Archaeological Research in Asia 25 (2021) 100238

Available online 12 January 20212352-2267/© 2020 Elsevier Ltd. All rights reserved.

Full length article

Insights into Lake Baikal’s ancient populations based on genetic evidence from the Early Neolithic Shamanka II and Early Bronze Age Kurma XI cemeteries

N.M. Moussa a,*, H.G. McKenzie b, V.I. Bazaliiskii c, O.I. Goriunova c, F. Bamforth f, A. W. Weber d,c,e

a Department of Biological Sciences, MacEwan University, Edmonton, Alberta, Canada b Department of Anthropology, Economics and Political Science, MacEwan University, Edmonton, Alberta, Canada c Research Centre “Baikal Region”, Irkutsk State University, Karl Marx Street 1, Irkutsk 664003, Russia d Department of Anthropology, 13-15 H.M. Tory Building, University of Alberta, Edmonton, Alberta T6G 2H4, Canada e Laboratoire Mediterraneen de Prehistoire Europe Afrique (LAMPEA), UMR 7269, Aix-Marseille Universite, 5 rue du Chateau de l’Horloge - B.P. 647, 13094 Aix-en- Provence Cedex 2, France f Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, AB, Canada

A R T I C L E I N F O

Keywords: Lake Baikal Early Neolithic Early Bronze Age DNA Kurma XI Shamanka II

A B S T R A C T :

Although previous ancient DNA research has contributed to the investigation of middle Holocene culture history and population dynamics in the Cis-Baikal, most of this work has been limited to the Angara valley and southwest Baikal, with only restricted genetic analysis of skeletal materials from the Little Sea microregion. In this paper, we expand upon initial findings by analyzing new mtDNA results from the EN/EBA Kurma XI cemetery (Little Sea area) and the EN Shamanka II cemetery (southwest Baikal). Our results not only contribute to the regional dataset, but also challenge previous findings. First, haplogroup Z was found for the first time in the ancient population of Cis-Baikal. Second, our data provide tentative support for the idea that an exogamous and/or patrilocal marriage pattern might be detectable at the Early Bronze Age cemetery Kurma XI. Third, our results indicate that the EN population of Cis-Baikal may not be as homogeneous in maternal origin as was previously suggested. Similarly, there seems to be less continuity between the Late Neolithic and Early Bronze age samples than previously thought, which further justifies the separation of these groups for future analyses. Finally, our data indicate that the maternal genetic background of the Early Bronze Age sample from Kurma XI is closer to that of known Early Neolithic groups than it is to those from the Late Neolithic or Early Bronze Age. This observation is surprising and, if correct, would seem to directly contradict the previous suggestion of a Middle Neolithic genetic discontinuity. These new findings complicate our understanding of the relationships between middle Holocene populations in the Cis-Baikal.

1. Introduction

The Cis-Baikal region in Eastern Siberia (52◦–58◦ north latitude, 101◦–110◦ east latitude) is characterized by numerous cemeteries dating to the middle Holocene. Extensive archaeological research of these sites suggests that the area was home to two temporally distinct foraging populations from the Early Neolithic (EN: ~7560–6690 cal. BP) to the Late Neolithic-Early Bronze Age (LN–EBA, ~6050–3470 cal. BP). The EN group was separated from the LN–EBA group by an ~700-year Middle Neolithic (MN) period in which no cemeteries have been

identified (Weber et al., 2010). In addition to temporal changes, varia-tion in lifeways is also visible in the area’s four archaeological micro-regions: Southwest Baikal, Angara River Basin, Upper Lena River Basin, and the Little Sea (or Ol’khon) area along the Lake’s northwest coast (Fig. 1). Research by the Baikal Archaeology Project (BAP) over the last 25 years has endeavored to explain this culture history through exten-sive multidisciplinary work, focusing on the synthesis of biological, environmental, and archaeological data.

Although previous ancient DNA (aDNA) research has contributed to this project, most of it has been limited to the Angara Valley and

* Corresponding author. E-mail address: nmoussa@ualberta.ca (N.M. Moussa).

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Archaeological Research in Asia

journal homepage: www.elsevier.com/locate/ara

https://doi.org/10.1016/j.ara.2020.100238 Received 9 February 2020; Received in revised form 16 October 2020; Accepted 27 October 2020

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southwest Lake Baikal, with only restricted analysis of genetic materials from the Little Sea microregion. More specifically, from the Little Sea area, only the Y-chromosome from six males from the EBA site Kurma XI had been successfully analyzed (Moussa et al., 2016). In this paper, we expand upon previous research by analyzing mtDNA data from 14 in-dividuals from Kurma XI, plus additional mtDNA data from 10 in-dividuals from the Shamanka II cemetery, located in southwest Baikal.

2. Background

Since 1996, an international consortium of researchers, BAP, has attempted to reconstruct the lifeways of ancient foragers from the Lake Baikal region through the application of several groups of research methods including archaeology, human osteology, bone chemistry, and environmental studies (Weber et al., 2010). This work has demonstrated that the populations on either side of the Middle Neolithic differed in terms of diet, health, mobility patterns, demography, spatial distribu-tion, social organization, and mortuary protocols (Weber et al., 2010). To date, genetic evidence from four sites has contributed to this analysis: the EN cemetery Shamanka II, located at the southwestern tip of Lake Baikal; the EN cemetery Lokomotiv, located along the Angara river in what is now the city of Irkutsk; the LN–EBA cemetery Ust’-Ida I, also located on the Angara river, downstream of Lokomotiv; and a limited amount of data from six males from the EBA cemetery Kurma XI, located in the Little Sea microregion on the shores of Lake Baikal. Attempts to extract DNA from skeletal remains recovered at a fifth cemetery, the EN

and EBA Khuzhir-Nuge XIV, located in the Little Sea microregion, have been mostly unsuccessful due to poor preservation.

2.1. Previous results of ancient DNA studies in the Lake Baikal region

Ancient DNA studies were first conducted on the Cis-Baikal materials by Russian scholars (e.g. Naumova and Rychkov, 1998; Naumova et al., 1997). These initial studies compared mtDNA data from 19 individuals from the Ust’-Ida I cemetery with profiles of modern populations of Siberia, Mongolia, and the Urals. They reported that the Baikal Neolithic populations were ancestral to modern contemporary Siberian pop-ulations (Naumova and Rychkov, 1998; Naumova et al., 1997). In this regard, it is important to note that these results were based on lower- resolution data than is typically used today.

Later analysis by BAP scholars compared mtDNA from human skel-etal and dental remains recovered from three sites (Shamanka II, Lokomotiv, Ust’-Ida I) using restriction fragment length polymorphisms (RFLP) (Mooder et al., 2005; Mooder et al., 2006; Thomson, 2006; Mooder et al., 2003). These results suggested discontinuity between the EN and LN–EBA populations, as evidenced by the different frequencies of mtDNA haplogroups (Mooder et al., 2010; Mooder, 2004; Mooder et al., 2006). Specifically, 6 East-Eurasian mtDNA haplogroups (A, C, D, F, G2a, U5a) were identified in 31 out of 40 skeletal remains from the EN Lokomotiv and 21 out of 28 samples from the EN Shamanka II (Mooder, 2004; Mooder et al., 2005; Thomson, 2006; Mooder et al., 2010). Sta-tistical analysis demonstrated that the mtDNA haplogroup distribution

Fig. 1. Locations of Kurma XI and Shamanka II cemeteries on Lake Baikal. The base map was created by Dr. Christian Leipe.

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was similar in the 2 burial sites, with D and F accounting for 38.5% (n =20/52) and 27% (n = 14/52) at Lokomotiv and Shamanka II, respec-tively (Mooder et al., 2010). In contrast, mtDNA analysis of 39 out of 42 individuals (29 LN and 10 EBA) from the LN–EBA cemetery, Ust’-Ida I, revealed the same 6 haplogroups but at significantly different fre-quencies relative to the EN Lokomotiv (p = 0.001) and Shamanka II haplogroups (p = 0.008) (Mooder et al., 2010). At Ust’-Ida I, hap-logroups A and C accounted for 54% (n = 21/39) of mtDNA (Mooder et al., 2010; Mooder, 2004). The same mtDNA analysis also suggested that at least some attributes of mortuary practices, such as the spatial arrangement of graves and body treatment, might be related to maternal kinship (Mooder et al., 2005). These genetic data have further been used by BAP researchers to evaluate large scale regional and temporal pat-terns of population affinity across Siberia and Inner Asia (e.g., Mooder et al., 2006; Schurr et al., 1999). Beyond BAP, Sikora et al. (2019) also incorporated many of the genetic data from the Cis-Baikal in their own analysis of the population history of northeastern Siberia since the Pleistocene.

Moussa et al. (2016) extended this research by including Y-chro-mosomal DNA from Lake Baikal’s middle Holocene populations, giving the first insights into paternal diversity. This study evaluated Y chro-mosomes from 7 EN Lokomotiv males, 9 EN Shamanka II males, 14 LN–EBA Ust’-Ida I males, and 6 EBA Kurma XI males. The results sug-gested paternal discontinuity between Lake Baikal’s EN and LN–EBA populations, consistent with Mooder et al. (2010) results. In addition, the similarity in Y-chromosome haplogroup distribution between the two EN populations (Lokomotiv and Shamanka II, haplogroup K) could indicate a common paternal source, which suggests a possible social interaction between the EN populations. However, these Y-chromo-somal findings are limited by the small number of males and the fact that they are based solely on haplogroup distributions (Moussa et al., 2016). A further limitation, as noted above, is that the six males from Kurma XI are the only samples representing the entire Little Sea microregion.

The primary aim of the current study is to expand the geographical and temporal coverage of aDNA research in the Cis-Baikal region by presenting new mtDNA results from Kurma XI (Little Sea area) to complement the existing Y-chromosome data from the same cemetery. Specifically, we used modified techniques to sequence directly the mtDNA hypervariable 1 region (HV1) from base pair (bp) 16,191 to 16,367, which contains most but not all of the SNP markers that identify Asian-specific haplogroups. We used the same modified techniques to reanalyze individuals from the Shamanka II cemetery (southwest Baikal) as well as to increase the sample size from that site. Our results provide new insights into the maternal lineage of Lake Baikal’s middle Holocene populations.

2.2. The Kurma XI cemetery

Kurma XI is located on the northwest coast of Lake Baikal’s Little Sea (Fig. 1). The archaeological and osteological materials from this site are described by Weber et al. (2012). Here, we present only a short summary for context. The 26 graves within the cemetery are distributed along the southeastern slope of a small hill approximately 500 m from the lake’s shoreline (Fig. 2). They have been radiocarbon dated to the Late Mesolithic/Early Neolithic1 and the Early Bronze Age. Eighteen graves, all EBA, were arranged over ~200 m along the base of the hill (6–16 m above the lake), while 8 graves (6 LM/EN and 2 EBA) were arranged on small terraces approximately 18–32 m above the lake. These two main clusters largely, although not entirely, correspond to the two different periods of cemetery use.

Based on both typological and radiocarbon data, foragers first

interred their dead at Kurma XI between ~8000–7500 cal. BP, and these initial 6 graves were built exclusively on the upper terraces (graves No. 20–24, 27). Owing to the small number of burials, the poor preservation of skeletal material, and the paucity of grave inclusions, relatively little can be said about the six graves that comprise this early component at Kurma XI except that they appear to resemble contemporaneous graves from the Little Sea microregion more than those from the Upper Lena, and much more than those in Angara valley or on Southwest Baikal (Goriunova et al., 2020). On typological grounds, they have been designated as belonging to the ‘Kurma’ mortuary group that is distinct from other EN traditions (e.g., Khotoruk, Kitoi) documented in the Cis- Baikal (see Goriunova et al., 2020).

Subsequent EBA foragers began to inter their dead at the site ~4600 cal. BP and continued to use the site until ~4000 cal. BP. Two of the individuals examined for ancient DNA variations (EBA KUR_2003.025, EBA KUR_2003.026) were interred on the upper terraces alongside the EN burials, while the rest were arranged along the base of the hill. While the demographic profile of Kurma XI was narrower than the largest neighboring EBA cemetery, Khuzhir-Nuge XIV, in that it lacked any children, the mortuary treatment of individuals as a whole was char-acterized by considerable diversity. It included both males and females ranging from older adolescents (17–19 years) to old adults (50+ years). These individuals received a range of different burial protocols (e.g., two different body positions, single and double burials, some covered in ochre, diverse grave disturbance patterns, unique grave goods, and variation in their number and diversity).

What this set of burials represents socially is, at present, unclear, and will likely require more extensive understanding of the broader cultural context of the EBA including comparison with neighboring sites. On the basis of the much narrower demographic profile of the site, along with the presence of several individuals with large artifact assemblages and rare goods, McKenzie (2012) has suggested that EBA Kurma XI might have acted as a specialized or exclusive burial ground in comparison to the neighboring Khuzhir-Nuge XIV, which seems to exhibit a more diverse representation of the microregional population.

3. Materials and methods

3.1. Contamination control

Contamination by modern DNA is one of the most difficult challenges researchers face trying to extract aDNA from prehistoric specimens. aDNA is highly degraded, and the extremely sensitive PCR techniques preferentially amplify intact contaminant modern DNA (O’Rourke et al., 2000). Rigorous excavation and laboratory protocols were essential to detect and eliminate contamination sources. Published criteria for verifying authenticity of aDNA results have been established through multiple analyses of the same sample and were followed for all labora-tory procedures in this study (Cooper and Poinar, 2000).

A laboratory at the University of Alberta dedicated to aDNA analysis was used to process, extract, and amplify aDNA specimens. Researchers dressed in protective suits, booties, sterile sleeves, masks, and goggles before entering the laboratory. All equipment and reagents were decontaminated either by bleaching, UV irradiation, and/or autoclaving before entering the laboratory depending on the suitability of the ma-terial. All equipment and bench surfaces were cleaned with a 10% (v/v) bleach solution. Sterile filtered tips were used for each analysis. Nega-tive controls (reagents with no added DNA) were introduced during all the extraction and PCR amplification steps. Positive modern DNA con-trols were analyzed only in the dedicated Post-PCR laboratory.

3.2. Samples

Vertebral bone or tooth samples were available for aDNA analyses from 10 EN Shamanka II (6 newly analyzed and 4 re-analyzed), 13 EBA Kurma XI, and 4 EN Kurma XI individuals (Tables 1 & 2). Vertebral

1 The earliest graves at Kurma XI are typologically contentious, and C14 dates from Burial 24 suggest that at least some of these EN graves may date to the Late Mesolithic.

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bones were selected in our study for two main reasons. Firstly, vertebrae contain a high proportion of spongy bone tissues, which have a higher DNA yield compared with other types of bone tissue (Lee et al., 1991). Secondly, because multiple vertebrae were available from one individ-ual, destruction of tissue for DNA extraction and analysis was seen as more justifiable.

Intact teeth are also a preferred source of DNA. They are embedded within bone, where they are largely protected from environmental fac-tors that might accelerate the DNA decomposition process (Schwartz et al., 1991), and their composition makes them less prone to contam-ination with extraneous DNA (Gilbert et al., 2005). Specifically, the acellular enamel covering the tooth crown (Nanci, 2003) protects the

cellular tooth pulp, which is the richest source of DNA in teeth (Malaver and Yunis, 2003). In this study, molars were the preferred samples because they have more than one root available for DNA extraction. Multi-rooted teeth provide more potential DNA than single rooted teeth (De Leo et al., 2000) due to the larger amount of pulp (Higgins and Austin, 2013). A total of 30 teeth and 5 vertebral bone samples were

Fig. 2. Distribution of graves in Kurma XI cemetery with mtDNA (orange) and Y-chromosomal (blue) haplogroups assigned to the individuals with their molecular sex indicated with male/female symbols. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 Summary of archaeological (Weber et al., 2011), osteological and demographic data (Lieverse, 2010) for Shamanka II samples used in ancient DNA analyses.

No Master ID Sample Age (years)

Morphological Sex

Period

1 SHA_2004.052 Vertebra 20–24 Probable Male EN 2 SHA_2004.044.02 Vertebra 20+ Undetermined EN 3 SHA_2005.059.01 Vertebra 35–39 Male EN 4 SHA_2006.083.01 Vertebra 20–22 Male EN 5 SHA_2007.090 Vertebra 18–20 Male EN 6 SHA_2007.096.02 Vertebra 30–35 Female EN 7 SHA_2001.012a Vertebra 25–35 Undetermined EN 8 SHA_2002.021.02a Vertebra 25–35 Male EN 9 SHA_2002.021.03a Vertebra 15–20 Undetermined EN 10 SHA_2002.023.04a Vertebra 20+ Undetermined EN

a Re-analyzed (for mtDNA) Shamanka II samples.

Table 2 Summary of archaeological, osteological and demographic data (Weber et al., 2011) for Kurma XI individuals used in ancient DNA analyses.

No. Master ID Sample Age (years)

Morphological Sex

Period

1 KUR_2002.001 Tooth 25–30 Male EBA 2 KUR_2002.007.01 Vertebra 20+ Undetermined EBA 3 KUR_2002.007.02 Vertebra 20–29 Male EBA 4 KUR_2002.010 Tooth 18–25 Probable Male EBA 5 KUR_2002.012 Tooth 20+ Undetermined EBA 6 KUR_2002.013 Tooth 40+ Male EBA 7 KUR_2002.014 Tooth & Vertebra 30–39 Female EBA 8 KUR_2002.015 Vertebra 17–18 Probable Male EBA 9 KUR_2002.016 Vertebra 20–30 Probable Female EBA 10 KUR_2003.017 Tooth 20+ Probable Male EBA 11 KUR_2003.018 Tooth 17–19 Probable Female EBA 12 KUR_2003.019 Tooth 20–30 Probable Male EBA 13 KUR_2003.021 Tooth 20–35 Probable Female EN 14 KUR_2003.022 Tooth 50+ Probable Female EN 15 KUR_2003.024 Tooth 20–35 Probable Female EN 16 KUR_2003.026 Tooth 35–50 Probable Male EBA 17 KUR_2003.027 Tooth 20–35 Undetermined EN

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available for aDNA analysis from the Kurma XI cemetery. Three Kurma XI individuals had both bone and tooth samples analyzed (KUR_2002.013, KUR_2002.014 and KUR_2002.015), and the rest of the Kurma XI individuals had more than one tooth sample available for analysis.

3.3. Sample preparation, decontamination, and DNA extraction

Decontamination protocols for the vertebral and tooth samples differed slightly, and these protocols are described in Moussa et al. (2016). For DNA extraction from bones and teeth, the silica-guanidium thiocyanate extraction protocol was adapted from Rohland and Hofreiter (2007a, 2007b), with some minor modifications that are described fully in Moussa et al. (2016).

3.4. Mitochondrial DNA haplogroup analysis and molecular sex determination

mtDNA was amplified as previously described by Mooder et al. (2005). We used modified techniques to examine direct sequencing of mtDNA hypervariable 1 region (HV1) from base pair (bp) 16,191 to 16,367 on the mtDNA described by Moussa et al. (2016).

Molecular sex determination using amelogenin was previously re-ported for males from Kurma XI and Shamanka II as part of an earlier analysis of Y-chromosome data (Moussa et al., 2016). In our study, molecular sex was analyzed for both males and females using the same protocols.

3.5. Evaluation of authenticity

Authenticity of results was established through several criteria. First, each sample was subjected to multiple analyses (Cooper and Poinar, 2000). In this paper, results are reported only if they were reproduced either by extracting DNA twice from the same sample or from two different samples belonging to the same individual on two independent occasions. DNA was extracted either from a bone or tooth sample depending on the availability of the samples for each individual. Sam-ples that did not show consistent results, or from which no DNA was obtained, were excluded from further analysis. Because only Eastern Siberian populations were studied, any results indicating a non-Asian haplogroup would have been excluded, although we observed no such instances. All researchers working with the samples gave consent to have their mtDNA characterized to facilitate early detection of modern DNA contamination.

3.6. Statistical analysis

mtDNA haplogroup distributions were compared using Fisher’s exact test with a two-by-two contingency table. The table extended to the size described by the number of populations and the number of haplogroups examined (see below). Fisher’s exact test is analogous to a two-way contingency chi-square test, but it is always chosen when dealing with a small sample size as in this study (The data analysis for this paper was generated using [SAS/STAT] software, Version [9] for [Linux]. Copy-right ©, 2014). All statistical analyses were performed using the SAS/ STAT® software.

The newly obtained results from the EN Shamanka II individuals (n = 10) were included, as were the results from the EBA individuals from Kurma XI (n = 12). These data were incorporated with published genetic data (Mooder et al., 2005; Mooder et al., 2006; Thomson, 2006) to make statistical comparisons between five groups from four prehistoric cem-eteries (EBA Kurma XI, LN Ust’-Ida I, EBA Ust’-Ida I, EN Lokomotiv, and EN Shamanka II). Owing to small sample size, the new results from the two EN individuals from Kurma XI were not included in statistical an-alyses; however, these data are still presented below.

It is important to note that previous genetic studies in Cis-Baikal have

treated the LN–EBA cemetery Ust’-Ida I as a single unit. However, recent refinement of C14 dating and observed differences in mortuary practices now support analysis of the LN and EBA components of this site sepa-rately (e.g., Bronk Ramsey et al., 2020; Weber et al., 2016; Weber et al., 2020). Therefore, our analysis treats the LN (n = 29) and EBA (n = 10) samples at Ust’-Ida I independently, which is the first time this has been done for genetic work in the region. As we discuss below, this approach has important ramifications for the interpretation of the genetic results for these samples.

4. Results

4.1. mtDNA results from Kurma XI and Shamanka II

For Kurma XI, mtDNA data were obtained from 2 out of 4 EN in-dividuals and 12 out of 13 EBA individuals (~82% success rate overall; Table 3). Both EN individuals were assigned to haplogroup F, while the 12 EBA individuals were assigned to 4 mtDNA haplogroups (A, D, F, Z). One of the haplogroup D individuals additionally carried a 16,224 ‘C >T’ transition, which has not been found previously to be associated with haplogroup D in the prehistoric population of Cis-Baikal. It is worth noting that our study produced mtDNA data for all five males for whom there are published Y-chromosome data (Moussa et al., 2016). Fig. 2 illustrates the spatial distribution of all mtDNA and Y-chromosomal DNA haplogroups (from both our study and previous research) plus the molecular sex assignment (see below) across the Kurma XI cemetery map. The 10 new mtDNA results from Shamanka II all came from EN individuals and are summarized in Table 4.

4.2. Molecular sexing

For Kurma XI, molecular sexing using amelogenin analysis was successful in 9 out of 12 EBA individuals (6 males and 3 females) and for 2 out 4 EN individuals (both female). In both EN individuals and in 7 of the 9 EBA individuals, molecular sex was concordant with the morphological sex. For one EBA individual (KUR_2003.018), molecular sex assignment of a male was discordant with a probable female morphological sex. One EBA individual (KUR_2002.007.01) with un-determined morphological sex was established to be female through molecular sexing (Table 3).

At Shamanka II, amelogenin analysis was only conducted on the six new EN individuals, as the other four had already been given molecular sex assignments (Thomson 2016). In all six cases, the analysis was successful, with four males and two females being identified. For one individual (SHA_2007.090), molecular sex assignment was a female, discordant with its male morphological sex. One individual (SHA_2004.044.02) with undetermined morphological sex was deter-mined to be male through molecular sexing (Table 4).

4.3. Comparison of mtDNA haplogroup frequencies between cemeteries

Haplogroup frequencies from each site can be found in Fig. 3. Because we considered the LN and EBA samples from Ust’-Ida I sepa-rately, it is worth highlighting these results. Likewise, it is useful to note the changes to the EN Shamanka II distribution with the addition of 10 new samples.

Once examined separately, the 29 LN individuals from Ust’-Ida I showed considerably greater diversity of haplogroups than the EBA sample (n = 10) from the same site (a point originally noted by Mooder et al., 2006 before they combined the data). Specifically, while only two haplogroups were represented in the EBA individuals (70% haplogroup C; 30% haplogroup A), at least 6 different haplogroups were represented in the LN group, with haplogroups A and ‘Others’ being the most populous (24% and 27.6% respectively). The designation ‘Others’ is assigned when a group of reproducible single nucleotide polymorphic sites on the mtDNA HV1 region do not relate to a specific haplogroup

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(Derenko et al., 2003). When our 10 new individuals from Shamanka II were combined with published data, the expanded Shamanka II dataset showed a higher frequency of haplogroup D (40.7%) and a lower fre-quency of haplogroup U5a (3.7%) than indicated previously (e.g. Mooder et al., 2010).

Table 5 summarizes comparisons between all the cemetery samples using the Fisher’s exact test results with p < 0.05 as the statistical sig-nificance level. For the most part, the mtDNA haplogroup distributions between the two EN cemeteries (Lokomotiv and Shamanka II), the LN and EBA components of Ust’-Ida I, and EBA Kurma XI were significantly different from each other. Interestingly, the exception to this pattern was that the EBA Kurma XI mtDNA haplogroup distribution was not signif-icantly different from that of either the EN Lokomotiv or the EN Sha-manka II populations (Table 5). EBA Kurma XI showed a higher frequency of haplogroup D (50%), a lower frequency of haplogroups A (8.3%) and Z (16.7%) (not previously found in Cis-Baikal prehistoric population), and the absolute absence of haplogroup C (Fig. 3). Another important result was that the expanded EN Shamanka II dataset was now statistically different from the EN Lokomotiv, making it inconsistent with previous findings (Mooder et al., 2010). As discussed below, it is possible that this is a result of small samples sizes.

5. Discussion

This study’s primary contribution is new information about the mtDNA genetic background of the Kurma XI cemetery in the Little Sea

microregion and the EN component of Shamanka II, located in South-west Baikal. Here, we discuss the implications of these new data within the context of what is already known about Lake Baikal’s middle Ho-locene populations. First, we discuss our new mtDNA results from EBA Kurma XI in conjunction with previously published data on Y-chromo-somal DNA from the same site. This will, for the first time in the Baikal region, permit analysis of both maternal and paternal lineages at a single site, thus enabling some tentative interpretations of marriage patterns. Second, we discuss the results of our comparison of mtDNA haplogroup frequencies between cemeteries (EBA Kurma XI, LN Ust’-Ida I, EBA Ust’- Ida I, EN Lokomotiv, and EN Shamanka II). This comparison provides new insights into the maternal background of the Baikal region as a whole, and it also challenges previous understandings (Mooder et al., 2005, 2006) of the apparent Middle Neolithic genetic discontinuity.

5.1. mtDNA and Y-Chromosome haplogroup distributions at EBA Kurma XI

As noted in the results, the mtDNA haplogroup distribution at EBA Kurma XI was mostly characterized by haplogroups frequently observed in the EBA populations of Cis-Baikal as a whole. However, two in-dividuals belonged to haplogroup Z, which has not been found previ-ously in the prehistoric populations of Cis-Baikal, despite being widespread in both ancient and contemporary populations of Asia (Derenko et al., 2003; Der Sarkissian et al., 2013). In addition, hap-logroup C was absent from Kurma XI, despite its presence at other

Table 3 Summary of the mtDNA haplogroups and their HSV1 sequence, Y-chromosomal haplogroups, and molecular sex assignment for Kurma XI individuals.

No Master ID Period mtDNA Haplogroup (this study)

HV1 Variants +16000 (this study)

Molecular Sex (this study and Moussa et al., 2016)

Morphological Sex

(Weber et al., 2011)

Y-Chr Haplogroup (Moussa et al., 2016)

1 KUR_2002.001 EBA D 223 319 XY Male m.d. 2 KUR_2002.007.01 EBA D 223 XX Undetermined 3 KUR_2002.007.02 EBA D 223 319 XY Male Q1a3 4 KUR_2002.010 EBA Z 223 260 m.d. Probable Male Q1a3 5 KUR_2002.012 EBA m.d. N/A m.d. Undetermined m.d. 6 KUR_2002.013 EBA A 223 290 319 m.d. Male m.d. 7 KUR_2002.014 EBA F 232A 249 304 311 XX Female 8 KUR_2002.015 EBA Z 223 260 XY Probable Male Q1a3 9 KUR_2002.016 EBA F 232A 249 304 311 XX Probable Female 10 KUR_2003.017 EBA D 223 XY Probable Male Q 11 KUR_2003.018 EBA D 223 224 XY Probable Female Q 12 KUR_2003.019 EBA F 232A 249 304 311 XY Probable Male m.d. 13 KUR_2003.021 EN m.d. N/A m.d. Probable Female m.d. 14 KUR_2003.022 EN F 232A 249 304 311 XX Probable Female 15 KUR_2003.024 EN F 232A 249 304 311 XX Probable Female 16 KUR_2003.026 EBA D 223 m.d. Probable Male m.d. 17 KUR_2003.027 EN i.r. m.d. m.d. Undetermined m.d.

m.d. = Missing data, no data obtained from the marked individuals. i.r. = Inconsistent results.

Table 4 Summary of the mtDNA haplogroups and their HSV1 sequence, Y-chromosome haplogroups and molecular sex assignment for Shamanka II individuals.

No Master ID Period mtDNA Haplogroup (this study)

HV1 Variants +16,000 (this study)

Molecular Sex

Morphological Sex (Lieverse et al., 2010)

Y-Chr Haplogroup (Moussa et al., 2016)

1 SHA_2004.052 EN D 223 XY Probable Male K 2 SHA_2004.044.02 EN C 223 298 327 XY Undetermined K 3 SHA_2005.059.01 EN C 223 298 327 XY Male K 4 SHA_2006.083.01 EN C 223 298 327 XY Male K 5 SHA_2007.090 EN A 223 227 290 XX Male 6 SHA_2007.096.02 EN D 223 319 XX Female 7 SHA_2001.012 EN D 223 XYa Undetermined K 8 SHA_2002.021.02 EN G2a 223 227 278 XYa Male K 9 SHA_2002.021.03 EN A 223 290 319 XYa Undetermined K 10 SHA_2002.023.04 EN D 223 XYa Undetermined K

a Molecular sex from these 4 Shamanka II individuals were determined by Thomson (2016).

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prehistoric cemeteries in the region (Mooder et al., 2006; Thomson, 2006).

5.1.1. Haplogroup Z Haplogroup Z is a subcluster from the macro-haplogroup M (Kivisild

et al., 2002). Its sister clade, haplogroup C, has higher frequency and diversity in eastern Eurasian populations (Schurr et al., 1999; Derenko et al., 2003). Haplogroup C is present in the Cis-Baikal prehistoric populations as described above (Mooder et al., 2006; Thomson, 2006). Both haplogroups C and Z are represented in Bronze Age south Siberian Kurgan people, with frequencies at 7.6% and 3.8%, respectively (Keyser et al., 2009). Haplogroup Z is also found among contemporary Central/

East Siberian populations (Der Sarkissian et al., 2013). In modern populations, haplogroup Z is found in northeast Asia (e.g.,

Itel’mens, 6.3% and (Koryaks, 5.8%) (Schurr et al., 1999), but it is also present in south Siberian populations such as the Altai-Sayan population (ranging between 1.1 and 6.5%) (Dulik et al., 2012; Gubina et al., 2013). It is present at low frequencies among numerous Finnic- and Turkic- speaking people in the Volga-Ural region (e.g. Udmurts, 5%, Maris, and Komis, 1.6%) (Ingman and Gyllensten, 2007; Bermisheva et al., 2002). Haplogroup Z also appears in other Siberian populations: the Evens, Yukaghirs and Dolgans, and in the Northern Yakuts and Evenks (Schurr and Wallace, 2003; Fedorova et al., 2013). The age of sub- haplogroup Z1a (~,9400 years BP), would imply its existence in the

Fig. 3. mtDNA haplogroup frequency distributions for EBA Kurma XI (this study), LN Ust’-Ida I and EBA Ust’-Ida I (Mooder et al., 2006), EN Lokomotiv (Mooder et al., 2005), and EN Shamanka II (this study and Thomson, 2006).

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northern Siberian region before the Neolithic period (Fedorova et al., 2013). Haplogroup Z is also present in European populations (e.g. Scandinavian-Saami, 1.3%), which might be due to its recent expansion and admixture with Saami (Tambets et al., 2004).

Given this information, it seems somewhat surprising that this hap-logroup has not been previously seen in ancient samples from Cis-Baikal. It is difficult to say whether this is simply a product of sampling or whether it reflects genuine population differences.

5.1.2. mtDNA and Y-chromosomal DNA haplogroup distribution in EBA Kurma XI: patrilocal/exogamous marriage?

The mtDNA and Y-chromosomal results from Kurma XI reveal four different mtDNA haplogroups (A, D, F and Z) but only one Y-chromo-somal haplogroup. More specifically, all five males that produced Y- chromosomal data belonged to haplogroup Q-M242 or its sub- haplogroup Q1a3-M346. Furthermore, all five carried either mtDNA haplogroup D or Z. Sample sizes are small; however, the higher level of diversity of mtDNA haplogroups in females and the lower level of di-versity of Y-chromosomal haplogroups in males could be suggestive of an exogamous marriage pattern, where men marry women from outside their social group. In addition, these data might also suggest a patrilocal post-marital residence pattern, where women move to their husbands’ social group after marriage and men stay within their own group. Pat-rilocal societies are usually characterized by a high level of diversity in mtDNA haplogroups and low level of diversity in Y-chromosomal hap-logroups within the same residential group, with the reverse situation observed between groups (Kumar et al., 2006; Oota et al., 2001; Perez- Lezaun et al., 1999; Seielstad et al., 1998). This pattern is the norm in about 70% of the world’s populations (Burton et al., 1996), and it is the most common form of post-marital residence for hunter-gatherers, found in ~65% of such societies (Kelly, 1995; Kelly, 2013). This is the first instance in which such patterns might be evident at a single site in this region.

5.2. New insights into mtDNA haplogroup distributions in Cis-Baikal

In the Cis-Baikal, previous studies have discussed mtDNA results from two EN cemeteries (Lokomotiv, and Shamanka II) and one LN–EBA cemetery (Ust’-Ida I). These analyses demonstrated that EN Lokomotiv individuals showed a higher distribution of haplogroup F (48.4%) and a lower distribution of haplogroup C (3.2%) (Mooder et al., 2005), while EN Shamanka II showed a higher frequency of haplogroups D and F (28.6%, and 23.8%, respectively) and lower frequency of haplogroup G2a (4.8%) (Thomson, 2006). Originally, Mooder et al., 2006 evaluated the genetic results from Ust’-Ida I as a combined LN–EBA population (Fig. 3), which showed a high frequency of haplogroup A and C (25.6%, and 28.2%, respectively) and low frequency of haplogroups D and F (5%, and 7.7% respectively) (Mooder et al., 2006). Overall, this work suggested that the two EN cemeteries, Lokomotiv and Shamanka II, were

similar to each other (p = 0.600), but both were statistically different from the combined LN–EBA cemetery Ust’-Ida I (p = 0.001 and p =0.008, respectively) (Mooder et al., 2010; Mooder et al., 2006).

However, as noted above, recent refinement of C14 dating and observed differences in mortuary practices now support analysis of the LN and EBA samples from Ust’-Ida I separately rather than combined. And when we add to this our new mtDNA results from EBA Kurma XI and EN Shamanka II, we are able to offer new insights into the maternal background of the Cis-Baikal middle Holocene populations.

First, the statistically different mtDNA haplogroup distributions of Lokomotiv and Shamanka II indicate that the EN population of Cis- Baikal might not be as homogeneous in maternal origin as was previ-ously suggested (Mooder et al., 2006). Similarly, the statistical differ-ence between the LN and EBA components of Ust’-Ida I indicate less continuity than previously thought, which further justifies the separa-tion of these groups for future analyses. The low haplogroup diversity in EBA Ust’-Ida I compared to the LN sample is notable and was previously discussed by Mooder et al. (2006) (Table 5). However, it is particularly striking once we consider that the LN component of Ust’-Ida was extremely short in duration (~400 years) compared to the EBA component (~1500 years) (Bronk Ramsey et al., 2020). We should also acknowledge that the unusual demographic structure of the LN popu-lation at Ust’-Ida (~60% children) might play a role, although it is difficult to interpret.

Second, the statistical differences between mtDNA haplogroup dis-tributions at EBA Kurma XI, LN Ust’-Ida I, and EBA Ust’-Ida I, and the similarities in the mtDNA profile between EBA Kurma XI, EN Lokomotiv, and EN Shamanka II individuals (Fig. 3; Table 5) seems to indicate that the maternal genetic background of EBA Kurma XI is closer to that of the known EN groups than to those from the LN or EBA. This is surprising given the chronological relationships between these sites, and it would seem to contradict the previous suggestion of a Middle Neolithic genetic discontinuity (Mooder et al., 2006, 2010). However, as noted above, it has been suggested that Kurma XI is an exclusive or specialized cemetery which might not be representative of the entire population that inhabited the Little Sea area during the EBA (McKenzie, 2010; McKen-zie, 2012). Nevertheless, these results do complicate the picture.

It is unfortunate that DNA analysis of samples from Khuzhir-Nuge XIV has thus far been unsuccessful, as it is the largest EBA cemetery in the Little Sea area and located only 15 km away from Kurma XI (McKenzie, 2006; McKenzie, 2012; McKenzie et al., 2008). mtDNA haplogroup analysis from this cemetery would help to confirm one of three hypotheses. First, if Khuzhir-Nuge XIV were similar to either LN Ust’-Ida I or EBA Ust’-Ida I, then it would suggest that EBA Kurma XI might be an outlier, which would be interesting given its unique mor-tuary record. Second, if Khuzhir-Nuge XIV were similar to EBA Kurma XI, then it might suggest that there was limited interaction between the Angara and the Little Sea microregions during this period. Third, if Khuzhir-Nuge XIV exhibited a different haplogroup distribution from all of EBA Kurma XI, LN Ust’-Ida I, and EBA Ust’-Ida I, then it would indicate that LN–EBA populations were far more heterogeneous with respect to maternal origins than previously assumed, or that each cemetery was being used by different population subsets.

Overall, the analysis of Kurma XI and additional material from Shamanka II now suggest that the maternal origins of EN, LN and EBA populations are more complicated than suggested in previous analyses. However, as noted, these results must be tempered by the fact that our sample sizes are still small and not evenly distributed across the Cis- Baikal in either time or space. Therefore, further investigation is still required to make stronger inferences about the genetic history and social structure of the ancient populations of the Cis-Baikal region.

6. Conclusion

This study contributes to our understanding of the genetic back-ground of middle Holocene Cis-Baikal populations. First, the analysis of

Table 5 Summary of Fisher’s exact tests comparing mtDNA haplogroup distributions between five Cis-Baikal cemetery samples.

Population (n = number of individuals) Fisher’s exact test results (p < 0.05)

EBA KUR (n = 12) and EBA UID (n = 10) p < 0.0001* EBA KUR (n = 12) and LN UID (n = 29) p = 0.0003* EBA KUR (n = 12) and EN LOK (n = 31) p = 0.1079 EBA KUR (n = 12) and EN SHA (n = 27) p = 0.1515 EBA UID (n = 10) and LN UID (n = 29) p = 0.0239* EBA UID (n = 10) and EN LOK (n = 31) p < 0.0001* EBA UID (n = 10) and EN SHA (n = 27) p = 0.0151* LN UID (n = 29) and EN LOK (n = 31) p = 0.0007* LN UID (n = 29) and EN SHA (n = 27) p = 0.0066* EN LOK (n = 31) and EN SHA (n = 27) p = 0.0174*

KUR = Kurma XI, UID = Ust’-Ida I, LOK = Lokomotiv, SHA = Shamanka II. * Statistically significant at p < 0.05.

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maternal genetic diversity of EBA Kurma XI revealed four different mtDNA haplogroups (A, D, F and Z), with haplogroup Z identified for the first time in the ancient populations of Cis-Baikal. These data supple-ment the previously published analysis of Y-chromosome haplogroup distributions, which showed only one Y-chromosomal haplogroup Q- M242 or its sub-haplogroup Q1a3-M346 (Moussa, 2015; Moussa et al., 2016). The higher level of diversity of mtDNA haplogroups in females and the lower level of diversity of Y-chromosomal haplogroups in males could be suggestive of exogamous and patrilocal marriage patterns. Second, comparing the mtDNA results from Kurma XI, the newly analyzed Shamanka II samples, and the two separate Ust’-Ida I compo-nents (LN and EBA) reveal new insights into the maternal background of the ancient Cis-Baikal population. Specifically, they suggest that the EN Cis-Baikal population was not as homogenous as previously suggested. In addition, the LN and EBA Ust’-Ida I populations show less continuity than previously believed, which further justifies their separate assess-ment, proposed on the basis of C14 dating and mortuary typology. Similarly, the maternal background of the EBA component at Kurma XI shows closer affinity to EN Shamanka and Lokomotiv individuals than to either LN and or EBA Ust’-Ida I individuals, giving rise to a number of hypotheses that will require further data to test. These new insights into the maternal background of ancient Cis-Baikal populations complicate our understanding of the relationships between the EN, LN and EBA populations and the previously reported Middle Neolithic genetic discontinuity. Future genetic data from other Little Sea cemeteries, including the large EBA Khuzhir-Nuge XIV, would greatly assist in the interpretation of these unexpected results.

Author statement

Contribution of Each Author. N.M. Moussa: Corresponding author, Conception and design of the

study, Laboratory Acquisition of data, data analysis and interpretation, Drafting of manuscript and critical revisions, Approval of final version of manuscript.

H.G. McKenzie: Co-author, Drafting of manuscript and critical re-visions, Approval of final version of manuscript.

V.I. Bazaliiskii: Conception and design of the study, Approval of final version of manuscript.

O.I. Goriunova: Conception and design of the study, Approval of final version of manuscript.

F. Bamforth: Conception and design of the study, data analysis and interpretation, Drafting of manuscript and critical revisions, Approval of final version of manuscript.

A.W. Weber: Conception and design of the study, data analysis and interpretation, Drafting of manuscript and critical revisions, Approval of final version of manuscript.

Declaration of Competing Interest

The authors declared that there is no conflict of interest and each contributed to the manuscript. If you have any further questions, please contact the corresponding author N.M. Moussa or any other the manu-script authors as their contact information was provided through the submission processes.

Acknowledgments

Our sincere acknowledgments to the Baikal Archaeology Project team for their continuous support, encouragement and their contribu-tion in the fieldwork in Russia. We would like to extend our thanks to the Social Sciences and Humanities Research Council of Canada (Major Collaborative Research Initiative Nos. 410-2000-1000, 412-2005-1004, and 412-2011-1001; and Partnership Grant No. 895-2018-1004) as well as numerous matching funding provided by the University of Alberta and other partner organizations. Also, we would like to acknowledge

Ms. Susan Kenny and Simona Veniamin from the Applied Genomic Centre at the University of Alberta for processing the sequencing ex-periments on their facility Sequencing Machines. Likewise, we would like to express our deep appreciation to Dr. Rong-Cai Yang (ARD Pro-fessor of Statistical Genomics, Department of Agricultural, Food and Nutritional Science, University of Alberta) for his help with statistical analysis in this paper. Furthermore, we thank Dr. Christian Leipe from Institute for Space-Earth Environmental Research (ISEE), Nagoya Uni-versity, Nagoya, Japan for creating a map for Lake Baikal (Fig. 1).

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