area of the landscape not yet adequately sampled.This study shows that humans constructing the cemeteries usedmarmot incisors collected from different areas, at least based onthe small number of teeth analyzed here. As previously described,on the other hand, the stable isotope values of humans buried atthe Shamanka II and Lokomotiv-Raisovet cemeteries showed someoverlap, indicating the possibility that some humans buried atthese two cemeteries had overlapping foraging ranges. Thesharing of mtDNA haplotype S03 by both cemeteries may indicatethe partial overlap in marmot hunting areas, but the broad dis-tribution range of this haplotype still leaves open the possibilitythat two entirely spatially distinct marmot hunting areas wereutilized.

The first possible cause for overlap in stable isotope values forsome individuals at the two cemeteries is the use of overlappingfishing areas (Weber et al., 2011). A productive fishery possiblyused by both groups is the section of the Angara River betweenthe Lokomotiv-Raisovet cemetery and Lake Baikal. This portion ofthe river was home to its own resident fish, but also receivedseasonal influxes of spawning fish from Lake Baikal (see Loseyet al., 2012). Further, this region was easily accessible to in-dividuals buried in both cemeteries by overland and water routes(Fig. 1). The widespread use of marmot teeth at these two ceme-teries clearly suggests interaction and shared practices, and thesharing of this fishery was one possible focal event for suchinteraction. A second possibility is that the fishery was not shared,and the overlap in isotope values is due to some individuals buriedat both sites consuming isotopically similar fish, as mentionedearlier. A third possibility is that the interaction between the tworegions involved exchanges of people through marriages, traderelations, visiting, and other forms of permanent migration.Isotope values for human bone can take up to a decade to fullyturn over, and as a result, people involved in such movementscould have retained the dietary signatures of their original areaslong after migrating (and dying), resulting in some overlappingisotope values for the two cemeteries. All three of the abovepossibilities require additional studies before they can beconfirmed or refuted.

Finally, the genetic confirmation of the presence of M. sibiricain the two cemeteries indicates the presence of steppe or forest-steppe ecosystems somewhere in the region from ~7550 e

6800 cal. BP, as such landscapes are known suitable habitats forM. sibirica. This is significant because the broader region appearsto have been wetter and warmer than present during the generalperiod in which the two cemeteries were created (Demske et al.,2005; Tarasov et al., 2007, 2009; Shichi et al., 2009; Bezrukovaet al., 2010, 2013; White and Bush, 2010). Forests of the Siberianspruce (Picea obovata) were dominant in the region during thisperiod, with the pine (Pinus sibirica) becoming progressively morecommon here only after ~7000 cal. BP with the onset of colderand drier conditions. In such forests, M. sibirica likely could nothave survived. Regardless, despite the wetter and warmer con-ditions here prior to ~7000 cal. BP, open landscapes had alreadyformed. Where those landscapes were located is not presentlyknown, but they could have been located in the same regionswhere steppe and forest-steppe are found today, namely theTunka, Kuda, and Angara valleys, and in these landscapes severalmtDNA (maternal) lineages of M. sibirica could have occurredallopatrically.


5. Conclusions

Overall, our mtDNA analyses of marmot teeth from the Sha-manka II and Lokomotiv-Raisovet cemeteries indicate that thesespecimens were clearly from tarbagan marmots, a species that in-habits steppe and forest-steppe habitats. These animals were likelya late-summer to early fall food source for some of the individualsburied at both cemeteries. Further, our results suggest that theseteeth likely did not originate from a single population, and that thepeople buried at Shamanka II and Lokomotiv-Raisovet probably didnot share a single marmot hunting region. The hunting of thesemarmots does not appear to have been a focal point of interactionfor the two groups, despite the fact that remains of this animal wereabundant at both cemeteries. Only a small number of teeth wereanalyzed from each site, and future study of a larger sample isnecessary to confirm our present findings. Isotopic and traceelement characterization of the marmot teeth, and those of othersmall non-migratory mammals in the sites, should also help tobetter define foraging ranges and how such fauna were involved inhuman interactions.

Acknowledgments

We thank Prof. A.W. Weber (University of Alberta) and Prof. H.Kato (Hokkaido University) for invaluable suggestions, and Dr. F.Khenzykhenova (Russian Academy of Sciences) and Prof. T. Sato(Keio University) for helpful support. We are also grateful to Ms. S.Ida and Ms. S. Isono (Hokkaido University) for technical assistance.Aaron Coons is thanked for his assistance with the map figure. Thisstudy was in part supported by the Baikal-Hokkaido ArchaeologyProject funded by the Social Sciences and Humanities ResearchCouncil of Canada (Major Collaborative Research Initiative GrantsNo. 412-2011-1001), and the Japan Society of the Promotion ofScience (JSPS) Core to Core Program (Advanced Research Networks)“Advanced Core Research Center for the History of Human Ecologyin the North”.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.quaint.2015.03.050.

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Ancient DNA analysis of marmot tooth remains from the Shamanka II and Lokomotiv-Raisovet cemeteries...

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