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УДК 575.1 636.5 904(56.562/569)
https://doi.org/10.24852/pa2021.L35.216.231
A ZOOARCHAEOLOGICAL AND MOLECULAR ASSESSMENT OF ANCIENT CHICKEN REMAINS FROM RUSSIA1
© 2021 Ophelie Lebrasseur, Dilyara Shaymuratova, Arthur Askeyev, Gulshat Asylgaraeva, Laurent Frantz, Greger Larson, Oleg Askeyev, Igor Askeyev
We here conduct ancient DNA analyses on 58 chicken bones from 15 archaeological sites (from the 9th to the 18th century AD) across the Volga region, the Leningrad region, the Pskov region, and the north of the Krasnoyarsk region to investigate genetic diversity of past chicken populations within this geographical area. We find all samples belong to sub-haplogroup E1, ubiquitous throughout the world and dominant in Europe, Africa and the Americas. This supports an introduction of chickens from the west, rather than a direct introduction from East Asia. Our study also demonstrates good endogenous DNA content, confirming species identification and sex of the individuals, thus highlighting the potential of genetic studies on archaeological remains in that region.
Keywords: zooarchaeology, Volga region, Russia, Medieval Period, chicken, ancient DNA, dispersal.
Introduction
Chickens play a major role in economies worldwide and provide an efficient source of animal protein to billions of people. Archaeological and palaeogenomics studies have shed light on the mystery surrounding their domestication, but the dispersal of chickens west and introduction to Russia remains relatively unknown.
Chicken domestication
A recent genomic study indicated that chickens were first domesticated in South/Southeast Asia from a Red Jungle fowl subspecies (Gallus gallus spadiceus) currently indigenous to Southwestern China, Thailand and Myanmar (Wang et al., 2020). Archaeological evidence suggests chickens were already domesticated by the 2nd millennium BC (Eda et al., 2019; Peters et al., 2016), while molecular data suggest that G. g. spadiceus and the domestic lineage diverged around 9500
+/- 3300 years BP (Wang et al., 2020). Altogether, this suggests that chickens were domesticated between ~9500-4000 years ago, although the earliest date is unlikely to reflect the beginning of the domestication process, especially given the lack of archaeological evidence.
Introduction and dispersal of chickens in Russia
Chickens spread across Southeast and South Asia before being introduced further afield. The first evidence of their presence outside of Asia was found on the site of Maresha in Israel dated to the Hellenistic period (4th-2nd century BC) (Perry-Gal et al., 2015). This date also coincides with the appearance of chickens in the Greek colonies of the northern Black Sea region (middle to late 1st millennium BC), where during the existence of the Bosporus kingdom (5th century BC - 6th century AD), domestic chickens were widespread (Burchak-Abramovich, 1962, p. 438-
1 OL and GL were supported by Arts and Humanities Research Council (grant number AH/L006979/1). OL, GL and LF were supported by ERC-2013-StG-337574-UNDEAD. GL and LF were supported by Natural Environmental Research Council grants (NE/ K005243/1, NE/K003259/1, NE/S007067/1, and NE/S00078X/1). LF was supported by the Wellcome Trust (210119/Z/18/Z). The zooarchaeological research was funded by RFBR, project number 20-09-00004.
Fig. 1. A map of the distribution of domestic chickens in the territory of European Russia and Siberia according to zooarchaeological studies in the 1st millennium BC - 17th century AD.
Рис. 1. Карта распространения домашних кур на территории Европейской части России и Сибири по данным зооархеологических исследований в I тыс. до н. э. - XVII в. н. э.
440; Burchak-Abramovich, Tsalkin, 1971, p. 54-56, 61).
Later, in the middle of the 1st millennium AD, domestic chickens appear in settlements located at the centre of the European part of Russia and the Volga region (Burchak-Abramovich,
Tsalkin, 1971, p. 54-56, 61; Askeyev et al, 2011, p. 161-163). From the 9th to the 18th century AD, bone remains of domestic chickens become numerous in archaeological sites throughout the European part of Russia (Burchak-Abramovich, Tsalkin, 1969, p. 49-50,
1971, p. 58; Zinoviev, 2009; Askeyev et al, 2011, 2013; Gorobets, Kovalchuk, 2016 p. 5; Shaymuratova et al, 2019, p. 97). This increase in abundance indicates the widespread distribution of domestic chickens amongst past populations, as well as the gradual development of poultry farming and an increasing importance of chickens in the diet (meat and eggs) of historical populations of this region. Finally, the appearance of chickens in Siberia is associated with the establishment of Russian trading settlements on the territory of Western Siberia in the 16th-17th century (Nekrasov, 2003, p. 163-168; Martynovich, 2013, p. 1131) (Figure 1).
Mitochondrial DNA analyses have previously been used to infer domestication and genetic diversity of modern chicken populations worldwide (Liu et al., 2006; Miao et al., 2013). These showed chickens clustered in nine main haplogroups (A-I) and six sub-haplogroups (C1-C3, E1-E3) based on full mitochondrial genomes (Miao et al., 2013), with E1 being ubiquitous throughout the world and dominant in Europe, Africa, and the Americas. Other haplogroups are more geographically restricted: A and B are widely distributed but absent in Africa, C, F and G are found throughout Asia, D is found throughout Asia and Africa but dominates the Pacific, and H and I are restricted to East Asia and South Asia respectively. In addition, wild fowl harboured haplogroups W-Z which are absent in domestic chickens (Miao et al., 2013).
A genetic study conducted on ancient chicken individuals from Germany, Austria, the United Kingdom, and Greece, and spanning the last two millennia, showed E1 was the sole sub-haplogroup found in the past (Girdland-
Flink et al., 2014). More recently, Dyomin and colleagues (2017) sequenced a fragment of the D-loop hypervariable region of the mitochondrial genome of five archaeological chicken bones from the medieval sites of Veliky Novgorod, Pskov, St. Petersburg, Staraya Ladoga, and Azov in the European part of Russia, and dating from the 9th to the 18th century. All but one sample belonged to sub-haplogroup E1, confirming previous findings. The non-E1 haplogroup, C1, was found in a sample from Pskov (18th century) which the authors suggested could have been an exotic import.
Aims and Objectives
The purpose of this paper is to investigate the dispersal of chickens in the Volga region, Russia, based on genetic analyses from samples recovered from 16 archaeological sites spanning the 9th to the 18th centuries AD. More specifically, our aims were the following:
1) Assessment of the endogenous DNA content present in the selected bones,
2) Confirmation of taxonomic identification and sexing of the individuals,
3) Exploration of the genetic diversity of ancient chicken populations in the Volga region.
This is the first genetic study conducted on ancient chicken remains from the vast territory of the Volga region, and will provide valuable information on the dispersal route(s) and settlement process of domestic chickens in this area.
Materials and Methods
Samples
A total of 75 ancient chicken bones spanning the 9th to the 18th centuries AD were obtained from 16 archaeological sites across Russia (Volga region, n=13; Leningrad region, n=1; Pskov, n=1; the north of the Krasnoyarsk region, n=1;
Fig. 2. Map of the archaeological sites with chicken remains.
Рис. 2. Карта с обозначением археологических памятников с остатками домашних кур.
Figure 2, Table 1). The material was recovered during excavations carried out in 2000-2015 mostly from household pits, and, to a lesser extent, from a free cultural layer. All bird remains were hand collected. The identification of bird bones was performed using comparative skeletal collections of birds kept in the Laboratory of Biomonitoring (Institute of Problems in Ecology and Mineral Wealth, Kazan), and following guidelines by Serjeantson (2009).
Coracoids were selected for this study due to their higher bone density (Figure 3). To reduce the likelihood of sampling a same individual twice,
coracoids from one side of the body were used, unless metrics or age proved otherwise.
Ancient DNA extraction Ancient DNA (aDNA) analyses were undertaken in the dedicated ancient DNA laboratory of the 'Palaeo-BARN', School of Archaeology, University of Oxford (UK). To prevent contamination and ensure the generation of authentic data, strict measures were applied in compliance with standard contamination precautions (Cooper & Poinar, 2000; Gilbert et al., 2005).
Of the 75 samples, we selected 58 for aDNA analyses. Fourteen coracoids
Table 1
List of samples in this study
Sample ID Chicken Project ID Site name Province/Region Side Dating/Phasing Information
1 OSO1196 Ostolopovo settlement The Republic of Tatarstan Left end of 9« - 12« cc. AD
2 OSO1197 Ostolopovo settlement The Republic of Tatarstan Left end of 10« - 12« cc. AD
3* OSO1198 Ostolopovo settlement The Republic of Tatarstan Left end of 10« - 12« cc. AD
4 OSO1199 Ostolopovo settlement The Republic of Tatarstan Right end of 10« - 12th cc. AD
5 MUM1200 Muromsky gorodok Samara region Left 10« - 12th cc. AD
6 MUM1201 Muromsky gorodok Samara region Right 10th - 12th cc. AD
7 BOG1202 Bogdashkino hillfort The Republic of Tatarstan Left 10th - 12th cc. AD
8 BOG1203 Bogdashkino hillfort The Republic of Tatarstan Left 10th - 12th cc. AD
9 BOG1204 Bogdashkino hillfort The Republic of Tatarstan Left 10th - 12th cc. AD
10** BIL1205 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
11** BIL1206 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
12 BIL1207 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
13 BIL1208 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
14 BIL1209 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
15** BIL1210 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
16* BIL1211 Bilyar fortified settlement The Republic of Tatarstan Left 11th - early 13th cc. AD
17 BIL1212 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
18 BIL1213 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
19 BIL1214 Bilyar fortified settlement The Republic of Tatarstan Right 11th - early 13th cc. AD
20** ELA1215 Elabuga hillfort The Republic of Tatarstan Left layers of 12th - 13th cc. AD
21 ELA1216 Elabuga hillfort The Republic of Tatarstan Left layers of 12th - 13th cc. AD
22 ELA1217 Elabuga hillfort The Republic of Tatarstan Left layers of 17th - 18th cc. AD
23 BUG1218 Bulgar fortified settlement The Republic of Tatarstan Right 13th - 14th cc. AD
24 BUG1219 Bulgar fortified settlement The Republic of Tatarstan Left 13th - 14th cc. AD
25 BUG1220 Bulgar fortified settlement The Republic of Tatarstan Left 13th - 14th cc. AD
26 BUG1221 Bulgar fortified settlement The Republic of Tatarstan Left 13th - 14th cc. AD
27 TOR1222 Toretskoe settlement The Republic of Tatarstan Left 15th c. AD
28 TOR1223 Toretskoe settlement The Republic of Tatarstan Right 15th c. AD
29 TOR1224 Toretskoe settlement The Republic of Tatarstan Right 15th c. AD
30 TOR1225 Toretskoe settlement The Republic of Tatarstan Right 15th c. AD
31* TOR1226 Toretskoe settlement The Republic of Tatarstan Left 15th c. AD
32* TOR1227 Toretskoe settlement The Republic of Tatarstan Left 15th c. AD
33* TOR1228 Toretskoe settlement The Republic of Tatarstan Left 15th c. AD
34 TOR1229 Toretskoe settlement The Republic of Tatarstan Right 15th c. AD
35 TOR1230 Toretskoe settlement The Republic of Tatarstan Right 15th c. AD
36* KAZ1231 Kazan, territory of Kazan State University The Republic of Tatarstan Left 16th - 17th cc. AD
37 KAZ1232 Kazan, territory of Kazan State University The Republic of Tatarstan Right 16th - 17th cc. AD
38 KAZ1233 Kazan, territory of Kazan State University The Republic of Tatarstan Right 16th - 17th cc. AD
39 CHB1234 Cheboksary The Republic of Chuvashia Right 16th - 18th cc. AD
40 CHB1235 Cheboksary The Republic of Chuvashia Left 16th - 18th cc. AD
41 CHB1236 Cheboksary The Republic of Chuvashia Left 16th - 18th cc. AD
42 CHB1237 Cheboksary The Republic of Chuvashia Left 16th - 18th cc. AD
43 CHB1238 Cheboksary The Republic of Chuvashia Left 16th - 18th cc. AD
44 CHB1239 Cheboksary The Republic of Chuvashia Right 16th - 18th cc. AD
45* CHB1240 Cheboksary The Republic of Chuvashia Right 16th - 18th cc. AD
46 PSK1241 Pskov Pskov Region Right 16th - 18th cc. AD
47 NNK1242 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
48 NNK1243 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
49 NNK1244 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
50* NNK1245 Nizhny Novgorod Kremlin Nizhny Novgorod Region Right second half of 13th - 14th cc. AD
51* NNK1246 Nizhny Novgorod Kremlin Nizhny Novgorod Region Right second half of 13th - 14th cc. AD
52* NNK1247 Nizhny Novgorod Kremlin Nizhny Novgorod Region Right second half of 13th - 14th cc. AD
53 NNK1248 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
54* NNK1249 Nizhny Novgorod Kremlin Nizhny Novgorod Region Right second half of 13th - 14th cc. AD
55 NNK1250 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
56* NNK1251 Nizhny Novgorod Kremlin Nizhny Novgorod Region Right second half of 13th - 14th cc. AD
57* NNK1252 Nizhny Novgorod Kremlin Nizhny Novgorod Region Right second half of 13th - 14th cc. AD
58 NNK1253 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
59 NNK1254 Nizhny Novgorod Kremlin Nizhny Novgorod Region Left second half of 13th - 14th cc. AD
60* STD1255 Staraya Ladoga Leningrad region Left 9th - 10th cc. AD
61 STD1256 Staraya Ladoga Leningrad region Right 9th - 10th cc. AD
62 STD1257 Staraya Ladoga Leningrad region Right 9th - 10th cc. AD
63* STD1258 Staraya Ladoga Leningrad region Left 9th - 10th cc. AD
64 STD1259 Staraya Ladoga Leningrad region Right 9th - 10th cc. AD
65 STD1260 Staraya Ladoga Leningrad region Right 9th - 10th cc. AD
66 STD1261 Staraya Ladoga Leningrad region Right 9«" - 10th cc. AD
67 STD1262 Staraya Ladoga Leningrad region Right 9th - 10th cc. AD
68 STH1263 Staroturukhanskoe hillfort Krasnoyarsk region Right 17th - 18th cc. AD
69 STH1264 Staroturukhanskoe hillfort Krasnoyarsk region Left 17th - 18th cc. AD
70* BAG1265 Bagaevka settlement Saratov region Right 13th - 14th cc. AD
71* BAG1266 Bagaevka settlement Saratov region Left 13th - 14th cc. AD
72 KIR1267 Kirmen settlement The Republic of Tatarstan Right 13th - 14th cc. AD
73 KIR1268 Kirmen settlement The Republic of Tatarstan Right 13th - 14th cc. AD
74 MAO1269 Maly Sundyr' hillfort Republic of Mari El Right end of 13th - 15th cc. AD
75 MA01270 Maly Sundyr'hillfort Republic of Mari El Right end of 13th - 15th cc. AD
* Samples excluded for ancient DNA analyses. ** Samples for which amplification has failed.
were thus excluded as neither their side, metrics nor age could confirm individual status (Table 1). A few others were rejected due to theirjuvenile age (samples 70 and 71). To reduce environmental contamination, 0.5 mm of the bone surface was removed using a Dremel 3000 electric hand-drill. A fragment weighing 150-450 mg was excised from each bone based on good macroscopic preservation. Each sample was grounded to fine powder using a Retsch MM400 microdismembrator. Ancient DNA extractions were performed following the Dabney protocol (Dabney et al., 2013).
Illumina Sequencing Library preparation and capture
Double-strand Illumina libraries were built following Meyer & Kircher (2010), with the addition of a six base-pair barcode added to the IS1_adapter. P5 adapter. The libraries were amplified on an Applied Biosystems StepOnePlus Real-Time PCR system to confirm the library had been successfully built and to determine the optimal number of cycles for the indexing amplification PCR reaction. A six base-pair barcode was added during the indexing amplification reaction resulting in each library being double-barcoded. Following amplification, the samples were run on an Agilent Tape Station 2200 to confirm successful amplification, then pooled equimolarly prior to cleaning with a QIAquick PCR purification
Kit (QIAGEN Ltd, UK) following the manufacturer's instructions.
In addition, we captured a total of 36 amplified libraries using MYcroarray mitochondrial MYbaits with 24 hours hybridisation at 55°C, following the MYbaits manual V3 instructions (2016). All libraries were sequenced on an Illumina HiSeq 2500 (Single End 80bp) sequencer at the Danish National High-Throughput Sequencing Centre, Copenhagen, Denmark.
Initial Quality Control and FastQ Screen
Adapters were removed using AdapterRemoval v2.0.0 (Schubert et al., 2016). Samples were run through FastQ Screen (Wingett & Andrews, 2018) using the BWA algorithm (Li & Durbin, 2009) for species identification based on a database comprising the full mitochondrial genomes of chicken (KX987152), turkey (NC010195), peacock (NC024533), Guinea fowl (KY865420) and goose (MK133021). Data processing
Reads were aligned using Burrows-Wheeler Aligner (BWA) version 0.7.5ar405 (Li & Durbin, 2009) to Galgal4, with default parameters apart from disabling the seed option ("l 1024") (Schubert et al., 2012). FilterUniqueSAMCons (Kircher, 2012) was then used to remove duplicates. BAM files from different libraries were merged using the MergeSamFiles tool from Picard v1.129 (http://broadinstitute.
M - Sample ID 1 1 ft к К Sample ID 2 i Sample ID
Sample ID 4 ] Sample ID / I Sample ID
_ en- ш - tfi В —— Sample !D 7 1 Sample ID i| II Sample ID
Sample ID • 10 i Sample ID 1 11 I Sample ID I,
Fig. 3. Photos of selected coracoids used in this study. Details of each bone can be found in
Table 1.
Рис. 3. Фото коракоидов домашних кур, которые были отобраны для данного исследования. Подробное описание каждой кости находится в Таблице 1.
github.io/picard/). We generated coverage excluding bases within 5bp of
mitochondrial genome consensus by the start and end of a read, and using
obtaining a majority consensus sequence reads with BQ>=20 and MAPQ>=30 as
for all samples that had at least 3x average implemented in htsbox: https://github.
com/lh3/htsbox. Sexing was conducted using a read depth-based method based on Skoglund et al. (2013), comparing alignment of sequencing reads post filtering for a mapping quality q30 to the W chromosome and chromosome 1.
Phylogenetic analysis
Fifty modern chicken mitochondrial genomes representing each haplogroup from Miao et al., (2013) were downloaded from Genbank for haplogroup identification (GU261674-GU261719, HQ857209-HQ857212). Multiple sequence alignments of both these published samples and samples from our study were completed using MAFFT v7.123b, and verified manually through Aliview v1.26 (Larsson, 2014). A phylogenetic tree was produced using IQ tree, where the best fitting-model was identified as TIM2+F+I+G4 (Hoang et al., 2018; Kalyaanamoorthy et al., 2017; Minh et al., 2020).
Results
Zooarchaeological assessment of species abundance
We investigated the bird remains from 20 archaeological sites in the Middle Volga region dating from the Medieval to the Post-Medieval period. Starting from the 10th century AD, domestic chickens were dominant amongst remains: out of 6463 bird bones, 3982 bones (61,6%) belonged to domestic chickens (Askeyev et al., 2013, p. 120-123; our unpublished data). In the two archaeological sites of North-West Russia - in the layers of medieval Staraya Ladoga (9th-10th cc. AD) and Pskov (12th-13th cc. AD) - a substantial number of domestic chicken bones have also been found. However, for Staraya Ladoga, wild bird remains still predominate over domestic birds, with a ratio of 1079 bones of wild birds (80,8%) to 225 bones of domestic chickens (16,8%) (Shaymaratova et al,
2019, p. 96-97). In the Pskov strata, the remains of domestic chickens prevail in number over the remains of wild birds: this was noted both for the early urban layers (12th-13th cc. AD) and for later layers (end of 13th - early 18th cc. AD) (Shaymuratova, Askeyev, 2017, p. 64, tab. 1). Thus, according to the zooarchaeological material we have analysed for the territory of the Volga region and North-West Russia, by the 13th century, domestic chickens were becoming one of the main domestic animals. Regarding the Staroturukhanskoe hillfort (17th -18th cc. AD) geographically located on the territory of Western Siberia, 29 bones of domestic chickens belonging to adults were identified, placing them as the fourth most abundant bird remains after wild species. Such a relatively small number of bones of domestic birds is typical for northern Post-Medieval settlements (Nekrasov, 2003; Martynovich, 2013), which is associated with the difficult climatic conditions and limited opportunities for raising and keeping domestic animals. However, the discovery of bones from adult individuals of domestic chickens indicates their existence and importance in the diet and economic life of the population of that time.
We have previously published in detail the results of a zooarchaeological study of chicken remains from the Volga region, their numbers on individual archaeological sites, size, age and sex composition of the flock, as well as their relationship with other identified bird species (see Askeyev et al., 2011, 2013; Galimova et al., 2014). This is thus not considered in detail in this article.
Species Identification
Of the 58 samples selected for genetic analyses, 54 were successfully
Table 2
Genetic Results
Sample ID Site Genetics ID Species ID (Genetics) Screening Results Mitochondrial Capture Results
Total Reads % Mapped Reads Once Duplicates Removed Haplogroup Sex Calls Depth Coverage % Mapped Reads Once Duplicates Removed
1 Ostolopovskoe settlement OL1676 Gallus gallus 827472 0,79 E1 N/A 27,2 1,0 14,04
2 Ostolopovskoe settlement OL1677 Gallus gallus 1107822 59,14 E1 Female 24,3 1,0 79,32
4 Ostolopovskoe settlement OL1679 Gallus gallus 1202591 5,04 E1 Female 12,5 1,0 15,95
5 Muromsky gorodok OL1572 Not enough reads 7111 0,62
6 Muromsky gorodok OL1576 Gallus gallus 1365216 44,60 E1 Female 59,5 1,0 77,51
7 Bogdashkinskoe hillfort OL1655 Gallus gallus 692574 3,62 E1 Male 40,3 1,0 50,19
8 Bogdashkinskoe hillfort OL1656 Gallus gallus 325533 2,00 E1 Male 19,8 1,0 38,21
9 Bogdashkinskoe hillfort OL1675 Gallus gallus 617277 3,75 E1 Female 9,7 1,0 35,06
12 Bilyar hillfort OL1680 Gallus gallus 796628 3,29 E1 Female 10,7 1,0 33,25
13 Bilyar hillfort OL1673 Gallus gallus 572301 10,64 E1 Female 16,9 1,0 47,31
14 Bilyar hillfort OL1672 Not enough reads 891 0,22
17 Bilyar hillfort OL1594 Gallus gallus 1117975 3,16 E1 Female 38,9 1,0 33,61
18 Bilyar hillfort OL1595 Gallus gallus 648762 7,56 E1 Female 38,3 1,0 58,05
19 Bilyar hillfort OL1596 Gallus gallus* 675564 0,37
21 Elabuga hillfort OL1598 Gallus gallus 2343501 1,03 E1 Female 10,9 1,0 11,29
22 Elabuga hillfort OL1599 Gallus gallus 455829 5,05 E1 Male 11,7 1,0 38,88
23 Bulgar OL1593 Gallus gallus* 487747 0,48 E1 N/A 3,5 1,0 10,51
24 Bulgar OL1587 Gallus gallus 1206097 0,86 E1 Male 4,5 1,0 12,31
25 Bulgar OL1591 Gallus gallus 460718 11,59 E1 Female 25,2 1,0 65,55
26 Bulgar OL1590 Gallus gallus 1508172 11,09 E1 Female 70,5 1,0 59,39
27 Toretskoe settlement OL1581 Gallus gallus 891445 2,32 E1 Male 23,6 1,0 34,30
28 Toretskoe settlement OL1578 Gallus gallus* 148175 2,81
29 Toretskoe settlement OL1583 Gallus gallus 1458885 1,84 E1 Female 4,5 1,0 6,55
30 Toretskoe settlement OL1582 Not enough reads 19199 1,68
34 Toretskoe settlement OL1577 Gallus gallus* 24933 26,04 E1 Female 1,3 0,7 48,80
35 Toretskoe settlement OL1654 Gallus gallus 2522615 0,72 E1 Female 12,6 1,0 14,97
37 Kazan city, territory of Kazan State University OL1584 Gallus gallus 2591876 4,96 E1 Female 59,0 1,0 43,78
38 Kazan city, territory of Kazan State University OL1659 Gallus gallus 1578223 0,83 E1 Male 8,7 1,0 10,47
39 Cheboksary city OL1588 Gallus gallus 1860607 38,39 E1 Female 71,7 1,0 71,01
40 Cheboksary city OL1585 Gallus gallus 1175959 16,46 E1 Female 22,0 1,0 49,19
41 Cheboksary city OL1589 Gallus gallus* 71971 5,28 E1 Female 0,4 0,3 23,52
42 Cheboksary city OL1586 Gallus gallus* 670751 0,20 E1 N/A 0,2 0,1 4,34
43 Cheboksary city OL1651 Gallus gallus* 818368 0,97
44 Cheboksary city OL1592 Gallus gallus* 394859 1,40 E1 Male 3,6 0,9 16,47
46 Pskov city OL1662 283728 0,64
47 Nizhny Novgorod Kremlin OL1665 Gallus gallus* 57583 3,92
48 Nizhny Novgorod Kremlin OL1573 Gallus gallus* 244507 2,00
49 Nizhny Novgorod Kremlin OL1658 Gallus gallus* 1599846 0,24 E1 Female 1,6 0,7 1,63
53 Nizhny Novgorod Kremlin OL1574 Gallus gallus 432125 13,98 E1 Female 12,6 1,0 55,30
55 Nizhny Novgorod Kremlin OL1575 Not enough reads 46067 1,39
58 Nizhny Novgorod Kremlin OL1666 Gallus gallus 639438 11,52 E1 Male 36,6 1,0 58,22
59 Nizhny Novgorod Kremlin OL1579 Gallus gallus 612521 13,82 E1 Female 7,1 1,0 38,21
61 Staraya Ladoga OL1670 Gallus gallus 3078340 1,23 E1 Female 6,8 1,0 3,86
62 Staraya Ladoga OL1678 Gallus gallus* 49068 2,16
64 Staraya Ladoga OL1669 Gallus gallus* 389184 0,60 E1 Female 1,3 0,7 10,47
65 Staraya Ladoga OL1657 Gallus gallus* 1191888 0,11
66 Staraya Ladoga OL1663 Gallus gallus 888972 0,67
67 Staraya Ladoga OL1664 Gallus gallus* 705244 0,16
68 Staroturukhanskoe hillfort OL1667 Gallus gallus* 70906 1,09
69 Staroturukhanskoe hillfort OL1661 Gallus gallus* 77652 2,72 E1 Female 2,6 0,9 36,34
72 Kirmenskoe settlement OL1580 Gallus gallus* 257085 0,27
73 Kirmenskoe settlement OL1660 Gallus gallus* 786797 0,61
74 Malo-Sundyrskoe hillfort OL1653 Gallus gallus 1265238 2,16 E1 Male 27,2 1,0 31,95
75 Malo-Sundyrskoe hillfort OL1650 Not enough reads 2659774 0,01
* <50 reads
amplified and screened for an initial assessment of DNA content and quality (Table 2, Figure 4). All samples with sufficient mitochondrial DNA sequences were identified as Gallus gallus through FastQ Screen, confirming morphological identification (Table 2).
Full mitochondrial genomes
For each sample, the total number of reads, and percentage of reads aligned to the chicken genome after duplicate removal are listed in Table 2. The latter ranged up to 59% with no distinct pattern based on the samples' age (Figure 4). It is worth noting though that samples from Staraya Ladoga (samples 61-67), Staroturukhanskoe hillfort (samples 68, 69), Kirmen settlement (samples 72, 73) and Maly Sundyr' hillfort (74, 75) yielded very little DNA.
Based on these results, 36 samples were then selected for mitochondrial
capture (BioProject Accession number PRJEB40810), for which the depth, coverage and percentage of reads aligned to the chicken genome are listed in Table 2. Thirty-four samples had good coverage; the percentage of sequenced reads mapping to the genome once duplicates were removed ranged from 10% to 79% with four samples below 10% (samples 29, 42, 49 and 61). Coverage depth ranged from 1.3x to 71.7x, with the exception of samples 41 and 42 which had both low coverage (<0.5) and low depth (<1x).
For phylogenetic analysis, the full mitochondrial genome sequences generated were first aligned to 50 sequences previously published by Miao et al. (2013) and representing the 19 mtDNA haplogroups and sub-haplogroups the authors identified. We also included the current chicken
Fig. 4. Screening results - Percentage of reads aligned to the chicken genome once
duplicates removed.
Рис. 4. Результаты скрининга - процент считываний, совпадающих с геномом домашней курицы после удаления дубликатов.
full mitochondrial genome reference NC_040970. The haplogroup nomenclature follows Miao et al. (2013). The results clearly show all samples fall within the E1 sub-haplogroup (Figure 5).
Sexing Identification
With the exception of three samples which did not possess enough reads to confidently infer the sex (samples 1, 23, 42), we found a large majority of the individuals (72.7%) were females. The sites containing more than one individual, the Ostolopovo settlement, Bilyar fortified settlement and Staraya Ladoga consisted solely of female samples.
Discussion
Fifty-eight samples from 15 sites and spanning the last millennium were assessed for DNA content, with 36 of these (62%) containing good endogenous DNA content for mitochondrial DNA capture. No correlation between DNA preservation and age of the samples was observed. This suggests good endogenous DNA preservation in archaeological bird remains, at least over the last 1200 years, and demonstrates the potential of aDNA analyses on ancient bones from birds and other animals from this region.
Species identification was positively confirmed for half of the individuals (53%). Nineteen samples contained <50 mitochondrial reads which nevertheless aligned to Gallus gallus, suggesting that these samples were indeed chickens. The remaining five samples did not yield enough reads for conclusive species identification. The quality of endogenous DNA permitted sex identification, demonstrating the value of aDNA analyses in providing additional information on the individual and consequently the population when no diagnostic features can be identified morphologically.
Interestingly, all samples sequenced in this study belonged to the E1 sub-haplogroup, ubiquitous throughout the world and dominant in the West. In fact, E1 was previously shown to be fixed in ancient European chickens (Dyomin et al., 2017; Girdland Flink et al., 2014). This finding suggests that chickens from the Volga region were introduced from Western Europe rather than Asia. This fits well with the zooarchaeological data suggesting that chickens were first introduced in the mid-first millennium BC through the Greek colonies in the Northern Black Sea region, after which,
Fig. 5. Maximum-likelihood tree based on full mitochondrial genomes from 50 published samples (denoted by their GenBank Accession number) and samples from this study (OLXXXX). Haplogroups are indicated by the letter following the GenBank Accession number in the published samples. The E1 sub-haplogroup is coloured in blue. BIL = Bilyar fortified settlement. BOG = Bogdashkino hillfort, BUG = Bulgar fortified settlement. CHB = Cheboksary. ELA = Elabuga hillfort. KAZ = Kazan, MAO = Maly Sundyr' hillfort. MUM = Muromsky gorodok, NNK = Nizhny Novgorod Kremlin, OSO = Ostolopovo settlement, STD = Staraya Ladoga, STH = Staroturukhanskoe hillfort, TOR = Toretskoe
settlement. Рис. 5. Дерево максимального правдоподобия, созданное на основе полных митохон-дриальных геномов из 50 опубликованных образцов (обозначены их регистрационные номера в ГенБанке) и образцы из данного исследования (OLXXXX). Гапло-группы обозначаются буквой
после регистрационного номера ГенБанка в опубликованных образцах. Под-гаплогруппа Е1 окрашена в синий цвет. BIL = Билярское городище, BOG = Богдаш-кинское городище, BUG = Булгар, CHB = Чебоксары,
ELA = Елабуга, KAZ = Казань, MAO = Мало-Сун-дырское городище, MUM = Муромский городок, NNK = Нижний Новгород (Кремль), OSO = Остолоповское селище, STD = Старая Ладога, STH = Старотуруханское городище, TOR = Торецкое поселение.
beginning of the first millennium AD and with the development of trade between the Bosporus kingdom and neighbouring regions, they expanded throughout the Northern Black Sea region, the Don region, the Dnieper region, the North Caucasus, and the centre of the European part of Russia and Volga region.
Chicken remains, however, are rare during these periods and their distribution across settlements is uneven. They only become ubiquitous by the end of the 9th century to the beginning of the 12th century AD, in almost all settlements in the centre of the European part of Russia and in the Middle Volga. This increase in chicken consumption is likely the result of migration and expansion of the Slavic population into the central and north-western parts of the Russian Plain, and of the Bulgar population into the Volga region from the lands of the Khazar Kaganate (Don and Kuban region), and which began during the 9th century AD. During the 12th and 15th centuries AD, chickens headed east and northeast to the Vyatka Territory, the Urals, the Middle Urals and the Sursky region with the Slavic-Russian (Novgorod and Vladimir-Suzdal) colonisation movement. Chickens were also widespread within large state formations including the Golden Horde and the Kazan Khanate. By the end of the 14th - 15th centuries AD, domestic chickens were present and abundant throughout the European part of Russia. The active development of the Russian territory of Western Siberia at the end of the 16th century - 17th century AD, which accompanied the development of trading settlements, led to the introduction of chickens in Siberia.
The lack ofnon-E1 (sub)-haplogroups amongst our assemblages does not
confirm nor refute the hypothesis of a secondary introduction of chickens by Slavic and Bulgar populations around the 11th century as chickens become more abundant in archaeological layers. However, it does support an introduction from the Black Sea region or Europe into the Volga region as a direct introduction from the East would most likely have resulted in the presence of non-E1 haplogroups as suggested by Dyomin and colleagues (Dyomin et al., 2017). Indeed, the latter identified an 18th century sample from Pskov belonging to the C1 sub-haplogroup, and suggested it may have been brought from Western Europe into Russia as Pskov was part of the Hanseatic league. Alternatively, the sample could have been introduced into Europe from China via Russia given the close economic ties between the last two.
Conclusion
Our research has shown a successful combination of zooarchaeological and genetic tools for studying the bones of domestic chickens. Through the large amount of chicken remains found on archaeological sites and the research methodology applied, we have reconstructed more than a thousand-year history of the evolution of the domestic chicken in Russia with special attention to the Volga region. We have established that all our samples belong to sub-haplogroup E1, which is ubiquitous throughout the world. This sheds some light on the monogenic origin of ancient chickens from the study area. The good preservation of endogenous DNA demonstrates the potential of ancient genetic research on bird bones from medieval and post-medieval archaeological sites in this region.
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About the Authors:
Ophelie Lebrasseur. PhD, Postdoctoral Researcher. Department of Archaeology, Classics and Egyptology, University of Liverpool. 12-14 Abercromby Square. Liverpool, L69 7WZ. UK; Palaeo-BARN, School of Archaeology, University of Oxford. 1 South Park Road. Oxford OX1 3TG. UK; ophelie.lebrasseur@liverpool.ac.uk
Shaymuratova Dilyara N. Researcher. The Institute of Problems in Ecology and Mineral Wealth, Tatarstan Academy of Sciences, Daurskaya St., 28, Kazan, 420087, Republic of Tatarstan, Russian Federation; galimovad@gmail.com
Askeyev Arthur O. Candidate of Biology Sciences, Researcher. The Institute of Problems in Ecology and Mineral Wealth, Tatarstan Academy of Sciences, Daurskaya St., 28, Kazan, 420087, Republic of Tatarstan, Russian Federation; art.regulus@mail.ru
Asylgaraeva Gulshat Sh. Candidate of Veterinary Sciences. Institute of Archaeology named after A.Kh. Khalikov, Tatarstan Academy of Sciences. Butlerov St., 30, Kazan, 420012, Republic of Tatarstan, Russian Federation; gul_shat@mail.ru
Laurent Frantz. Professor, Doctor. Department of Veterinary Sciences, Paleogenomic group, Ludwig Maximillians University of Munich, Kaulbach St., 37/III, Munich, 80539, Germany; Senior Lecturer. School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS, UK; laurent.frantz@lmu.de
Greger Larson. Professor. Director. Palaeo-BARN, School of Archaeology, University of Oxford. 1 South Park Road. Oxford OX1 3TG. UK; greger.larson@arch.ox.ac.uk
Askeyev Oleg O. Candidate of Biology Sciences. Head of Laboratory, The Institute of Problems in Ecology and Mineral Wealth, Tatarstan Academy of Sciences, Daurskaya St., 28, Kazan, 420087, Republic of Tatarstan, Russian Federation; parus.cyanus@rambler.ru
Askeyev Igor V. Candidate of Biology Sciences, Senior Researcher, Associate Professor, The Institute of Problems in Ecology and Mineral Wealth, Tatarstan Academy of Sciences, Daurskaya St., 28, Kazan, 420087, Republic of Tatarstan, Russian Federation; archaeozoologist@yandex.ru
ЗООАРХЕОЛОГИЧЕСКАЯ И МОЛЕКУЛЯРНАЯ ОЦЕНКА ДРЕВНИХ ОСТАТКОВ ДОМАШНИХ КУР ИЗ РОССИИ
О. Лебрассер, Д. Шаймуратова, А. Аськеев, Г. Асылгараева, Л. Франц, Г. Ларсон, О. Аськеев, И. Аськеев
В данной статье представлены результаты исследований авторами древней ДНК, извлеченной из 58 костей домашних кур, костные остатки которых происходили из 15 археологических памятников средневековья (ГХ-ХУШ вв. н.э.) 4-х регионов России - Поволжья, Ленинградской области, Псковской области и севера Красноярского края, чтобы исследовать генетическое разнообразие прошлых популяций кур в этой географической области. Нами выявлено, что все протестированные образцы костных остатков кур принадлежат к субгаплогруппе Е1, распространенной во всем мире и доминирующей в Европе, Африке и Америке. Этот вывод подтверждает факт прихода домашних кур в Европейскую часть России с Запада, а не прямую интродукцию из Восточной Азии. Наше исследование также демонстрирует хорошее содержание эндогенной ДНК, подтверждающее идентификацию вида и пола, тем самым подчеркивая потенциал генетических исследований костных остатков животных из археологических памятников исследуемой территории.
Ключевые слова: зооархеология, Поволжье, Россия, средневековье, домашняя курица, древняя ДНК, расселение.
Информация об авторах:
Офелия Лебрассер, доктор философии, постдокторант, кафедра археологии, классики и египтологии, Ливерпульский университет (г. Ливерпуль, Великобритания); Палео-БАРН, Школа археологии Оксфордского университета (г. Оксфорд, Великобритания); ophelie.lebrasseur@liverpool.ac.uk
Шаймуратова Диляра Наилевна, научный сотрудник, Институт проблем экологии и недропользования АН РТ (г. Казань, Россия); galimovad@gmail.com
Аськеев Артур Олегович, кандидат биологических наук, научный сотрудник, Институт проблем экологии и недропользования АН РТ (г. Казань, Россия); art.regulus@mail.ru
Асылгараева Гульшат Шарипзяновна, кандидат ветеринарных наук, старший научный сотрудник, Институт археологии им. А.Х. Халикова АН РТ (г. Казань, Россия); gul_shat@mail.ru
Лоран Франц, профессор, доктор философии, департамент ветеринарных наук, палеоге-номная группа, Мюнхенский университет имени Людвига и Максимилиана (г. Мюнхен, Германия); Школа биологических и химических наук, Лондонский университет королевы Марии (г. Лондон, Великобритания); laurent.frantz@lmu.de
Грегер Ларсон, профессор, директор, Палео-БАРН, Школа археологии Оксфордского университета (г. Оксфорд, Великобритания); greger.larson@arch.ox.ac.uk
Аськеев Олег Васильевич, кандидат биологических наук, заведующий лабораторией, Институт проблем экологии и недропользования АН РТ (г. Казань, Россия); parus.cyanus@rambler.ru
Аськеев Игорь Васильевич, кандидат биологических наук, доцент, старший научный сотрудник, Институт проблем экологии и недропользования АН РТ (г. Казань, Россия); archaeozoologist@yandex.ru
Статья принята в номер 01.12.2020 г
Генетические исследования O. Лебрессер и Г. Ларсена были поддержаны Советом по исследованиям в области искусства и гуманитарных наук (номер гранта AH/L006979 /1). Исследовательский проект О. Лебрессер, Г.Ларсена и Л. Франца был поддержан Европейским Исследовательским Советом и Объединением Одомашнивания и эволюционной биологии через древнюю ДНК ERC-2013-StG-337574-UNDEAD. Исследования Г. Ларсена и Л. Франца поддержаны грантами Совета по исследованиям окружающей среды (NE/ K005243/1, NE/K003259/1, NE/S007067/1, NE/S00078X/1). Исследования Лорана Франца были поддержаны Велком Траст (Wellcome Trust) (210119/Z/18/Z). Зооархеологические исследования выполнены при финансовой поддержке РФФИ в рамках научного проекта № 20-09-00004..
