Origin and ancestry of Egyptian clover (Trifolium alexandrinum L.) As revealed by AFLP markers

RESEARCH ARTICLE

Origin and ancestry of Egyptian clover (Trifoliumalexandrinum L.) As revealed by AFLP markers

Abdelfattah Badr Æ Hanaa H. El-Shazly ÆLinda E. Watson

Received: 3 October 2006 / Accepted: 10 January 2007 / Published online: 6 March 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 geo-

graphic 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. bery-

theum 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

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 begin-

ning 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

A. Badr (&)Botany Department, Faculty of Sciences, TantaUniversity, Tanta 31527, Egypte-mail: [email protected]

H. H. El-ShazlyDepartment of Biological Sciences and Geology,Faculty of Education, Ain Shams University, Cairo,Egypt

L. E. WatsonDepartment of Botany, Miami University, Oxford,OH 45056, USA

123

Genet Resour Crop Evol (2008) 55:21–31

DOI 10.1007/s10722-007-9210-0

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) men-

tioned 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 Leba-

non, 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 Ber-

seem. 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.

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 interpreta-

tion 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. alex-

andrinum, 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, espe-

cially 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,

22 Genet Resour Crop Evol (2008) 55:21–31

123

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 (Put-

iyevsky et al. 1975). These results also indicated

that the grouping of some species is contrary to

their subsectional classification proposed by Zoh-

ary (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 diver-

sity 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. alexandri-

num (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 glass-

house at Miami University, Oxford, Ohio, USA.

Leaves of actively growing seedlings were har-

vested on ice, frozen in liquid nitrogen, and stored

at –80�C 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 assign-

ment, 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 Accession ID number Other IDs Sourcea Origin

T. alexandrinum L. alex-02 PI 250659 K-608 SRPIS PakistanT. alexandrinum L. alex-03 PI 277510 Miscavi SRPIS IsraelT. alexandrinum L. alex-04 PI 250105 Berseem SRPIS EgyptT. alexandrinum L. alex-05 PI 383769 NL-1714 SRPIS TurkeyT. alexandrinum L. alex-06 PI 291550 Miscavi SRPIS TunisiaT. alexandrinum L. alex-07 PI 291549 Miscavi SRPIS MoroccoT. alexandrinum L. alex-08 PI 217543 13887 SRPIS PakistanT. alexandrinum L. alex-09 PI 251213 17154 SRPIS YugoslaviaT. alexandrinum L. alex-10 PI 445883 L-51 SRPIS PakistanT. alexandrinum L. alex-11 PI 445879 L-16 + 40 SRPIS PakistanT. alexandrinum L. alex-12 PI 378128 6007 SRPIS GreeceT. alexandrinum L. alex-13 PI 253582 K-2101 SRPIS Spain

Genet Resour Crop Evol (2008) 55:21–31 23

123

For DNA extraction, a modified CTAB meth-

od (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·

CTAB buffer containing 1% PVP, 1% Na-bisul-

phite, and 0.2% mercaptoethanol. The tubes were

incubated for 30 min in a 60�C water bath with

occasional gentle mixing of the tubes. Following

Table 1 continued

Species Accession ID number Other IDs Sourcea Origin

T. alexandrinum L. alex-14 PI 292967 SRPIS IraqT. alexandrinum L. alex-15 PI 445877 B-23 SRPIS PakistanT. alexandrinum L. alex-16 PI 164413 8363 SRPIS IndiaT. alexandrinum L. alex-17 PI 459105 SRPIS TunisiaT. alexandrinum L. alex-18 PI 445881 L- 64 + 13 SRPIS PakistanT. alexandrinum L. alex-19 PI 233811 SRPIS ItalyT. alexandrinum L. alex-20 PI 226284 52177 SRPIS KenyaT. alexandrinum L. alex-21 PI 291768 FAO-9329 SRPIS EgyptT. alexandrinum L. alex-22 Sero 1 Cultivar ARC EgyptT. alexandrinum L. alex-23 PI 517061 GR-7623 SRPIS MoroccoT. alexandrinum L. alex-24 PI 214205 SRPIS ItalyT. alexandrinum L. alex-28 Sakha 3 Cultivar ARC EgyptT. alexandrinum L. alex-29 Sakha 4 Cultivar ARC EgyptT. alexandrinum L. alex-34 PI 468402 ENMP-4428 SRPIS PortugalT. alexandrinum L. alex-51 PI 201954 Fahli SRPIS EgyptT. alexandrinum L. alex-57 Sakha 96 Cultivar ARC EgyptT. alexandrinum L. alex-94 IG 120953 IFTR 3679 ICARDA SyriaT. alexandrinum L. alex-99 IG 66553 IFTR 304 ICARDA SyriaT. apertum Bobr. aper-84 PI 516230 S 70-3 TaylorT. apertum Bobr. aper-102 PI 314117 SRPIS F S UnionT. berytheum Boiss. bery-59 PI 369019 NYT1667 SRPIS TurkeyT. berytheum Boiss. bery-121 PI 353412 S-153-5 Taylor TurkeyT. carmeli Boiss. carm-82 PI 353422 SRPIS IsraelT. carmeli Boiss. carm-158 TRIF 100/75 IPK IsraelT. clypeatum L. clyp-44 PI 292471 No.55-51 SRPIS IsraelT. clypeatum L. clyp-53 PI 241478 SRPIS IsraelT. clypeatum L. clyp-85 TRIF 129/96 IPKT. constantinopolitanum Ser. cons-52 PI 369028 45082 SRPIS JordanT. constantinopolitanum Ser. cons-130 IG 67731 IFTR 1382 ICARDA SyriaT. constantinopolitanum Ser. cons-134 IG 67739 IFTR 1490 ICARDA SyriaT. constantinopolitanum Ser. cons-156 IG 67543 IFTR 1294 ICARDA SyriaT. echinatum M. B. echn-60 PI 238159 G 2490 SRPIS TurkeyT. echinatum M. B. echn-66 PI 494720 T-41 SRPIS RomaniaT. echinatum M. B. echn-70 PI 419273 147 SRPIS GreeceT. echinatum M. B. echn-71 PI 238159 G 2490 SRPIS TurkeyT. echinatum M. B. echn-77 PI 238159 Mu-029 MU TurkeyT. latinum Seb. lat-192 Iowa StateT. latinum Seb. lat-218 NY 4724 NYBGT. meironense Zoh. meir-164 IG 69098 IFTR 2849 ICARDA AlgeriaT. plebeium Boiss. pleb-159 IG 67954 IFTR 1705 ICARDA SyriaT. plebeium Boiss. pleb-162 IG 67899 IFTR 1650 ICARDA SyriaT. plebeium Boiss. pleb-219 NY 5439 NYBGT. salmoneum Mout. salm-154 PI 179056 S-273-1 TaylorT. supinum Savi sup-81 TRIF 104/99 IPK Romania

a ARCE, Agricultural Research Center, Cairo, Egypt; ICARDA, International Center for Agricultural Research in DryAreas, Aleppo, Syria; Iowa State, Iowa State University Herbarium, Ames, Iowa, USA; IPK, Institut fur Pflanzengenetikund Kulturpflanzenforschung, Gatersleben, Germany; NYBG, New York Botanic Gardens, New York, USA; MU, MiamiUniversity, Turrell Herbarium, Oxford, Ohio, USA; SRPIS, Southern Regional Plant Introduction Station, USDA; Taylor,Dr. Norman Taylor, University of Kentucky, Lexington, Kentucky, USA


123

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 –20�C 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 sus-

pended in 0.2 ml TE buffer, and 1 ll RNase was

added and incubated at 37�C for 30 min. DNA

quantity in the TE buffer was estimated spectro-

photometrically, 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 dou-

ble-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

37�C. The restriction/ligation (R+L) product was

diluted to 200 ll and stored at 4�C for pre-

amplification, or stored at –20�C 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 thermocy-

cler for 25 cycles at 94�C denaturation (20 s), 56�C

annealing (30 s), and 72�C extension (2 min), with

initial hold at 72�C and a final old at 60�C for

30 min. The pre-selective amplification product

was diluted 15X in 0.1 TE buffer and stored at 4�C

for amplification, or stored at –20�C 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). Ampli-

fication was conducted in a total volume of 15 ll

for 9 cycles at 94�C (2 min), 56�C (30 s), and 72�C

(2 min), reducing the annealing temperature by

one degree per cycle, followed by 21 cycles at

94�C (2 min), 56�C (30 s) and 72�C (2 min), and a

hold at 60�C for 30 min. Of the amplified product,

2 ll were mixed with 20 ll of deionized formam-

ide and 0.5 ll of GeneScan 500 ROX internal size

standard in a 0.5-ml tube, denatured at 95�C 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 NTSYS-

pc 2.1 (Rohlf 1993). In addition, average distance

UPGMA and Neighbor joining trees were pro-

duced 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 addi-

tions. 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, alex-

22, 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

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. constanti-

nopolitanum (cons-134, cons-256). The third clus-

ter contains two subgroups: one comprised of

T. echinatum, T. meironense, and two accessions

of T. constantinopolitanum (cons-52 & cons-

130); 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 com-

prised of seven Egyptian and two Syrian acces-

sions, 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.

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 acces-

sions 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

Fig. 1 AFLP bandingprofile for nine accessionsof Trifoliumalexandrinum (1–9),T. salmoneum (10),T. apertum (11–12),and T. berytheum (13–14).DNA was digested withEcoRI and MseI, andfragments were amplifiedusing PCR in the presenceof the MseI adapter CAC,and the two EcoRIadapters (ACA (a) leftand AAG (b) respectively


123

(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 (Putiye-

vsky and Katznelson 1973; Katznelson and Put-

iyevsky 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 agree-

ment 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 spe-

cies. A close relationship for T. alexandrinum,

Fig. 2 UPGMA Dicecoefficient distance tree,based on AFLP data


123

T. berytheum, and T. apertum was also supported

by molecular phylogenies based on nuclear ribo-

somal ITS and chloroplast trnL nucleotide

sequences (Ellison et al. 2006; Badr et al. unpub-

lished 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 Putiye-

vsky et al. (1975) on T. alexandrinum and other

species of subsection Alexandrina, including cross-

ability, meiotic behavior of chromosomes, and

pollen fertility of hybrids, indicated that T. vavil-

ovii is distant to T. alexandrinum, T. meironense,

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

alex02alex03alex05alex06alex07alex17alex08alex10alex15alex11alex18alex16alex09alex14alex12alex13alex19alex24alex20alex23alex34alex04alex29alex21alex28alex22alex57alex94alex99salm154alex51aper84aper102bery59bery121clyp44clyp85clyp53pleb159pleb162pleb219lat192lat218meir164cons134cons156echn71echn60echn66echn70echn77cons52cons130carm82carm158supi81

Fig. 3 UPGMA averagedistance tree, based onAFLP data


123

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. al-

exandrinum. This idea was supported by Oppen-

heimer (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.

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). Trifo-

lium 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

alex02alex34alex03alex05alex06alex07alex17alex12alex13alex08alex10alex09alex11alex14alex16alex15alex18alex19alex20alex23alex24alex04alex21alex28alex29alex51alex22alex57alex94aper84aper102bery59bery121salm154alex99clyp44clyp85clyp53pleb159pleb162pleb219cons134cons156echn60echn66echn70echn71echn77carm82carm158supi81lat192lat218meir164cons52cons130

53

90

6963

97

77

73

89

86

56

9391

56100

86

99100

95

73

66

99

Fig. 4 Strict consensustree of 2149 equally mostparsimonious trees basedon AFLP data. Bootstrapvalues are abovebranches, Cl = 0.178,RI = 0.678, andRC = 0.121


123

evidence presented by Putiyevsky et al. (1975)

clearly indicated that T. salmoneum is the prob-

able 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. meiron-

ense from T. berytheum, T. salmoneum, and

T. alexandrinum (Putiyevsky et al. 1975). These

authors nominated T. berytheum and T. salmone-

um, 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 acces-

sions 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 distrib-

uted worldwide. The distinction between the

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 forBioinformatics and Functional Genomics at MiamiUniversity, Oxford, Ohio and technical advice ofDirector Chris Wood. We are also grateful to ProfessorDavid Francko, former Chair of the Botany Department atMiami University, for facilities and encouragement. ABacknowledges the financial support by Tanta Universityand the Fulbright Foundations in Washington and Cairo,and HH thanks Ain Shams University in Cairo and theInternational Office of Miami University for financing hervisit to Miami University.

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Documents

Origin and ancestry of Egyptian clover (Trifolium alexandrinum L.) As revealed by AFLP markers