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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: Abdelfattahbadr@yahoo.com
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
24 Genet Resour Crop Evol (2008) 55:21–31
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
Genet Resour Crop Evol (2008) 55:21–31 25
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
26 Genet Resour Crop Evol (2008) 55:21–31
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
Genet Resour Crop Evol (2008) 55:21–31 27
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
28 Genet Resour Crop Evol (2008) 55:21–31
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
Genet Resour Crop Evol (2008) 55:21–31 29
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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