UDK 575:633.1 (392/393)"6342"_
Documenta Praehistorica XXV (Poročilo o raziskovanju paleolitika, neolitika in eneolitika v Sloveniji XXV)
Einkorn vvheat domestication site mapped by DNA fingerprinting*
Manfred Heun1, Basilio Borghi2 and Francesco Salamini3
1. Institutt Bioteknologifag, Norges Landbrukshogskole, As, Norway, manfred.heun@ikb.nlh.no 2. Istituto Sperimentale Cerealicoltura, Sant' Angelo Lodigiano, ltaly 3. Max-Planck-lnstitut Zuchtungsforschung, Koln, Germany
ABSTRACT - Wild relatives of the founder' crops of the European agriculture, chickpea, lentil, pea, barley, Emmer and Einkorn ivheats, bitter vetch (Zohary and Hopf1993) continue to grow in the Fer-tile Crescent. The study of the genetic relationships beliveen cultivated types occuring outside their nat-ural habitat and their ivild relatives clarifies important aspects of plant domestication, For example, by comparing - based on DNA fingerprinting - cultivated lines ivith ivild relatives collected in defined areas, ive have been able to pinpoint precisely the plače of origin of Einkorn ivheat ivithin the Fertile Crescent (Heun etal. 1997), apuzzle ivhich archaeology alone has been unable to solve. Similar stud-ies of other Fertile Crescent crops might ansiver ivhether the Neolithic revolution in this part of the ivorld had a common origin, or ivhether the above mentioned other crops ivere domesticated inde-pendently. DNA analyses can contribute to archaeology; more interaction is needed.
POVZETEK - Divji sorodniki prvotnih pridelkov evropskega poljedelstva (čičerka, leča, grah, ječmen, žiti Emmer in Einkorn, grenka grašica (Zohary in Hopf 1993) še danes uspevajo v Rodovitnem polmesecu. Raziskave genskih povezav med gojenimi tipi, ki se pojavljajo izven njihovega naravnega okolja. in njihovimi divjimi sorodniki pojasnjujejo pomembne vidike udomačitve rastlin. Na primer, na osnovi primerjave prstnih odtisov DNK gojenih vrst in divjih sorodnikov, ki smo jih nabrali na znanih območjih, smo lahko natančno določili izvor žita Einkorn znotraj Rodovitnega polmeseca (Heun et al. 1997), in tako rešili uganko, kije sama arheologija ni mogla razrešiti, Podobne raziskave drugih pridelkov z Rodovitnega polmeseca bodo morda odgovorile na vprašanje, ali ima neolitska revolucija v tem delu sveta skupni izvor ali pa so bili zgoraj omenjeni pridelki udomačeni neodvisno drug od drugega. DNK analize lahko prispevajo k arheologiji; potrebno je večje sodelovanje.
INTRODUCTION
DNA techniques provide powerful tools for studying evolution and domestication. However, use of DNA techniques is limited when only small amounts of high quality DNA can be extracted, as is the čase with ancient samples. Although this limitation can be overcome to some extent (Broivn et al. 1994), an alternative approach to addressing questions about the domestication of plants is to use modern seed samples. Einkorn wheat is a forgotten crop, to which no modern breeding has been applied, and has been cultivated for several thousand years outside its nat-ural habitat.
Wild Einkorns stili occur in nature (Zohary and Hopf 1993), and large samples of these wild lines are stored in gene banks around the world. Therefore,
* see acknowledgements
a representative collection of cultivated Einkorns, geographically well isolated from their wild relatives, can be used to identify the closest wild relative in a defined geographic area. As a result, the possi-ble Einkorn wheat domestication site was pinpointed within the Fertile Crescent (Heun et al. 1997).
THE PLANT MATERIAL
Einkorn wheats are diploid, self-pollinating plants (2n = 2x = 14), belonging to the family Poaceae and carrying the A genome. Triticum monococcum ssp. monococcum (T. monococcum) and Triticum monococcum ssp. boeoticum {T. boeoticum) are the respective Latin names of the domesticated and the
-limits of Fertile Crescent O T.m. boeoticum
* sampling of Karacadag lines A. T.m. monococcum v (vvith number of samples) + archeological site	□ T.m. aegilopoides J
A - L: areas of wild T.m. boeoticum sampling in the Fertile Crescent
Fig. 1. Sampling sites of 538 Einkorn tiheats. Insert: the Karacadag region. For the area of the Fertile Crescent, tvhere Einkorn occurs in primary habitats, nine groups ivere formed (see Heun et al. 1997for details). Reprinted ivithpermissionfrom Science, 14 November 1997, Volume 278,p. 1313, Fig. 1. ©1998 American Association for the Advancement of Science.
wild Einkorn wheat. Triticum monococcum ssp. aegilopoides (T. aegilopoides) is another Einkorn wheat which is fully fertile with the two other Ein-korns. T. aegilopoides occurs in the wild mainly in the Balkans, and is of interest because it shows domestication traits similar to those of T. monococcum. Ten gene banks world-wide (see Heun et al. 1997 for details) provided Einkorn wheat samples. In total we obtained 1362 lines, then verified their taxonomic assignment and evaluated their agronom-ic performance. The collection sites for about 900 of the samples were provided by some gene banks. For the Fertile Crescent samples, as well as for most of the samples from Turkey, only lines for which the collection site was known within + 5 km were con-sidered. Outside the primary habitat of wild Einkorn, most lines are frequently known only by their coun-try of origin. Moreover, since agriculture led to the spread of cultivated types, consideration of their sites of collection could be misleading. The geogra-
phic distribution of the T. boeoticum and T. aegilopoides lines present in our collection is in agree-ment with the distribution of wild Einkorn as pub-lished in Harlan and Zohary (1966). In their Fig. 3, the primary habitats of T. boeoticum are shown to include the Taurus-Zagros region from South-eastern Turkey through North-eastern Iraq into Western Iran (i.e., the Eastern half of the Fertile Crescent). T. aegilopoides grows wild mainly in the Balkans and We-stern Anatolia, where it occupies marginal habitats. In Central Anatolia and Transcaucasia the two wild Einkorns occur in marginal habitats together with cultivated Einkorns (Zohary and Harlan 1966). West of the Balkans, only cultivated Einkorns occur.
FORMING GROUPS
The T. boeoticum samples collected in the Fertile Crescent were divided into nine geographic groups
(A, B, C, D, E, G, H, I and L). Ali T. aegilopoides sam-ples were included in the 'Aegi' group and the cultivated Einkorn in the 'Mono' group. To test for the monophyletic origin of the cultivated types, this last group was also separated into four subgroups based on their geographic origins (Central Europe, the Bal-kans, Mediterranean countries and Turkey). Figure 1 (from Heun et al. 1997) shows the sampling sites of the 338 Einkorns used for DNA fingerprinting. To re-duce our collection to 338, samples were randomly chosen within the above mentioned 11 groups.
DNA FINGERPRINTING DATA
Amplified fragment length polymorphism (AFLP) markers were generated (Vos et al. 1995) for ali 338 lines. A total of 288 stable and reliably readable AFLPs were scored for presence vs. absence. Dif-ferent genetic distance estimates were used to con-struct several phylogenetic trees based on neighbor-joining and restricted maximum likelihood estima-tion methods. Almost identical topologies were de-tected by ali methods employed. Finally, a consensus tree based on ten different tree-building procedures was obtained (see Heun et al 1997 for details).
WILD ANCESTORS OF CULTIVATED EINKORN
Figure 2A shows that the nine geographic groups of T. boeoticum collected in the Fertile Crescent can be distinguished genetically. Group D, originating from the Karacadag Mountains in Southeast Turkey, is the most distant group. By adding the cultivated Einkorns (Mono) and the wild Einkorns from the Bal-kans (Aegi) to these nine groups, we obtained the results in Figure 2B. Cultivated Einkorn appears clo-sely related to T. aegilopoides. Group D links 'Mono' and Aegi' with the remaining eight groups. This result is a major achievement, since for the first tirne cultivated Einkorns can be traced back to a group of wild Einkorns showing ali the characteristics of a wild species, whereas the lines that grow wild in the Balkans show clear signs of domestication. It is con-cluded that bolh T. monococcum and T. aegilopoides are derived from group D wheats. Figure 2C clearly demonstrates the monophyletic origin of the cultivated Einkorn and strongly suggests that T. aegilopoides is a derivative of the cultivated forms. Group D is again positioned between T. monococcum and ali other T. boeoticum forms. The second major result that emerges from our studies is that ali
group D lines were collected from a relatively small area on the slopes of the Karacadag Mountains. A gradient ranging from high to very high relation-ships within the 19 representatives of group D is evident (Fig. 2F).
CONNECTING DNA STUDIES WITH ARCHAEOLOGY
The localisation of the origin of cultivated Einkorn to the Karacadag Mountains stimulates questions concerning the human community which achieved this domestication: are there neighboring human settlements with signs of early Einkorn cultivation? It is known that Cafer Hoyiik, Nevali Cori and Cayonii are ali located in the vicinity of these mountains. These are among the oldest settlements at which pa-laeontologists have found wild and domesticated Einkorn seeds in different horizons. In Table 2 of Nesbitt and Samuel (1996) ali archaeological data relevant to the origin of agriculture are summarised. From these it becomes evident that the cultivation of Einkorn began between 7800 and 7500 BC in the settlements cited. At the excavated sites in the Jordan Valley mentioned by Jones et al. (1998), no deci-sive (concerning general identification problems see Hillman et al. 1993) earlier remains of cultivated Einkorn have been found (Heun et al 1998, Nesbitt 1998; Nesbitt and Samuel 1998), emphasising the importance of the Northern Fertile Crescent in Einkorn domestication. In the čase of other excavated sites, such as Abu Hureyra and Mureybit in Northern Syria, wild seeds of T. boeoticum seem to have not been collected locally (Zeist and Casparie 1968; Zeist and Bakker-Heeres 1984).
SUMMARY
Wild ancestors of cultivated Einkorn have been local-ized in the Karacadag Mountains of Turkey. The archaeological evidence from neighboring excavations implies that Einkorn domestication was initiated there about 9500 years ago. The genetic data also indicate that the domestication event was mono-phyletic (see also Zoliary in press) and that the cultivated lines differentiated to a limited extent (quick-ly achieved by domestication, Hillman and Davies 1990) during the spread of agriculture to Western Europe. T. aegilopoides is probably a feral form of the cultivated types which reached the Balkans as a result of the spread of agriculture.
Fig. 2 A, B and C: Unrooted trees with the nine T. boeoticum groups alone, ivith the same nine groupsplus T. monococcum (Mono) and T. aegilopoides (Aegi) and the tree resulting from splitting up the Mono group intofour distinct subgroups. D: Consensus tree summarising the results ivith the nine T. boeoticum groups and the groups Mono and Aegi. E: Unrooted tree u ith ali fingerprinted lines. red: cultivated Ein-korns, green: T. aegilopoides, orange: T. boeoticum from the Karacadag, blue: remaining T. boeoticum. F: Unrooted tree for the 19 Karacadag lines aligned to one consensus genotype of the remaining T. boeoticum and one consensus genotype of the cultivated Einkorn. For details on the tree building proce-dures see Heun et al. (1997). Reprinted ivithpermission from Science, 14 November 1997, Volume 278, p. 1314, Fig. 2. © 1998 American Association for the Advancement of Science.
ACKNOWLEDGEMENTS
This review is based on Heun et al. (1997 in SCIENCE) and is a shortened version of a review by Heun et al. (in press, ENCYCL O P EDI A GENETICS).
The persons acknowledged there provided seeds, data on collection sites and help. Please refer to the original SCIENCE article containing the originals of the figures. Thanks for corrections on the English are due to John Einset, NLH, Norway.
REFERENCES
BROWN T. A, ALLABY R. G, BROWN K. A, 0'D0N0G-HUE K. and SALLARES R. 1994. DNA in wheat seeds from European archaeological sites. Experientia 50: 571-575.
HARLAN J. R. and ZOHARY D. 1966. Distribution of wild wheats and barley. Science 153: 1074-1080.
HEUN M, SCHAFER-PREGL R, KLAWAN D, CASTAG-NA R, ACCERBI M, BORGHI B. and SALAMINI F. 1997. Site of einkorn wheat domestication identified by DNA fingerprinting. Science 278: 1312-1314.
HEUN M, BORGHI B. and SALAMINI F. 1998. Wheat domestication; response. Science 279:303-304.
HILLMAN G. C. and DAVIES M. S. 1990. Measured domestication rates in wild wheats and barley under primitive cultivation, and their archaeological impli-cations. Journal ofWorld Prehistory 4: 157-222.
HILLMAN G, WALES S, MCLAREN F, EVANS J. and BUTLER A. 1993. Identifying problematic remains of ancient plant foods: a comparison of the role of che-mical, histological and morphological criteria. World
Archaeologv 25: 94-121.
JONES M. K, ALLABY R. G. and BROWN T. A. 1998. Wheat domestication. Science 279: 302-303.
NESBITT M. 1998. Where was einkorn wheat domes-ticated? Trends Plant Sci. 3: 82-83.
NESBITT M. and SAMUEL D. 1996. From staple to extinction? The archaeology and history of hulled wheats. In Padulosi S, Hammer K. and Heller J. (eds). Hulled Wheats: 41-100.
1998. Wheat domestication: archaeological evidence. Science 279:1433-
VOS P, HOGERS R, BLEEKER M, REIJANS M., VAN DE LEE T, HORNES M, FRIJTERS A, POT J, PELE-MAN J, KUIPER M. and ZABEAU M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407-4414.
ZEIST W. VAN and CASPARIE W. A. 1968. Wild einkorn wheat amd barley from Teli Mureybit in north-ern Syria. Acta Botanica Nederlandica 17: 44-53■
ZEIST W. VAN and BAKKER-HEERES J. A. H. 1984. Archaeobotanical studies in the Levant 3. Late-Pa-laeolithic Mureybit. Palaeohistoria 26: 171-199.
ZOHARY D. in press. Monophyletic vs. polyphyletic origin of the crops on which agriculture was found-ed in the Near East. Genetic Resources & Crop Evo-lution.
ZOHARY D. and HOPF M. 1993. Domestication of plants in the Old World (2nd edition). Oxford.