Tracing the introduction of domestic small bovids in Austral Africa using palaeoproteomics

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Tracing the introduction of domestic small bovids in Austral Africa using palaeoproteomics

Louise Le Meillour, Antoine Zazzo, Sophie Cersoy, Marie Arul, Matthieu Lebon, Joséphine Lesur, Chrystelle Le Danvic, David Pleurdeau, Patricia

Nagnan-Le Meillour, Severine Zirah

To cite this version:

Louise Le Meillour, Antoine Zazzo, Sophie Cersoy, Marie Arul, Matthieu Lebon, et al.. Tracing the introduction of domestic small bovids in Austral Africa using palaeoproteomics. SMMAP 2017 (Spectrométrie de Masse, Métabolomique et Analyse Protéomique 2017)., Oct 2017, Marne-la-Vallée, France. �mnhn-03203212�

T RACING THE INTRODUCTION OF DOMESTIC SMALL BOVIDS IN A ^USTRAL A FRICA USING

P ALAEOPROTEOMICS

L ^E M ^EILLOUR L.

¹

, Z ^AZZO A.

¹

, C ^ERSOY S.

²

, M ^ARIE A.

³

, L ^EBON M.

⁴

, L ^ESUR J.

¹

, L ^E D ^ANVIC C.

⁵

, P ^LEURDEAU D.

⁴

, N ^AGNAN -L ^E M ^EILLOUR P.

⁶

& Z ^IRAH S.

⁷

1 Archéozoologie, Archéobotanique : sociétés, pratiques et environnements (AASPE) – Sorbonne Universités, Muséum national d’Histoire naturelle (MNHN), CNRS : UMR 7209 – CP 56, 55 rue Buffon, 75005 Paris

2 Centre de recherche sur la Conservation des Collections (CRC) – Muséum national d’Histoire naturelle (MNHN), Ministère de la Culture et de la Communication, CNRS : USR 3224 – CP 21, 36 rue Geoffroy Saint-Hilaire, 75005 Paris, France

3 Plateforme de spectrométrie de masse – CNRS : UMR 7245, 63, rue Cuvier 75005 Paris, France

4 Histoire naturelle de l’Homme préhistorique (HNHP) – Sorbonne Universités, Muséum national d’Histoire naturelle (MNHN), UPVD, CNRS : UMR 7194 – 17 Place du Trocadéro, 75116 Paris, France

5 Unité de Glycobiologie Structurale et Fonctionnelle (ALLICE) – Allice – Cité Scientifique, avenue Mendeleiev – 59655 Villeneuve d’Ascq cedex, France

6 Unité de Glycobiologie structurale et fonctionnelle - UMR 8576 CNRS/Univ. Lille1/USC INRA 1409 (UGSF) – CNRS : UMR 8576, Université Lille I - Sciences et technologies – Bâtiment C9 Cité Scientifique, avenue Mendeleiev – 59655 Villeneuve d’Ascq cedex, France

7 Molécules de Communication et Adaptation des Micro-Organismes (MCAM) – Sorbonne Universités, Muséum national d’Histoire naturelle (MNHN), CNRS : UMR 7245 – CP 54, 57 rue Cuvier, 75005 Paris, France

o In Austral Africa, no remains of domestic caprines (sheep Ovis aries and goat Capra hircus) has never been found in archaeological assemblages.

This suggests that those domestic animals have been introduced from the Near East to East Africa and then to the southern part of the continent (MacHugh, Larson & Orlando 2017, Vigne 2011).

o Classical morphological determination of caprines from bone remains is often tricky due to the similarities between close related species as well as between domestic and wild species, fragmentation of the remains and bone diagenesis.

o Paleoproteomics has revealed as a powerful approach for the distinction of domestic caprines (Buckley et al. 2010). We used this technique on remains from two Austral African sites considered as the oldest pastoral sites of the region: Leopard Cave in Namibia (Pleurdeau et al. 2012) and Toteng in Botswana (Robbins et al. 2008). Our objective was to ascertain the presence or absence of domestic animals at these sites.

o Three different sample preparation protocols were tested in order to identify the best conditons for remains from arid environment. Bottom-up proteomics analysis was performed on an ESI-Q-TOF device with a data dependent autoMS/MS method. Modern samples from referential collections (MNHN) were also analyzed to constitute a database of unreported diagnostic peptides for species identification.

I NTRODUCTION

Leopard Cave

Toteng

C ^ONCLUSION

We would like to thank the LabEx BCDiv for funding a Master’s grant on this topic; the MNHN for funding the analysis through the ATM grant

« ProtéArch »; the CNRS and the MNHN for funding the ISABIO equipment project; the UMR 8576 for accessing the lab and providing equipment for molecular biology manipulations; the « Plateforme de Caractérisation des Matériaux » for FT-IR analysis facilities and the « Plateau technique de Spectrométrie de Masse Bio-Organique » of the MNHN for access to the UHPLC-ESI-Q-TOF instrument.

R ^ESULTS

M ^ATERIALS

100 km

Gaborone Ghanzi

TOTENG Lac Ngami

BOTSWANA

100 km

Windhoek LEOPARD CAVE

NAMIBIE

3 dental remains morphologically identified as caprines

26 unidentified remains (due to taphonomy)

9 presented here

5 cm

M3 inf. R.

5 cm

M2 sup. R.

5 cm

½ mandible R.

TOT_26

2 cm 2 cm

TOT_03

2 cm

TOT_06

2 cm

TOT_07

2 cm

TOT_09

2 cm

TOT_11

2 cm

TOT_14 TOT_15

2 cm

TOT_18

2 cm

A

RCHAEOLOGICAL

M

^ODERN

Bones and teeth from domestic and wild African species

Sheep Ovis aries

Damara breed

Goat Capra hircus

Brandberg breed

Impala Aepyceros melampus

Collections Anatomie Comparée MNHN

Springbok Antidorcas marsupialis

Collections Anatomie Comparée MNHN

M ^ETHODS

E

STIMATION OF THE ORGANIC PHASE PRESERVATION

Fourier-Transform Infrared Spectroscopy (FT-IR)

FT-IR spectrumof one Leopard Cave sample

% N = !",$ &'()* +

,-_. + 0,31

% collagène =

//0,/0 &'()* +

,-_. + 1,69

Lebon et al. 2016, Cersoy et al. 2016

M

^ASS

S

PECTROMETRY ANALYSIS

S

AMPLE PREPARATION

NH₄CO₃50 mM 3 h, 65°C, 350 rpm

Trypsin « Proteomics Grade » (Promega)

1 μg/ μL, 18h, 37°C, 350 rpm

Demineralization Solubilization Digestion

Tris-EDTA 0.5 M, pH 7.4 HCl 1M, 20 min

HCl 0.6M, 4h

Bucker Maxis II

UHPLC-MS/MS (ESI-Q-TOF)

o UHPLC : Ultimate 3000-RSLC (ThermoSci), Acclaim RSLC Advantage II column (2,2 μm, 2,1x100 mm, ThermoSci);

o MS : ESI-Q-TOF positive mode, m/z 200-3000

o Analysis : AutoMS/MS data

14,37 13,45

11,24

5,72 5,38

3,58 3,34 3,11 3,10 2,94 2,73 2,66 2,57 2,45 2,44 2,40 2,21 2,18 2,03 1,99 1,91 0

5 10 15

%20

Modern ref. Leopard

Cave Toteng

E

STIMATION OF THE ORGANIC PHASE PRESERVATION

LC_N7_113_1M

260.164 342.218

430.235

540.285 600.343 637.340 671.381

799.441

b I

y I G A

b (4)

b (5)

y (4)

y (5) y (6) y (7)

300 400 500 600 700 m/z

Cmpd 55, +MS2(799.4415), 41.0eV, 2.4 min #156 [Cmpd 55, +MS2(799.4415), 41.0eV, 2.4 min #156]

x105

0.0 0.2 0.4 0.6 0.8 1.0

Intensity

175.121 361.188 501.201 686.284

754.395 2+

853.902

924.467

1078.543

1206.603

1334.665 1405.708 1532.685 R y

b (3) b (6)

y (15)++

y (9)

b (14)

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Cmpd 96, +MS2(853.9018), 34.2eV, 3.2 min #216 [Cmpd 96, +MS2(853.9018), 34.2eV, 3.2 min #216]

x106

0.0 0.2 0.4 0.6 0.8

Intensity

799.44

LC_N7_113_06M

171.079 260.164 342.218

430.235

543.320 600.343 671.380

799.441

b I

y I G A

b (4)

b (5)

y (4)

y (5) y (6) y (7)

y (9)

150 200 250 300 350 400 450 500 550 600 650 700 750 m/z

Cmpd 78, +MS2(799.4408), 41.0eV, 2.3 min #157-163 [Cmpd 78, +MS2(799.4408), 41.0eV, 2.3 min #157-163]

x106

0.0 0.2 0.4 0.6 0.8

Intensity

175.121 361.187 501.200 686.282

754.394 2+

853.901

924.465

1078.541

1206.602

1277.639 1346.615 1532.685 R y

b (6)++ b (6)

y (15)++

y (9)

b (14)

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Cmpd 128, +MS2(853.9009), 34.2eV, 3.2 min #228 [Cmpd 128, +MS2(853.9009), 34.2eV, 3.2 min #228]

x105

0.0 0.5 1.0 1.5

Intensity

LC_N6_176_1M

171.115 311.176 426.240 596.348 3+

763.379

3+

961.826

2+

1144.565 2+

1278.146 2+

1357.182 1444.726 1697.415

b G

b (4)++ y (5)++ b (7)

y (26)++

200 400 600 800 1000 1200 1400 1600 m/z

Cmpd 175, +MS2(961.8263), 35.0eV, 4.3 min #362-365 [Cmpd 175, +MS2(961.8263), 35.0eV, 4.3 min #362-365]

x106

0.0 0.5 1.0 1.5 2.0 2.5

Intensity

86.097 199.111 312.195 411.265

2+

608.317

2+

730.924

865.500

932.470 1050.582

1149.652

b I V G Q P P G

Ry P P Q G V I

b (2) b (3) b (4)

y (5)

b (6)

y (15)++

y (9)

y (11)

y (12)

100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z

Cmpd 208, +MS2(730.9245), 29.2eV, 4.6 min #409 [Cmpd 208, +MS2(730.9245), 29.2eV, 4.6 min #409]

x105

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Intensity

MS/MS spectrum of [M+H]⁺ ion of GAAGIPEGK peptide (m/z 799.44, 2,4 min) of one Leopard Cave’s sample, demineralized with Tris-EDTA.

MS/MS spectrum of [M+H]⁺ ion of LCPDCPLLANDSR peptide with a phosphorylation (S)

(m/z 853.90, 3,2 min) of one Leopard Cave’s sample, demineralized with Tris-EDTA. MS/MS spectrum of [M+H]⁺ ion of TPIVGQPSVPGGPVR peptide (m/z 730.92, 4,6 min) of one Leopard Cave’s sample, demineralized with Tris-EDTA.

644 GAAGIPEGK 652

m/z 799,44 (2⁺)

145 LCPDCPLLAPLNDSR 159

333 TPIVGQPSVPGGPVR 348

m/z 853,90 (2⁺)

m/z 730,41 (2⁺)

Alpha 2 chain collagen type I

Alpha 2 HS glycoprotein

TOT 14 TOT 09 TOT 11 & 15

🐖 🐗 🐎

🐑

TOT 14 TOT 09 TOT 11 & 15

🐑

O. aries

O. aries

Suids Equids

Biochronology &

geographic repartition

Kingdon 1997

Phacochoerus sp.

Potamochoerus sp.

Equus burchellii

or

o Identification of the best protocol for arid environments remains : Tris-EDTA

o Identification of specific markers of unpublished species (Antidorcas marsupialis) on 2 different proteins o Attribution of 4 morphologically undetermined remains

o Re-attribution of Leopard Cave remains ➜ wild species: no domestication at this site o Identification of sheep at Toteng ➜ confirmation of domestication at this site

Namibia

South Africa Lesotho

Swaziland

Toteng 3 to be dated Leopard Cave

Spoegrivier

II^e c. BCE Blombos Cave

I^st c. BCE Toteng 1

I^st c. BCE

Pleurdeau et al. 2012, Robbins et al. 2008

A

RCHAEOLOGICAL IMPLICATIONS

- MacHugh, Larson & Orlando 2017, Annual Review of Animal Bioscience, 5, 329-51.

- Vigne 2011, Comptes rendus biologies, 334(3), 171-181.

- Buckley et al. 2010, Journal of Archaeological Science, 37(1), 13-20.

- Pleurdeau et al. 2012, Plos One, 7(7), e40340.

- Robbins et al. 2008, Journal of African Archaeology, 6(1), 131-149.

- Lebon et al. 2016, Radiocarbon, 1-15.

- Cersoy et al. 2017, Radiocarbon, 1-16.

- Kingdon 1997, Princeton Univ. Press, London, 544 p.