Genet Resour Crop Evol DOI 10.1007/s10722-007-9210-0 R E S E A R C H A RT I C L E Origin and ancestry of Egyptian clover (Trifolium alexandrinum L.) As revealed by AFLP markers Abdelfattah Badr Æ Hanaa H. El-Shazly Æ Linda E. Watson Received: 3 October 2006 / Accepted: 10 January 2007
Springer Science+Business Media B.V. 2007 Abstract The origin and ancestry for Egyptian clover, Trifolium alexandrinum, was examined using AFLP data. The data support a close relationship of T. alexandrinum accessions from Syria and Egypt to T. apertum, T. berytheum, and T. salmoneum. However, crossability and geographic distributions suggest that T. apertum is an unlikely progenitor. In contrast, T. salmoneum appears to be the most probable progenitor for Syrian material of Egyptian clover, although a close relationship to T. berytheum was also revealed. The ability of these species to cross freely indicates that T. salmoneum and T. berytheum may be regarded as the primary ancestors from, which man domesticated Egyptian clover through artificial selection in Syria. Following domestication, the earlier forms of the crop species could have been taken into rain-fed cultivation in Palestine and irrigated cultivation A. Badr (&) Botany Department, Faculty of Sciences, Tanta University, Tanta 31527, Egypt e-mail: Abdelfattahbadr@yahoo.com H. H. El-Shazly Department of Biological Sciences and Geology, Faculty of Education, Ain Shams University, Cairo, Egypt L. E. Watson Department of Botany, Miami University, Oxford, OH 45056, USA in Egypt. In this regard, the domestication of Egyptian clover may be analogous to other crops, such as barley and wheat, which were also domesticated in the Fertile Crescent and taken into cultivation in the Nile Valley. It appears that genetic improvement of the crop occurred in Egypt after cultivation, and that the varieties that were developed in Egypt were later distributed worldwide. Keywords AFLP
Ancestry
Berseem
Egyptian clover
Origin
Trifolium alexandrinum Introduction Egyptian clover (also known as Berseem), Trifolium alexandrinum L., has been widely cultivated as a forage crop in western Asia and northern Africa. Its cultivation was extended into central Asia, particularly in Pakistan and India, and also into the United States since the beginning of the 20th century (Knight 1985). In their comprehensive monograph on Trifolium, Zohary and Heller (1984) recognized two varieties of T. alexandrinum: alexandrinum Boiss. and serotinum Zoh. et Lern. locally known as Fahli and Miscavi, respectively. The former variety exhibits apical branching only and produces one crop per cultivation. The most common cultivars of Miscavi are Sakha and Kohrawi, which exhibit 123 Genet Resour Crop Evol basal branching and produce 4–6 harvests per cultivation. A third variety, Saidi, produces both basal and apical branching and produces 2–3 crops per cultivation. In Egypt, where crop rotation is necessary to agricultural practice, the Fahli and Saidi cultivars are planted in October and harvested in January and February to enrich the soil prior to cotton cultivation, while the other varieties are cultivated from October to May. The origin and ancestry of Egyptian clover has been one of the longest debated issues in the history of cultivated plants. Delile (1824) mentioned that seeds were frequently imported into Egypt from Syria where it is cultivated and grows wild (Boissier 1856). Bobrov (1947) supported an earlier hypothesis proposed by Hegi (1923) that the Mamluks, rulers of Egypt from the 12th to 15th century AD, introduced clover into Egypt from Caucasus, whereas Becker-Dellingen (1922) suggested that it was introduced into Egypt in the 6th century AD. However, Putiyevsky et al. (1975) considered all of these views erroneous due to the relatively recent descriptions of the species related to Berseem (T. vavilovii Eig 1934, T. apertum Bobr. 1945, T. salmoneum Mout. 1953, T. meironense Zoh. et Lern. 1972), as well as the misidentification or taxonomic uncertainty of T. berytheum Boiss. They (1975) suggested that Berseem was probably the earliest forage crop to be sown during the first Egyptian dynasty (3500 – 3800 BC). Taylor (1985) also assumed that T. alexandrinum was probably native to the Nile Valley in the ancient Lower Egypt. Trabut (1910) put forward the idea that T. berytheum from the coastal plains of Lebanon, which he viewed as a wild form of T. alexandrinum, might be the progenitor of T. alexandrinum. This idea was supported by Eig (1934) who considered T. berytheum to be a separate, but related, species of T. alexandrinum. However, Aaronsohn (1910) allegedly reported the occurrence of wild T. alexandrinum in Palestine, and claimed that T. echinatum M. B. (syn.=T. supinum Savi) which also grows in Palestine, might be a probable ancestor of Berseem. Bobrov (1947), on the other hand, claimed that T. apertum is the progenitor of Berseem, he based his claims on morphological similarities of T. alexandrinum and T. apertum. 123 Oppenheimer (1959) accepted the view of Trabut (1910) that T. berytheum is a wild form of T. alexandrinum, and suggested that T. berytheum should be regarded as the main genetic resource from which man domesticated Egyptian clover through artificial selection in Syria (Damascus) and Palestine, and later in Egypt during or after the Bronze or Iron Age. Oppenheimer’s interpretation of T. alexandrinum var. berytheum as a wild form of T. alexandrinum focused the search for the wild progenitor of Berseem on T. berytheum. He felt that no distinct, wild plant species, such as T. apertum, could have given rise to the cultivated forms of the Egyptian clover. He rejected claims for T. echinatum M.B., and also for T. carmeli Boiss. and T. vavilovii Eig, both of which he considered distinct species closely related to T. alexandrinum. Oppenheimer (1959) also rejected claims for other species related to T. alexandrinum, particularly T. constantinopolitanum Ser., T. leucanthum M.B., T. phleoides Pourr. ex Willd., and T. salmoneum Mout. In his view, the origin of Egyptian clover was analogous to that of other clovers, which are known to occur both as wild and cultivated races. However, this view and that of Aaronsohn (1910) are contradicted by the recent view by Taylor (1985) that T. alexandrinum is unknown in the wild and that no living wild ancestor(s) is known. Comprehensive studies on the relationship between T. alexandrinum and its closest relatives were conducted by Putiyevsky and Katznelson (1973, 1974), Katznelson and Putiyevsky (1974) and Putiyevsky et al. (1975). Their studies included cytogenetic evidence, the ability of the species to cross, and pollen fertility of their hybrids. The species used in these studies included five that are placed with T. alexandrinum in subsection Alexandrina Zoh. by Zohary (1972) and an additional six species that were considered potential donors to its genome. Successful crosses were obtained between T. alexandrinum, T. berytheum and T. salmoneum (Putiyevsky and Katznelson 1973), and thus Putiyevsky et al. (1975) concluded that these two species, especially T. salmoneum, seemed to be the true progenitors of cultivated Berseem. Another group of closely related species, more distantly related to T. alexandrinum, includes T. echinatum, Genet Resour Crop Evol T. carmeli, T. latinum Seb., T. plebeium Boiss., and T. scutatum Boiss. (Putiyevsky and Katznelson 1973; Katznelson and Putiyevsky 1974). However T. vavilovii was considered more distantly related and placed in a different crossability group (Putiyevsky et al. 1975). These results also indicated that the grouping of some species is contrary to their subsectional classification proposed by Zohary (1972) and Zohary and Heller (1984). AFLP markers have been applied to a wide range of topics in botanical research and used extensively for the assessment of genetic diversity and characterization of germplasm collections (Maughan et al. 1996; Abdalla et al. 2000; Sharma et al. 2000; Coulibaly et al. 2002; Rouf-Mian et al. 2002; Fu et al. 2004; Fjellheim and Rognli 2005). AFLP analysis is also an attractive technique for studies in gene linkage (Thomas et al. 1995; Hartl et al. 1999) and systematics and evolution (Hill et al. 1996; Kardolus et al. 1998; Massa et al. 2001; Badr et al. 2002; El-Rabey et al. 2002), and for elucidating the origin and domestication history of some cultivated crops (Heun et al. 1997; Badr et al. 2000). In this paper, we use AFLPs to address the origin and ancestry of Egyptian clover by applying an approach similar to that of Heun et al. (1997) for einkorn wheat and by Badr et al. (2000) for barley. Each of these two crops has a known living wild ancestor, and the objective was to search for the area in which the cultivated crop was first domesticated. However, unlike these two grass crops Egyptian clover has no known living wild ancestor(s). Thus the objective of this study is best achieved by the analysis of genetic diversity in numerous accessions of T. alexandrinum from different sources and accessions of related species that have been regarded as putative ancestor(s) or donors to its genome. Material and methods Eleven species, in addition to T. alexandrinum, were examined and included five from section Alexandrina (Zohary and Heller 1984) and six with demonstrated crossability to T. alexandrinum (Putiyevsky and Katznelson 1973, 1974; Katznelson and Putiyevsky 1974; Putiyevsky et al. 1975). Seeds of accessions representing these species were soaked in tap water for two days and germinated in small pots in the glasshouse at Miami University, Oxford, Ohio, USA. Leaves of actively growing seedlings were harvested on ice, frozen in liquid nitrogen, and stored at –80C for DNA extraction. Seedlings from the same accessions were transferred to larger pots (2–3 plants per pot) and grown until flowering to confirm their identity. Over 120 accessions of the 12 species were planted, however the identity of only 50 accessions of T. alexandrinum and 56 accessions of the other 11 species were confirmed. A total of 30 T. alexandrinum accessions and 26 accessions of the other 11 species were used for AFLP analysis with confirmed identifications. A list of these accessions, their species assignment, source, ID number, and origin is provided in Table 1. In addition, it should be noted that two species, T. carmeli Boiss. and T. supinum Savi (Table 1), have been regarded as subspecies of T. echinatum (Zohary and Heller 1984). Table 1 A list of Egyptian clover accessions used in this study, their species assignment, ID number, source and origin Species T. T. T. T. T. T. T. T. T. T. T. T. alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum alexandrinum L. L. L. L. L. L. L. L. L. L. L. L. Accession ID number Other IDs Sourcea Origin alex-02 alex-03 alex-04 alex-05 alex-06 alex-07 alex-08 alex-09 alex-10 alex-11 alex-12 alex-13 PI PI PI PI PI PI PI PI PI PI PI PI K-608 Miscavi Berseem NL-1714 Miscavi Miscavi 13887 17154 L-51 L-16 + 40 6007 K-2101 SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS Pakistan Israel Egypt Turkey Tunisia Morocco Pakistan Yugoslavia Pakistan Pakistan Greece Spain 250659 277510 250105 383769 291550 291549 217543 251213 445883 445879 378128 253582 123 Genet Resour Crop Evol Table 1 continued Species T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. alexandrinum L. apertum Bobr. apertum Bobr. berytheum Boiss. berytheum Boiss. carmeli Boiss. carmeli Boiss. clypeatum L. clypeatum L. clypeatum L. constantinopolitanum constantinopolitanum constantinopolitanum constantinopolitanum echinatum M. B. echinatum M. B. echinatum M. B. echinatum M. B. echinatum M. B. latinum Seb. latinum Seb. meironense Zoh. plebeium Boiss. plebeium Boiss. plebeium Boiss. salmoneum Mout. supinum Savi Ser. Ser. Ser. Ser. Accession ID number alex-14 alex-15 alex-16 alex-17 alex-18 alex-19 alex-20 alex-21 alex-22 alex-23 alex-24 alex-28 alex-29 alex-34 alex-51 alex-57 alex-94 alex-99 aper-84 aper-102 bery-59 bery-121 carm-82 carm-158 clyp-44 clyp-53 clyp-85 cons-52 cons-130 cons-134 cons-156 echn-60 echn-66 echn-70 echn-71 echn-77 lat-192 lat-218 meir-164 pleb-159 pleb-162 pleb-219 salm-154 sup-81 PI 292967 PI 445877 PI 164413 PI 459105 PI 445881 PI 233811 PI 226284 PI 291768 Sero 1 PI 517061 PI 214205 Sakha 3 Sakha 4 PI 468402 PI 201954 Sakha 96 IG 120953 IG 66553 PI 516230 PI 314117 PI 369019 PI 353412 PI 353422 TRIF 100/75 PI 292471 PI 241478 TRIF 129/96 PI 369028 IG 67731 IG 67739 IG 67543 PI 238159 PI 494720 PI 419273 PI 238159 PI 238159 NY 4724 IG 69098 IG 67954 IG 67899 NY 5439 PI 179056 TRIF 104/99 Other IDs B-23 8363 L- 64 + 13 52177 FAO-9329 Cultivar GR-7623 Cultivar Cultivar ENMP-4428 Fahli Cultivar IFTR 3679 IFTR 304 S 70-3 NYT1667 S-153-5 No.55-51 45082 IFTR 1382 IFTR 1490 IFTR 1294 G 2490 T-41 147 G 2490 Mu-029 IFTR 2849 IFTR 1705 IFTR 1650 S-273-1 Sourcea Origin SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS SRPIS ARC SRPIS SRPIS ARC ARC SRPIS SRPIS ARC ICARDA ICARDA Taylor SRPIS SRPIS Taylor SRPIS IPK SRPIS SRPIS IPK SRPIS ICARDA ICARDA ICARDA SRPIS SRPIS SRPIS SRPIS MU Iowa State NYBG ICARDA ICARDA ICARDA NYBG Taylor IPK Iraq Pakistan India Tunisia Pakistan Italy Kenya Egypt Egypt Morocco Italy Egypt Egypt Portugal Egypt Egypt Syria Syria F S Union Turkey Turkey Israel Israel Israel Israel Jordan Syria Syria Syria Turkey Romania Greece Turkey Turkey Algeria Syria Syria Romania a ARCE, Agricultural Research Center, Cairo, Egypt; ICARDA, International Center for Agricultural Research in Dry Areas, Aleppo, Syria; Iowa State, Iowa State University Herbarium, Ames, Iowa, USA; IPK, Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany; NYBG, New York Botanic Gardens, New York, USA; MU, Miami University, Turrell Herbarium, Oxford, Ohio, USA; SRPIS, Southern Regional Plant Introduction Station, USDA; Taylor, Dr. Norman Taylor, University of Kentucky, Lexington, Kentucky, USA For DNA extraction, a modified CTAB method (Saghai-Maroof et al. 1984) was used. Leaflets were powdered in liquid nitrogen using a mortar and pestle, and homogenized in 0.75 ml hot 4· 123 CTAB buffer containing 1% PVP, 1% Na-bisulphite, and 0.2% mercaptoethanol. The tubes were incubated for 30 min in a 60C water bath with occasional gentle mixing of the tubes. Following Genet Resour Crop Evol incubation, the mixture was emulsified with 0.5 ml of chloroform-isoamyl alcohol (24:1) and centrifuged at 10,000g for 5 min. The aqueous layer was pipetted into a new tube, mixed with 0.5 ml cold isopropanol, kept at –20C for 30 min, and centrifuged at 12,000g for 10 min. The alcohol was discarded and the pellet was washed in 0.75 ml 76% EtOH/0.01 M NH4OAC for 5 min followed by washing in 0.75 ml 76% EtOH/ 0.01 M NaOAC. The pellet was dried and suspended in 0.2 ml TE buffer, and 1 ll RNase was added and incubated at 37C for 30 min. DNA quantity in the TE buffer was estimated spectrophotometrically, and its quality was evaluated by running 10 ll in 10 % agarose gel in Trsi-acetate buffer (TAE) buffer. The AFLP analysis was performed using the ABI PRISM fluorescent dye labeling and detection protocol (Perkin Elmer, USA) based on the method of Vos et al. (1995), with slight modifications. Genomic DNA (500 ng) was double-digested with EcoRI and MseI restriction enzymes and ligated to EcoRI and MseI adapters by incubating in a total volume of 11 ll for 4 h at 37C. The restriction/ligation (R+L) product was diluted to 200 ll and stored at 4C for preamplification, or stored at –20C for later use. Five microliter of the R+L product were pre-amplified with EcoRI + A and MseI + C primers in a total volume of 20 ll in a thermocycler for 25 cycles at 94C denaturation (20 s), 56C annealing (30 s), and 72C extension (2 min), with initial hold at 72C and a final old at 60C for 30 min. The pre-selective amplification product was diluted 15X in 0.1 TE buffer and stored at 4C for amplification, or stored at –20C for later use. Five microliter of the above solution were used as a template for selective amplification using three 5¢end labeled EcoRI + 3 primers (ACA, blue; AAG, green; and ACC, yellow) and three MseI + 3 primers (CAC, CTC, and CTT). Amplification was conducted in a total volume of 15 ll for 9 cycles at 94C (2 min), 56C (30 s), and 72C (2 min), reducing the annealing temperature by one degree per cycle, followed by 21 cycles at 94C (2 min), 56C (30 s) and 72C (2 min), and a hold at 60C for 30 min. Of the amplified product, 2 ll were mixed with 20 ll of deionized formamide and 0.5 ll of GeneScan 500 ROX internal size standard in a 0.5-ml tube, denatured at 95C for 5 min, and analyzed by capillary electrophoresis on an automated ABI 310 DNA sequencer (Perkin Elmer, Applied Biosystems) with an injection time of 12 s and a run time of 30 min. AFLP fragment profiles produced by the nine primer pair combinations were analyzed with GeneScan analysis software version 3.1 (Perkin Elmer, Applied Biosystems), as well as printed on photographic paper for manual scoring and confirmation. The presence (1) or absence (0) of bands from 50 to 350 bp was scored (Fig. 1). Only polymorphic bands scored in at least two accessions were considered for analysis; uncertain fragments were scored as unknown (?). In total, 192 polymorphic bands were scored across 30 accessions of T. alexandrinum and 26 accessions of the remaining 11 species. Distance trees were constructed using Dice and Jaccard similarity coefficients using UPGMA (Sokal and Michener 1958) and Neighbor-joining (Saitou and Nei 1987) tree building methods with the software NTSYSpc 2.1 (Rohlf 1993). In addition, average distance UPGMA and Neighbor joining trees were produced using PAUP* 4.0 (Swofford 2002). PAUP was also used to conduct a parsimony analysis using a heuristic search with MULTREES in effect, TBR branch swapping, and 100 replicate random additions. Bootstrap values were calculated for 1000 replicates, and plotted onto the strict consensus tree of 2149 most parsimonious trees. Results The nine primer pair combinations for EcoRI and MseI produced considerable variation in the AFLP banding profiles (examples are illustrated in Fig. 1). Distance trees based on Dice and Jaccard coefficients have identical topologies (Fig. 2). Accessions of T. alexandrinum form one distinct cluster comprised of two subgroups: one of seven Egyptian (alex-04, alex-29, alex-21, alex-28, alex22, alex-51, alex-57) and two Syrian accessions (alex-94, alex-99), and a large subcluster of the remaining 21 accessions of T. alexandrinum. In the latter subgroup, two accessions (alex-02 from Pakistan and alex-34 from Portugal) are distinct 123 Genet Resour Crop Evol Fig. 1 AFLP banding profile for nine accessions of Trifolium alexandrinum (1–9), T. salmoneum (10), T. apertum (11–12), and T. berytheum (13–14). DNA was digested with EcoRI and MseI, and fragments were amplified using PCR in the presence of the MseI adapter CAC, and the two EcoRI adapters (ACA (a) left and AAG (b) respectively from each other and from all other accessions. In both the Dice and Jaccard distance trees, the accessions of the remaining 11 species form three clusters. The first is comprised of T. berytheum, T. apertum, and T. salmoneum. The second is comprised of T. supinum, T. carmeli, and two accessions of T. constantinopolitanum (cons-134, cons-256). The third cluster contains two subgroups: one comprised of T. echinatum, T. meironense, and two accessions of T. constantinopolitanum (cons-52 & cons130); and the other subcluster comprised of T. clypeatum, T. plebeium, and T. latinum. The average distance UPGMA and NJ trees have similar topologies (UPGMA tree, Fig. 3). Both trees agree to some extent with the Dice and Jaccard trees in separating T. alexandrinum from the remaining 11 species. In the average distance UPGMA trees, the T. alexandrinum accessions similarly form two subgroups, a small one comprised of seven Egyptian and two Syrian accessions, and a larger one comprised of all other accessions. Similar to distance trees based on Dice and Jaccard coefficients, accessions alex-02 (Pakistan) and alex-34 (Portugal) are distinct. In the average distance trees, T. salmoneum is placed in the T. alexandrinum cluster comprised of the seven Egyptian and two Syrian accessions. 123 Accessions representing T. berytheum and T. apertum also occur in the T. alexandrinum cluster. In the UPGMA average distance tree (Fig. 3), two clusters are present: T. clypeatum, T. plebeium, T. latinum, and T. meironense, and T. echinatum, T. supinum, T. carmeli and T. constantinopolitanum. Parsimony analysis of the AFLP data (Fig. 4) produced similar topologies to the average distance trees. In this tree, the small clade of T. alexandrinum, comprised of seven Egyptian and two Syrian accessions, is placed with the accessions representing T. berytheum, T. apertum, and T. salmoneum. Of the remaining species, only accessions of T. clypeatum and T. plebeium form a clade. The bootstrap values for the branches in the parsimony tree are generally low (Fig. 4). Discussion The AFLP data clearly delimit the accessions of T. alexandrinum as a single cluster, distinct from all remaining species sampled. This confirms the monophyly of Egyptian clover, and supports its distinctness from its putatively related species. The relationships among the other 11 species is in general agreement with their crossability Genet Resour Crop Evol Fig. 2 UPGMA Dice coefficient distance tree, based on AFLP data (Putiyevsky and Katznelson 1973; Putiyevsky et al. 1975), but is contrary to their sub-sectional taxonomy (Zohary 1972; Zohary and Heller 1984) with the exception of a close relationship for T. berytheum, T. apertum, and T. salmoneum of subsection Alexandrina and for T. clypeatum and T. plebeium of subsection Clypeata Gib. et Belli. In agreement with crossability data (Putiyevsky and Katznelson 1973; Katznelson and Putiyevsky 1974), the AFLP data support a distant relationship of T. alexandrinum to T. echinatum, T. carmeli, T. supinum, T. latinum, and T. plebeium. Thus the AFLP data contradict the claims of Aaronsohn (1910) that T. echinatum (syn.=T. supinum) is a probable ancestor of Berseem clover and support the alternative view of Oppenheimer (1959) who rejected claims for T. echinatum, as well as for T. carmeli and T. vavilovii, as ancestors for T. alexandrinum. The data further indicate that T. carmeli and T. supinum may be regarded as two species distinct from T. echinatum. The AFLP data support a close relationship between T. alexandrinum, T. berytheum, T. apertum, and T. salmoneum. This is in agreement with the placement of these species together in subsection Alexandrina (Zohary and Heller 1984). However, T. meironense, also in subsection Alexandrina, appears more distant to these species. A close relationship for T. alexandrinum, 123 Genet Resour Crop Evol Fig. 3 UPGMA average distance tree, based on AFLP data T. berytheum, and T. apertum was also supported by molecular phylogenies based on nuclear ribosomal ITS and chloroplast trnL nucleotide sequences (Ellison et al. 2006; Badr et al. unpublished data). However, these phylogenies do not reflect an apparent close genetic affinity between these three species and T. salmoneum, as suggested by the AFLP data and their crossability (Putiyevsky and Katznelson 1973; Putiyevsky et al. 1975). Comprehensive cytogenetic studies by Putiyevsky et al. (1975) on T. alexandrinum and other species of subsection Alexandrina, including crossability, meiotic behavior of chromosomes, and pollen fertility of hybrids, indicated that T. vavilovii is distant to T. alexandrinum, T. meironense, 123 alex02 alex03 alex05 alex06 alex07 alex17 alex08 alex10 alex15 alex11 alex18 alex16 alex09 alex14 alex12 alex13 alex19 alex24 alex20 alex23 alex34 alex04 alex29 alex21 alex28 alex22 alex57 alex94 alex99 salm154 alex51 aper84 aper102 bery59 bery121 clyp44 clyp85 clyp53 pleb159 pleb162 pleb219 lat192 lat218 meir164 cons134 cons156 echn71 echn60 echn66 echn70 echn77 cons52 cons130 carm82 carm158 supi81 T. apertum, T. berytheum, and T. salmoneum. These authors concluded that the two latter species, and particularly T. salmoneum, seem to be the true progenitors of cultivated Berseem clover. Their conclusion is strongly supported by the AFLP data. However, the AFLP data place T. meironense distant to the species of subsection Alexandrina, and reveal a close relationship for T. berytheum, T. salmoneum, and T. apertum. This is congruent with apparent frequent gene flow between these species (Putiyevsky et al. 1975) and T. alexandrinum, and thus are possible genetic resources from which the Egyptian clover could have been derived. The close relationship of T. berytheum to T. salmoneum, T. apertum, and T. alexandrinum Genet Resour Crop Evol Fig. 4 Strict consensus tree of 2149 equally most parsimonious trees based on AFLP data. Bootstrap values are above branches, Cl = 0.178, RI = 0.678, and RC = 0.121 90 53 77 69 63 73 89 56 86 97 56 99 86 95 73 66 99 is congruent with previous reports on the origin and ancestry of Egyptian clover. Specifically, Trabut (1910) viewed T. berytheum as a wild form of T. alexandrinum, and assumed that material from the coastal plains of Lebanon might be a progenitor for cultivated Berseem. This idea was supported by Eig (1934) who considered T. berytheum closely related to T. alexandrinum. This idea was supported by Oppenheimer (1959) who believed that T. berytheum must be regarded as the main genetic resource from which man developed Egyptian clover by selection in Syria (Damascus) and Palestine, and later in Egypt in the Bronze or Iron age or later. However, neither Trabut (1910) nor Eig (1934) considered T. salmoneum and T. apertum as possible progenitors for Egyptian clover. 91 93 100 100 alex02 alex34 alex03 alex05 alex06 alex07 alex17 alex12 alex13 alex08 alex10 alex09 alex11 alex14 alex16 alex15 alex18 alex19 alex20 alex23 alex24 alex04 alex21 alex28 alex29 alex51 alex22 alex57 alex94 aper84 aper102 bery59 bery121 salm154 alex99 clyp44 clyp85 clyp53 pleb159 pleb162 pleb219 cons134 cons156 echn60 echn66 echn70 echn71 echn77 carm82 carm158 supi81 lat192 lat218 meir164 cons52 cons130 Furthermore, the view that these two latter species could have led to cultivated forms of Egyptian clover (Bobrov 1947) was also denied by Oppenheimer (1959) who considered T. apertum to be more closely related to T. carmeli and T. vavilovii but distinct from T. alexandrinum. This view is in contrast to the taxonomy of T. apertum and T. vavilovii in subsection Alexandrina (Zohary (1972; Zohary and Heller 1984), and the crossability of T. apertum with T. alexandrinum (Putiyevsky et al. 1975). Trifolium salmoneum was not yet identified at the time Trabut (1910) and Eig (1934) addressed the origin of the Egyptian clover, which also was not considered by Oppenheimer (1959) who focused his investigation on T. berytheum and on material from Palestine (Israel). However, cytogenetic 123 Genet Resour Crop Evol evidence presented by Putiyevsky et al. (1975) clearly indicated that T. salmoneum is the probable progenitor for T. alexandrinum. The AFLP data support a close relationship of T. berytheum, T. salmoneum, and T. apertum to T. alexandrinum accessions from Egypt and Syria. The crossability data of species in subsection Alexandrina separate T. apertum and T. meironense from T. berytheum, T. salmoneum, and T. alexandrinum (Putiyevsky et al. 1975). These authors nominated T. berytheum and T. salmoneum, particularly the latter species, to be the progenitor of T. alexandrinum. Since T. apertum is not known from Syria and is less able to cross with T. alexandrinum, compared to the other two species (Putiyevsky et al. (1975), it may be regarded as unlikely progenitor of Egyptian clover. The parsimony trees place T. berytheum, T. salmoneum, and T. apertum closest to the two Syrian accessions of T. alexandrinum (alex-99 and alex-94); however the average distance trees support only T. salmoneum closest to the Syrian and Egyptian (alex-57) accessions. These accessions are placed with the other six Egyptian accessions and form a major clade separate from accessions from other parts of the world. These results may therefore be taken to propose T. salmoneum as the most probable progenitor for Syrian material of Egyptian clover. However, the close relationship between the accession of T. salmoneum and the two accessions of T. berytheum, and the ability of these two species to cross freely, may indicate a contribution by material of this species from Syria to the genome of T. alexandrinum. Thus T. salmoneum and T. berytheum may be regarded as the ancestors from, which man developed Egyptian clover by artificial selection in Syria. In this regard, the domestication of the Egyptian clover may be analogous to other crops, such as barley and wheat that were domesticated in the Fertile Crescent and taken into cultivation in the Nile Valley. After domestication, the early forms of the crop may have been taken into rain-fed cultivation in Syria and Palestine, and later into irrigated cultivation in Egypt. It seems that genetic improvement of the crop has occurred in Egypt after cultivation, and that the varieties developed in Egypt were distributed worldwide. The distinction between the 123 Syrian and Egyptian accessions as one cluster, separate from the accessions from other parts of the world, may be due to changes that occurred following the introduction of the crop into North America and central Asia at the beginning of the 20th century. Acknowledgements We thank the Center for Bioinformatics and Functional Genomics at Miami University, Oxford, Ohio and technical advice of Director Chris Wood. We are also grateful to Professor David Francko, former Chair of the Botany Department at Miami University, for facilities and encouragement. AB acknowledges the financial support by Tanta University and the Fulbright Foundations in Washington and Cairo, and HH thanks Ain Shams University in Cairo and the International Office of Miami University for financing her visit to Miami University. References Aaronsohn A (1910) Agricultural explorations in Palestine. 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