chloroplast dna non-coding sequences variation in aegilops tauschii coss.: evolutionary history of...
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Genetic Resources and CropEvolutionAn International Journal ISSN 0925-9864Volume 59Number 5 Genet Resour Crop Evol (2012)59:683-699DOI 10.1007/s10722-011-9711-8
Chloroplast DNA non-coding sequencesvariation in Aegilops tauschii Coss.:evolutionary history of the species
Alexander Ju. Dudnikov
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RESEARCH ARTICLE
Chloroplast DNA non-coding sequences variationin Aegilops tauschii Coss.: evolutionary history of the species
Alexander Ju. Dudnikov
Received: 17 February 2011 / Accepted: 23 May 2011 / Published online: 18 June 2011
� Springer Science+Business Media B.V. 2011
Abstract Sequences of four chloroplast DNA non-
coding regions, about 3,000 bp in total, were ana-
lysed in 112 Aegilops tauschii accessions, 56 of ssp.
tauschii and 56 of ssp. strangulata, representing all of
the species range. One inversion, 8 insertions/dele-
tions, 18 base pair substitutions and 5 microsatellite
loci were found. The data revealed that Ae. tauschii
originated in Caucasia. Neither of the two
Ae. tauschii subspecies was an ancestor to one
another. Aegilops tauschii divided into ssp. tauschii
and ssp. strangulata at the very beginning of its
existence as a species. Subspecies tauschii was the
first to start geographic expansion and relatively
rapidly occupied a vast area from Caucasia—east-
ward up to central Tien Shan and western Himalayas.
In contrast to ssp. tauschii, geographic spread of ssp.
strangulata was a complicated, multi-stage and slow
process. At the beginning of ssp. strangulata evolu-
tionary history its major phylogenetic lineage for a
lengthy time span had existed as a small isolated
population. Several forms of ssp. strangulata, better
adapted to relatively moister and cooler habitats, had
originated. Each of these forms has gradually forced
out ssp. tauschii from some part of its area in the
west, up to central Kopet-Dag.
Keywords Aegilops tauschii � Chloroplast DNA �Evolutionary history � Intraspecies phylogeny
Introduction
Aegilops tauschii Coss. (syn. Aegilops squarrosa
auct. non L.) is a diploid (2n = 14, genome DD)
goat-grass species which donated its genome D to
common wheat, Triticum aestivum L. It is considered
as the most important prospective donor of agricul-
turally important genes for the improvement of
common wheat (Kimber and Feldman 1987; Kilian
et al. 2011). Aegilops tauschii is a self-pollinating
species with cross-pollination occasionally taking
place (Dudnikov 1998, 2009). According to Zhukov-
sky (1928), the origin of the species took place in the
Eastern Mediterranean at the end of Tertiary period.
Hammer (1980) considers Caucasia as a place of
Ae. tauschii origin. Aegilops tauschii occupies a vast
range in central Eurasia, from Turkey to Kirgizia (van
Slageren 1994; Kilian et al. 2011). Its primary
habitats are patches of dwarf-shrub steppe-like for-
mations in hilly or mountainous regions. Such
shrubby slopes are practically useless for any kind
of agriculture and present examples of non-disturbed
environments. Aegilops tauschii is presented by two
subspecies, Ae. tauschii Coss. ssp. tauschii and
Ae. tauschii Coss. ssp. strangulata (Eig) Tzvelev
A. Ju. Dudnikov (&)
Institute of Cytology and Genetics, 630090 Novosibirsk,
Russia
e-mail: [email protected]
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Genet Resour Crop Evol (2012) 59:683–699
DOI 10.1007/s10722-011-9711-8
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(Eig 1929), which considerably and distinctly differs
genetically (Dudnikov 1998, 2000; Dudnikov and
Kawahara 2006). Genetic differentiation between ssp.
tauschii and ssp. strangulata was pointed out to be of
interspecies level (Jaaska 1980, 1981), and it is
completely in line with morphological and ecological
variation. Subspecies strangulata ‘‘prefers’’ more
moist and higher located habitats. It is relatively
much more abundant in comparison with ssp. tauschii
in south and south-western Precaspian region and was
not found to the east of central Kopet-Dag in
Turkmenistan (Dudnikov 1998, 2000; Dudnikov and
Kawahara 2006). In Afghanistan, Pakistan, Uzbeki-
stan, Kirghizia, Kazakhstan, Tajikistan and northern
India Ae. tauschii is represented only by ssp. tauschii
(Jaaska 1980, 1981; Dudnikov 2000). Numerous
experimental crosses between ssp. tauschii and ssp.
strangulata revealed not a trace of fertility loss in
‘‘hybrids’’ (Dudnikov 2009). Genetic exchange
between the subspecies in nature was found to be
rather common but with no evolutionary conse-
quences for the species: intermediate genotypes being
eliminated by natural selection as disadvantageous
(Dudnikov 1998). Aegilops tauschii is presented by
numerous, small, fairly well isolated local popula-
tions ‘‘scattered’’ throughout the area. The level of
genetic differentiation among local populations of
Ae. tauschii is very high: the value of Nei’s coefficient
of genetic differentiation, GST, makes up 0.67 for ssp.
tauschii and 0.64 for ssp. strangulata, indicating low
migration capacity of the species (Dudnikov 1998).
Aegilops tauschii is of great interest (1) as a model
object for the studies of genetic basis of adaptive
speciation and (2) as an important natural resource
for improvement of cultivated wheats. Investigation
of chloroplast DNA (cpDNA) non-coding sequences
in Ae. tauschii could help to reveal peculiarities of its
intraspecies divergence and geographic expansion.
Chloroplast microsatellites DNA variation was used
for intraspecies studies of Ae. tauschii by Matsuoka
et al. (2005, 2008, 2009). Although some geographic
patterns in cpDNA variation were identified, the
chosen set of cpDNA sites failed to reflect
Ae. tauschii intraspecies divergence (Matsuoka
et al. 2008).
A study of intra- and interspecies phylogenetic
relationships among diploid Triticum-Aegilops spe-
cies was carried out with the help of non-coding
cpDNA sequences (Yamane and Kawahara 2005).
Among the other, ten accessions of Ae. tauschii were
analysed and this species was found to be the most
polymorphic. Obviously the four cpDNA non-coding
regions used in the study could be an effective tool
for the investigation of Ae. tauschii evolutionary
history if only to increase tenfold the number of
accessions analysed, which has been done in my
study and the results are presented below.
Materials and methods
Plant materials
Since Ae. tauschii genetic variation is of particular
economic importance, the seeds of this species were
collected by many expeditions through all of the
species range. This seed material is being preserved
and regenerated in the world gene-banks and could be
obtained by request.
112 Ae. tauschii accessions were chosen for the
study: 56 of ssp. tauschii and 56 of ssp. strangulata,
representing well each subspecies range (Table 1).
The sources of the plant material are as follows: (1)
N.I. Vavilov All-Russian Institute of Plant Industry
(VIR), St.-Petersburg (‘‘k’’); (2) Kyoto University
(‘‘KU’’); (3) IPK Gatersleben (‘‘AE’’) and (4) the
collection of Dudnikov (1998) (‘‘t’’). The data for
seven of these accessions was taken from the DDBJ/
EMBL/GenBank database (http://www.ncbi.nlm.nih.
gov/, accession numbers GBAN-AB207274 to
GBAN-AB207765) provided by Yamane and Kawa-
hara (2005) (Cited below in the text as ‘‘DDBJ’’).
One plant from each of the remaining 105 accessions
was grown and cpDNA analysis was done.
Subspecies attribution of each accession was
carried out according to Dudnikov (1998, 2000).
Considerable genetic difference between the subspe-
cies is key to distinguish them. The researcher has to
determine genotypes at a set of different loci in a set
of plants, representing accessions the researcher
studying. Multivariate analysis of this genetic varia-
tion will distinctly divide Ae. tauschii accessions into
ssp. tauschii and ssp. strangulata. We used a set of 11
polymorphic enzyme-encoding loci for this purpose
(Dudnikov 1998, 2000; Dudnikov and Kawahara
2006). Gliadin proteins (Khakimova 2010), AFLP
(Saeidi et al. 2008; Mizuno et al. 2010) and SSR
genetic markers (Pestsova et al. 2000; Takumi et al.
684 Genet Resour Crop Evol (2012) 59:683–699
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Table 1 Ae. tauschii accessions
Accession Subspeciesa Countryb Localityc Longitude
(decimal)
Latitude
(decimal)
t 1 t Russia Dagestan, 3 km from Gedzhuh v. to Ersy v., 120 m 48.07 42.09
t 2 s Dagestan, 4 km from Ersy to Gedzhuh, 200 m 48.04 42.07
t 6s s Dagestan, vicinity of Rukel v., 380 m 48.24 41.98
t 9(1)s s Dagestan, eastern slope of hill ‘‘336’’, 250 m 48.29 42.00
k 362 t Dagestan, vicinity of Novolakskoye v. 46.49 43.11
k 1025 s Dagestan, Derbent—Kuba road, 19 m 48.31 42.00
k 1770 s Dagestan, vicinity of Kasumkent v., 630 m 48.11 41.67
AE 725 t N Ossetia, 10 km S of Ordzhonikidze 44.65 42.87
k 608 t Georgia Marneuli d., Marneuli—Tbilisi road, 530 m 44.77 41.55
k 612 s Gori d., vicinity of Rene v., 750 m 44.09 41.91
k 1216 t Aspindza d., 1,000 m 43.25 41.58
k 1792 s Ambrolauri d. 43.14 42.51
AE 929 s Mccheta, Dzvari, S slope, 550 m 44.68 41.84
AE 1037 s 20 km NW Citeli-Ckaro, 1,000 m 45.89 41.62
KU 2139 t Turkey 76.7 km NNE from Van to Erics, 1,780 m 43.62 38.98
t 18 t Armenia 50 km from Sisian to Azizbekov, turn to Dzhermuk, 1,400 m 45.56 39.69
t 34 s 5 km from Kafan to Goris, 1,000 m 46.31 39.22
t 39 s At SE entrance to Goris, 1,300 m 46.35 39.48
k 1185 s Abovyan d., vicinity of Gehard, 1,660 m 44.78 40.15
AE 476 t Erebuni 44.65 40.19
KU 2824 t 1 km S of Bjurakan 44.26 40.32
t 13 s Azerbaijan 1.5 km from Maraza v. to Hilmilli v., 1,000 m 48.88 40.64
t 15 s Vicinity of Tirdjan v. (the road to Ismailli), 760 m 48.34 40.76
t 26 s At entrance to Dzhebrail (from Fizuli), 580 m 47.03 39.41
k 106 s Pushkino d., Bellusvar frontier post, 130 m 48.38 39.38
k 108 s Massali d., Muskyudzha v., Chapayev collective farm, 100 m 48.65 39.00
k 109 s Astrahanbazar d., Novogolovnya v., 90 m 48.57 39.21
k 124 s Shemaha d., collective farm No 3, 630 m 48.64 40.63
k 141 s Near Kutkashen, 600 m 47.83 40.95
k 169 s Zakatali experiment station, 570 m 46.65 41.64
k 296 s Vartashen d., Yakubli v., 600 m 47.45 40.91
k 336 s Lachin d., Karnkaha v., 1,300 m 46.54 39.66
k 493 s Stepanokert d., vicinity of Chanahchi v., 1,100 m 46.83 39.70
k 616 t SE of Shamhor 46.07 40.80
k 1038 s Kuba d., Velvelichay river valley, 800 m 48.62 41.31
k 1099 s 10 km from Lerik to Lenkoran 48.50 38.80
k 1552 s Dzhalilabad d., Lyaken frontier post, 410 m 48.30 39.11
k 1782 t Kusari d., Hudat v. 48.42 41.46
k 1866 s Zangelan d., Ordakni v., 800 m 46.61 39.04
AE 224 s Divichi d., Bilidzhi v. 48.86 41.19
k 1954 t Iran (c) 26 km from Shirat, 1,300 m 47.20 37.91
k 1956s s Near Maku, 850 m 44.83 39.29
k 1960 s Baijan, 1,100 m 52.29 35.97
k 1961 s Tage-Boston, 1,400 m 47.07 34.28
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Table 1 continued
Accession Subspeciesa Countryb Localityc Longitude
(decimal)
Latitude
(decimal)
KU 20-7 s 2 km N of Karaj (Suburbs of Teheran) 51.00 35.82
KU 2071 t 2 km N of Karaj (Suburbs of Teheran) 51.00 35.82
KU 20-8 s 25 km WSW of Firuzkuh 52.53 35.65
KU 2112 s 19 km SW of Ardabil (Ardabil—Surab) 48.14 38.14
KU 2113 t Suburbs of Mahabad 45.73 36.86
KU 2116 t 32 km S of Khoy (Rezaiyeh—Khoy) 44.83 38.32
KU 2118 s Khoy 44.96 38.55
KU 2121 t 42 km NW of Tabriz (Khoy—Tabriz) 45.91 38.33
KU 2123 s Tabriz 46.29 38.08
KU 2142 t 16.4 km E from Maku to Khoy, 1,200 m 44.67 39.27
KU 2145 t 71.2 km SE from Maku to Khoy, 1,440 m 45.01 38.96
KU 2150 t 44.9 km N from Marand to Jolfa, 1,360 m 45.60 38.76
KU 2151 t 22.8 km WNW from Mianeh to Tabriz, 1,340 m 47.48 37.36
KU 2154 t 68.7 km SW from Qazvin to Hamadan, 1,500 m 49.45 38.85
KU 2156 s 7.3 km SW Awej to Hamadan, 2,160 m 49.15 35.55
KU 2157 t 17.6 km SE from Karand to Shahabad, 1,480 m 46.41 34.16
KU 20-10 s Iran (wp) 9 km NW of Ramsar (Chalus—Resht) 50.55 36.96
KU 2096 s 51 km W of Babulsar (Babulsar—Chalus) 52.10 36.57
KU 2102 s 52 km NW of Ramsar (Chalus—Resht) 50.17 37.18
KU 2103 s 13 km SSE of Resht (Chalus—Resht) 49.73 37.27
KU 2105 s 12 km NW of Pahlavi (Pahlavi—Astara) 49.34 37.50
KU 2107 s 12 km of Hashtpar (Pahlavi—Astara) 48.90 37.89
KU 2109 t Astara (Pahlavi—Astara) 48.86 38.41
KU 2110 s 32 km SW of Astara (Astara—Ardabil) 48.55 38.40
KU 2126 s Techalousse (near Chalus) 51.42 36.64
KU 2159 s Ramsar 50.62 36.91
k 1959 s Iran (ep) Sari-Mazandaran, 10 m 53.04 36.57
KU 20-9 s 5 km W of Behshahr (Sari—Behshahr) 53.46 36.66
KU 2075 s 15 km E of Behshahr (Behshahr—Gorgan) 53.70 36.70
KU 2076 s 8 km W of Gorgan 54.35 36.82
KU 2080 s Gharaghaj near Shahpesend (Gorgan—Hoshyeylak) 55.18 37.09
KU 2081 s NE of Koshyailagh (Gorgan—Koshyailagh) 55.45 36.94
KU 2083 s Koshyailagh 55.35 36.82
KU 2087 t 15 km NE of Sari (Sari—Behshahr) 53.19 36.48
k 426 t Turkmenistan Kara-Kala d., Bahchi, 540 m 56.62 38.41
k 427 t Kizil-Atrek d., Meshhet-Missarian plateau 54.78 37.64
k 428 t Kara-Kala d., the road from Yari-Kala v. to Shermop, 320 m 56.32 38.38
k 429 t Kara-Kala d., W Kopet-Dag, Kara-Su gorge, 720 m 56.75 38.42
k 433 s Central Kopet-Dag, Baharden d., Nuhur, Koyne-Gumberg v.,
1,200 m
57.03 38.47
k 1345 t Chardzhou region, Charshanga d., K. Marks collective farm,
1,600 m
66.67 37.96
k 1560 t Ashkhabad d., Firyuza gorge 58.09 37.92
k 1561 t Bolshoy Balhan mountain ridge 54.40 39.58
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2008) gave the same positive result. Apparently,
RFLP (Lubbers et al. 1991) and many other types of
genes or genetic markers could be successfully used
for this purpose. Subspecies attribution determination
of a single plant could be done morphologically (Eig
1929): ssp. tauschii has a cylindrical spike with low
SI index (which is a ratio between spikelet glume
width and rachis segment width of the spikelet
from the middle of the spike; Dudnikov 1998, 2000)
and ssp. strangulata has a moniliform appearance of
the spike (high SI). SI has a bimodal distribution in
Ae. tauschii, reflecting its differentiation into the two
subspecies, and vary from 0.9 to 1.9 (Dudnikov 2000;
Dudnikov and Kawahara 2006). Morphological sub-
species distinguishing will not be reliable if the plant
has SI value about 1.3 which is an approximate
dividing line between the subspecies. For such cases
a very reliable criteria based on allelic variation of
electrophoretically ‘‘fast’’ acid phosphatase encoded
by Acph1 gene (Dudnikov 2003a, 2007; Kirby et al.
2005) could be used: its ‘‘fast’’ allele, Acph1100, is
fixed in ssp. tauschii whereas Acph195 is fixed in ssp.
Table 1 continued
Accession Subspeciesa Countryb Localityc Longitude
(decimal)
Latitude
(decimal)
k 1563 t Maliy Balhan mountain ridge, the river-head of Chal-Su spring 55.02 39.31
k 1903 t Kara-Kala d., Keshi-Yol mountain 56.50 38.45
AE 213 s Syunt-Hasardag mountain ridge, Mezetli mountains, 1,400 m 56.58 38.54
k 963 t Afghanistan Kabul, the road Kabul—Lonchar, 1,850 m 69.14 34.33
k 964 t Kabul—Kunduz road, S slope of Gindikush, before Salang pass,
1,850 m
69.22 35.17
k 967 t Kabul—Kunduz road, N of Pulihumri, 640 m 68.67 36.06
k 982 t From Akkupruk upstream along Balh river, 760 m 66.84 36.08
k 989 t Badgis d., 640 m 63.70 35.76
k 995 t Gerat—Kushka road, 1,390 m 62.13 34.58
k 998 t Doshi—Bamian road, 940 m 68.44 35.57
KU 20-6 t Pakistan 8 km SW of Quetta 66.97 30.10
KU 2002 t Suburbs of Quetta 67.00 30.21
KU 2009 t 2 km E of Chaman 66.42 30.91
k 912 t India Kashmir, near Srinagar 74.86 34.07
k 394 t Uzbekistan Sir-Darya region, Zaamin d., the place Uyulma 68.39 39.94
k 421 t Tashkent region, VIR experiment station 69.25 41.33
k 1346 t Kahka-Darya region, 370 m 65.92 38.47
k 1352 t Surhan-Darya region, Baysun, 1,250 m 67.19 38.21
k 1356 t Surhan-Darya region, Sherabad d., 750 m 66.86 37.74
k 1805 t Kahka-Darya region, Karshi d. 65.79 38.85
AE 955 t Tajikistan S Tajikistan, Gandzhina low mountain region, Gazimajlik
valley, dry slope, 900 m
68.57 37.96
k 677 t Kirgizstan Osh region, Karasu d., Kenegi collective farm, department No 4
Bogara
72.89 40.70
k 1261 t Osh region, 39 km from Arslanbob, 890 m 72.83 41.11
k 1322 t Kazakhstan Chimkent region, Turkestan d., 5 km W of Kentau 68.44 43.50
k 1336 t Dzhambul region, Lugovoy d., 8 km from Angabas, 600 m 72.39 42.97
k 1611 t Alma-Ata region, Talgar d., 15 km from Novo-Alekseevka v.,
800 m
77.22 43.41
a ‘‘t’’—ssp. tauschii; ‘‘s’’—ssp. strangulatab ‘‘(c)’’—continental; ‘‘(wp)’’—western precaspian; ‘‘(ep)’’—eastern precaspianc ‘‘d.’’—district; ‘‘v’’ village
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strangulata (Dudnikov 1998, 2000; Dudnikov and
Kawahara 2006).
DNA extraction; PCR amplification
and sequencing
Genomic DNA was extracted from young leaves of
individual plants according to Bender et al. (1983).
PCR amplification and sequencing was carried out as
in Yamane and Kawahara (2005). Four chloroplast
fragments were amplified: the trnC-rpoB intergenic
region, the trnF-ndhJ intergenic region, the ndhF-
rpl32 intergenic region, and the atpI-atpH intergenic
region. PCR was carried out with ExTaq polymerase
(Takara, Japan) using primers based on the chloro-
plast sequence of wheat (GenBank accession no.
AB042240). Five pairs of primers (‘‘A’’-‘‘E’’) were
used for PCR amplification and for sequencing (an
additional fifth pair ‘‘E’’ was necessary to achieve
complete sequencing of the long intergenic region
trnC-rpoB) (Table 2). PCR conditions were as fol-
lows: 25 cycles of 45 s at 96�C for denaturation,
1 min at 56�C for annealing, and 1 min at 72�C for
polymerization; final extension of 3 min at 72�C.
PCR fragments were washed at 70% ethanol. PCR-
products were sequenced using BigDye Terminators
version 3.0 at ABI PRISM equipment at the sequenc-
ing centre ICG and IChBFM SD RAS, Novosibirsk.
Data analyses
Median network was used for the data analysis (Bandelt
et al. 1995; Network software version 4.5.1.6 available
at http://www.fluxus-engineering.com).
The major results were obtained through the
analysis of the raw data table presenting haplotype
of each of the 112 Ae. tauschii accessions studied.
The huge table of 112 rows presenting accessions and
of 32 columns presenting cpDNA mutations, was
rearranged manually with the help of Microsoft
Access in a way that (1) made it compact and (2)
revealing the consequence of mutations in the course
of Ae. tauschii evolutionary history. The more
frequent the occurrence of the mutation has been,
the more to the left its column was placed; and all the
rows with the accessions having this mutation were
arranged together. As a result, a compact table
presenting genetic constitution of each accession at
each of 27 cpDNA sites with inversion, insertion/
deletion and base pair substitution was made (with
some of the microsatellites being reflected also).
Going through the table from the left to the right, one
can find the haplotype of each accession. The
consequence of mutations in evolution is also
reflected in the table, since a ‘‘descendant’’ mutation
is expected to be included exclusively and entirely as
a subset in a set of accessions with the ‘‘ancestor’’
mutation (Table 4).
Results
All mutations found through the study are presented
in Table 3. 32 polymorphic loci were pointed out:
one inversion, 8 insertions/deletions, 18 base pair
substitutions and 5 microsatellites. The first two
letters in a locus designation display an intergenic
region where the locus was found. The letter in
brackets, ‘‘i’’, ‘‘d’’, ‘‘s’’, ‘‘m’’, shows the type of
Table 2 The primers used
(Yamane and Kawahara
2005)
Region Pair Primer
trnC-rpoB A trnC/22F (50-TGGGGATAAAGGATTTGCAG)
rpoB/16R (50-ATTGTGGACATTCCCTCGTT)
E IGR (trnC-rpoB) 517F (50-CCTACCCAAGTGAAATTACG)
IGR (trnC-rpoB) 717R (50-GAGGGTTCTTTTTTATTTCGG)
trnF-ndhJ B trnF/1F (50-GTCAGGATAGCTCAG TTGGT)
ndhJ/61R (50-TGCCTGAAAGTTGGATAGGC)
ndhF-rpl32 C ndhF/139F (50-GACGAAGATTTTTTGTTGCTG)
IGR (50-ndhF-rpl32) 643R (50-TAT GGTATGGAAGCCTATCC)
atpI-atpH D atpI/643F (50-CCGGTCATGTTTCTTGG ATT)
atpH/18R (50-CAATAACAGAAGCAGCAGCA)
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Table 3 Mutations found in the four intergenic regions of cpDNA among 112 accessions of Aegilops tauschii
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mutation: inversion, insertion/deletion, base pair
substitution and microsatellite, respectively. Next,
the number is the locus position, calculated starting
from the first nucleotide in the cpDNA sequence
studied, as it is presented in ‘‘DDBJ’’. An allelic
variant is shown as a small letter. The alleles which
occur more frequently among Ae tauschii accessions
are designated as ‘‘a’’ in all the loci (Table 3).
Reduced median network based on inversions,
insertions/deletions and base pair substitutions (II/
D&S), 27 in total, was constructed for the 112
Ae. tauschii accessions (Fig. 1). It can be seen that in
the case of Ae. tauschii (the species, presented by
numerous small, fairly well isolated populations),
such a marker genetic system as non-coding cpDNA
sequences, with low mutation rates and no recombi-
nation, fail to separate subspecies tauschii and
strangulata as completely as enzyme-encoding genes
allelic variation (Jaaska 1981; Dudnikov 1998).
Nevertheless, the separation of the subspecies with
cpDNA looks rather satisfactory: ssp. tauschii and
ssp. strangulata are situated, respectively, below and
above the diagonal, coming from the upper left to the
lower right corners of Fig. 1. Also it can be seen that
mutation at the locus Tc(s)384 (displayed as broad
lines on Fig. 1) occurring sporadically several times
throughout the network, causing considerable com-
plications but do not reveal any genealogy (Fig. 1).
Figure 2 presents the same network but with Ae.
searsii Feldman et Kislev ex Hammer added as an out-
group. It reveals that Ae. tauschii ‘‘AE725’’ and
‘‘AE929’’ gaplogroups are the most similar to the
cpDNA gaplogroups of the ancestor species. Compar-
ative analysis of ‘‘DDBJ’’ and Ae. tauschii cpDNA data
(Table 4) explains the position of the out-group among
haplogroups of Ae. tauschii in Fig. 2. All the diploid
Aegilops and Triticum species presented in ‘‘DDBJ’’
have exclusively the alleles ‘‘b’’ at Tc(s)380 and
Tc(s)472 loci. In Ae. tauschii the alleles Tc(s)380b and
Tc(s)472b are very rare and were only found in AE929
and t9(1)s accessions (Table 4). At all the other
cpDNA loci studied, the alleles ‘‘a’’ (i.e. the most
common in Ae. tauschii) are the most common among
the diploid Aegilops and Triticum species presented in
‘‘DDBJ’’, and ‘‘AE725’’ gaplogroup of Ae. tauschii has
the alleles ‘‘a’’ at all cpDNA loci studied (Table 4).
The most important results of this study were
obtained through the analysis of the original raw data
table. As a rule, original genetic data can’t be directly
used for phylogenetic reconstructions ‘‘as they are’’:
they fail to form a ‘‘tree’’; a ‘‘net’’ is formed instead
due to homoplasy. Therefore statistical approaches
such as distance methods, maximum parsimony,
maximum likelihood or median networks are neces-
sary (Bandelt et al. 1995; Hedrick 2005). Statistical
program forms thousands of ‘‘trees’’ and choose one.
Still is doubtful, whether the statistically chosen
Fig. 1 Reduced median network for the chloroplast DNA
haplogroups of Ae. tauschii constructed using biallelic
polymorphisms. The network based on inversion ? inser-
tions/deletions ? base pair substitutions (microsatellites were
not used for the analysis). The circles represent haplogroups
with diameters proportional to the number of accessions.
Subspecies tauschii is presented in white color; ssp. strangu-lata is presented in grey color. The name of one representative
of the haplogroup is given by the circle (the full list of the
accessions from each haplogroup can be found in Table 3).
Bold lines between the circles display mutation at Tc(s)384locus
690 Genet Resour Crop Evol (2012) 59:683–699
123
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variant of a tree actually reflects the real evolutionary
process (Bandelt et al. 1995).
Ae. tauschii a real phylogenetic tree, directly
reflecting the raw genetic data is desirable. Such a
single possible and consistent tree could be con-
structed only if the following conditions are met: (1)
each mutation used for the study is unique, it
originated only once; and (2) there is no recombina-
tion. The data obtained through this study do not
differ considerably from this ideal case and was used
for direct construction of the real phylogenetic tree.
Although some artefacts are inevitable, there were
only a few of them. In contrast to statistical methods
of tree construction, we are able to comment on them
all in ‘‘Discussion’’ section.
Table 4 is arranged in a form which enables to carry
out manual construction of Ae. tauschii genealogical
tree directly reflecting cpDNA variation raw data. The
construction of the tree is based on the following
principle: as time passes, the mutations originate and
form new haplotypes; and the ancestor haplotype
entirely contains the descendant haplotype as a subset.
The procedure of the phylogeny tree construction is as
follows: Microsoft Office Access helps to rearrange the
data of the original data table to the form revealing the
consecutive mutations which is presented then in a
form of a tree with real phylogenetic lineages
(‘‘branches’’) being marked with genetic markers.
Using this basic principle, the huge original data
table was transformed to a very compact form
(Table 4): 28 from 32 polymorphic loci were per-
fectly arranged as a set of consecutive mutations
which have originated in the course of Ae. tauschii
evolutionary history. Variations at only four loci was
unsuitable for phylogenetic reconstruction, among
them there were the three microsatellites, Nf(m)300,
Ai(m)340 and Tc(m)357 (Table 3); which is not
surprising: homoplasy of chloroplast microsatellite
alleles is known to be rather common (Provan et al.
2001), therefore these loci are often more misleading
then helpful in phylogenetic analysis (Yamane and
Kawahara 2005) and worthwhile to be omitted from
consideration. Nevertheless, the two other microsat-
ellite loci, Tc(m)310 and Tc(m)917, and the allele
Tc(m)357c of the locus Tc(m)357 were incorporated
well into the tree construction (Tables 3, 4; Fig. 3). In
contrast to microsatellites—inversions, insertions/
deletions and base pair substitutions (II/D&S) were
expected to be useful and reliable in the study, which
was evident, but with one surprising exception which
is Tc(s)384 locus displaying uninterpretable variation
(Table 4). It is even more striking since Tc(s)384 is
completely monomorphic among other diploid Trit-
icum and Aegilops species presented in ‘‘DDBJ’’.
Sporadical allele variation at Tc(s)384 locus among
Ae. tauschii accessions (Table 4) explains the above
mentioned negative role of this locus in the reduced
median network construction (Fig. 1).
The following results come from the original raw
data presented in Table 4. But it is much easier to
follow them looking at Fig. 3, which is a graphical
scheme representing Table 4. (Table 4 is entirely
presented in Fig. 3, only the few artifacts (underlined
in Table 4) are not shown and will be specifically
discussed later.) Consecutive mutations in cpDNA
which finally led to modern haplotypes in Ae. tauschii
Fig. 2 Reduced median network for the chloroplast DNA
haplogroups of Ae. tauschii and Ae. searsii. Ae. tauschii ssp.
tauschii, Ae. tauschii ssp. strangulata and Ae. searsii are
presented in white, grey and black colors, respectively. (The
data for Ae. searsii were taken from the database of Yamane
and Kawahara 2005)
Genet Resour Crop Evol (2012) 59:683–699 691
123
Author's personal copy
Ta
ble
4P
oly
mo
rph
ism
of
chlo
rop
last
DN
An
on
-co
din
gse
qu
ence
sam
on
g1
12
acce
ssio
ns
of
Aeg
ilo
ps
tau
sch
ii
Tf(
s)22
5a
Tc(
s)38
0a,
Tc(
m)3
10a,
N
f(d)
199a
, T
f(d)
114a
, T
c(s)
390a
A
E72
5NO
, t18
AR
, KU
2150
CI,
k96
3AF
*, k
1336
KZ
, k16
11K
Z*
Tc(
s)38
0b
Tc(
s)47
2a
Tc(
s)47
2b
Tc(
s)21
a
Tc(
s)21
b N
f(s)
291a
N
f(s)
291b
T
c(m
)357
a,b
T
c(m
)357
c A
i(d)
351a
T
f(d)
53a
T
f(d)
53b
Tf(
s)26
9a
Tf(
s)26
9b
t9(1
)sD
G
Ai(
d)35
1b
AE
929G
ET
c(m
)310
b T
c(s)
697a
Tc(
s)69
7b
Tc(
s)49
2a,
Nf(
d)23
7a,
Tc(
d)13
2a
Tc(
s)49
2b
t3
4AR
, t39
AR
, t2D
G*,
t6s
DG
*,k1
770D
G*,
k1
961C
I(N
f(i)
225b
) , K
U21
12C
I, t
13A
Z, t
15A
Z, k
106A
Z*,
k1
24A
Z, k
169A
Z, k
336A
Z, k
493A
Z, k
1038
AZ
, k15
52A
Z*,
k1
866A
Z, A
E22
4AZ
*, K
U21
26W
I(N
f(i)
225b
), K
U20
83E
I
Nf(
d)23
7b
Tf(
s)16
3a
t26A
Z, k
108A
Z, k
1099
AZ
, KU
20-1
0WI,
KU
2102
WI,
K
U21
03W
I, K
U21
05W
I, K
U21
07W
I, K
U20
96W
I(T
f(s)
225b
) ,
KU
20-9
EI,
KU
2076
EI,
KU
2081
EI(
Ai(
d)35
1b)
Tf(
s)16
3b
k118
5AR
, KU
2080
EI
Tc(
d)13
2b
Nf(
i)22
5a,
Nf(
d)25
a k6
12G
E, k
141A
Z, k
296A
Z, K
U20
-7C
I
Nf(
i)22
5b
k195
6sC
I, k
1960
CI*
, KU
20-8
CI,
KU
2118
CI,
KU
2123
CI,
K
U21
56C
I(T
f(s)
225b
), k
433T
N
b52)d(fN
AE
213T
N
Nf(
d)19
9b
Ai(
d)42
0a
Ai(
d)42
0b
k195
4CI,
KU
2824
AR
T
f(d)
114b
KU
2002
PA
* T
c(s)
390b
k912
IN(T
c(m
)310
b)T
f(s)
225b
N
f(s)
226a
, T
c(s)
978a
, T
c(s)
270a
, N
f(s)
567a
, N
f(s)
510a
, T
c(m
)917
a
k102
5DG
*, k
1959
EI*
, KU
2159
WI;
AE
476A
R, t
1DG
, k36
2DG
, K
U20
71C
I, K
U21
13C
I, K
U21
21C
I, K
U21
42C
I, K
U21
45C
I,
KU
2151
CI,
KU
2154
CI*
, KU
2157
CI,
k61
6AZ
, k17
82A
Z,
KU
2109
WI,
k15
60T
N*,
k15
61T
N*,
k15
63T
N*,
k19
03T
N,
k964
AF
, k96
7AF
, k98
9AF
*, k
998A
F, K
U20
-6P
A*,
KU
2009
PA
*,
k394
UZ
, k42
1UZ
*, k
1345
UZ
(Tc(
m)3
10b)
, k13
52U
Z, k
1356
UZ
, k1
805U
Z, A
E95
5TJ,
k67
7KI,
k12
61K
I*N
f(s)
226b
T
c(m
)310
a
k179
2GE
, AE
1037
GE
, k10
9AZ
*, K
U21
10W
I T
c(m
)310
cK
U20
75E
I(T
c(m
)917
b)T
c(s)
978b
E
G6121kT
c(s)
270b
KU
2116
CI
Nf(
s)56
7b
Tf(
s)23
7a
T
f(s)
237b
K
U21
39T
Y
Nf(
s)51
0b
Tf(
s)12
9a
T
f(s)
129b
k4
26T
N*
Tc(
m)9
17b
k6
08G
E, K
U20
87E
I*, k
427T
N, k
428T
N, k
429T
N*,
k98
2AF
*,
k995
AF
*, k
1346
UZ
*, k
1322
KZ
*
Inv
ersi
on
(i)
?in
sert
ion
s/d
elet
ion
s(d
)?
bas
ep
air
sub
stit
uti
on
s(s
)at
27
loci
tota
lly
are
pre
sen
ted
and
thre
em
icro
sate
llit
elo
ci(m
)ar
ein
clu
ded
.S
sp.
tau
sch
iiis
sho
wn
init
alic
s,
ssp
.str
an
gu
lata
—in
bo
ldty
pe.
Th
etw
ole
tter
saf
ter
anac
cess
ion
nu
mb
erd
isp
lay
the
cou
ntr
yo
fo
rig
in(s
eeF
ig.
3fo
rd
esig
nat
ion
s).C
on
seq
uen
tm
uta
tio
ns,
pre
sen
ted
fro
mth
ele
ftto
the
rig
ht,
hav
efo
rmed
hap
loty
pes
of
the
acce
ssio
ns.
Th
eco
mp
lete
hap
loty
pe
of
each
acce
ssio
nis
pre
sen
ted
:th
eac
cess
ion
has
anal
lele
‘‘a’
’at
each
locu
s,if
no
tsh
ow
nth
atit
has
ano
ther
alle
le.A
llel
esw
hic
ho
ccu
rren
ces
fall
ou
to
fth
esc
hem
e,ar
ep
rese
nte
dsp
ecia
lly
inb
rack
ets
and
un
der
lin
ed.A
cces
sio
ns
hav
ing
Tc(
s)3
84
bal
lele
are
mar
ked
wit
has
teri
sk(*
)
692 Genet Resour Crop Evol (2012) 59:683–699
123
Author's personal copy
accessions are given from the left to the right in
Table 3, and from the ‘‘root’’ to the ‘‘leaves’’ in
Fig. 3. The subdivision of Ae. tauschii into subspe-
cies tauschii and strangulata, which is displayed in
Table 4 in different types, is presented in Fig. 3 as
the two primary branches.
The following peculiarities of non-coding cpDNA
sequences polymorphism in Ae. tauschii are outlined.
(1) Within the four cpDNA regions studied, II/
D&S mutations were found to occur at 26 sites (not
mentioning Tc(s)384 which was omitted from the
consideration). 10 of these II/D&S were characteris-
tical for ssp. tauschii, 15 were characteristical for ssp.
strangulata, and only one, Tf(s)225b, was found in
both subspecies (Table 4). From this set of II/D&S,
Tf(s)225b was the first characteristical for
Ae. tauschii mutation to originate in the course of the
species evolution history (Table 4; Fig. 3). (All diploid
Aegilops/Triticum presented in ‘‘DDBJ’’ are mono-
morphic at this locus having Tf(s)225a allele only).
(2) A distinct difference in the level of cpDNA II/
D&S loci polymorphism between ssp. tauschii and
ssp. strangulata was identified. Considering a locus
with a frequency of the common allele less then 0.95
(i.e. having uncommon allele(s) in at least three
accessions, 3/56 = 0.054) as ‘‘essentially polymor-
phic’’, we have in ssp. strangulata six essentially
polymorphic loci from the 15 afore mentioned,
specific for the subspecies polymorphic cpDNA II/
D&S loci (40%). No single essential polymorphic II/
D&S locus from the 10 characteristic for ssp. tauschii
was found in this subspecies (0%) (Table 4).
(3) Subspecies strangulata phylogenetic lineage
Tc(s)21b has a set of sequential mutations without
‘‘branching’’ (Fig. 3). Subspecies strangulata lineage
Tc(s)697b (the major one in this subspecies, contain-
ing 84% of all ssp. strangulata accessions (Table 4))
has sequential mutations Tc(m)310b, Tc(s)697b with-
out branching (i.e. the ancestor allelic variants,
Tc(m)310a, Tc(s)697a, were lost in this lineage (in
Table 4 it is reflected as empty cells in the right
column with no accessions)) (Fig. 3). This lineage
further divided into the three lineages, Tc(s)492b,
Nf(d)237b and Tc(d)132b, and again the ancestor
alleles, Tc(s)492a, Nf(d)237a, Tc(d)132a, were lost
(Table 4; Fig. 3). In ssp. strangulata only the lineage
Tf(s)225b, containing only 14% of the subspecies
accessions, branches well without the loss of ancestor
haplotypes (Table 4; Fig. 3).
In contrast to ssp. strangulata, in ssp. tauschii its
both primary lineages, Tf(s)225b and Tf(s)225a,
branch well. The consequent mutations without
branching were found only in the small secondary
lineages Nf(s)567b, Nf(s)510b and Nf(d)199b
(Fig. 3).
Fig. 3 A general presentation of the raw data of Aegilopstauschii cpDNA non-coding sequences polymorphism from the
Table 3 in the form of a graphical scheme of Ae. tauschiiintraspecies phylogeny. Mutations that have taken place in the
lineages are shown in rectangles on ‘‘branches’’. The circlesrepresent haplogroups. The area of a circle is proportional to
the number of accessions. The area of a shaded sector is
proportional to the number of accessions originated from
Azerbaijan or Precaspian Iran—the regions where ssp. stran-gulata is relatively much more abundant than ssp. tauschii.Abbreviations near the circles show the countries where from
the accessions originated (TY-Turkey, NO-North Ossetia,
DG-Dagestan, GE-Georgia, AR-Armenia, AZ-Azerbaijan,
CI-Continental Iran, WI-Western Precaspian Iran, EI-Eastern
Precaspian Iran, TN-Turkmenistan, AF-Afghanistan, PA-Paki-
stan, IN-India, UZ-Uzbekistan, TJ-Tajikistan, KZ-Kazakhstan,
KI-Kirgizstan). According to the database of Yamane and
Kawahara (2005), the two mutations marked with black circles
already existed in the ancestor species
Genet Resour Crop Evol (2012) 59:683–699 693
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(4) In ssp. tauschii, geographic occurrences of II/
D&S mutations characteristic for this subspecies is
uneven throughout its area. Six out of 10 such II/D&S
were found to the west of Caspian Sea, and only two
were found to the east from central Kopet-Dag
(Fig. 4).
Caucasus is the only region where all the five ssp.
strangulata lineages, Tc(s)21b, Tc(s)492b, Nf(d)237b,
Tc(d)132b and Tf(s)225b, were found (Table 4).
(5) In ssp. strangulata the geographic patterns of
occurrences of different phylogenetic lineages dis-
tinctly differ. Tc(s)492b lineage mostly occupies
eastern Caucasia (Fig. 5). Nf(d)237b lineage extended
about 800 km eastward along narrow Precaspian zone
but was not found 50 km to the south (Fig. 6).
Tc(d)132b lineage occur widely through ssp. strangu-
lata area: from Caucasia to central Kopet-Dag, but only
inland. Not a single example of its occurrence in
Precaspian regions was found—neither in Iran nor in
Azerbaijan or Dagestan (Fig. 7). (The probability of
such a pattern to happen by chance is very low, only
about 0.0006). The lineage Tf(s)225b is scattered
widely throughout the species range (Fig. 8). Finally,
the lineage Tc(s)21b of ssp. strangulata is represented
Fig. 4 Geographic occurrences of characteristic for ssp.
tauschii substitutions and indels in chloroplast non-coding
sequences
Fig. 5 Geographic occurrences of Tc(s)492 alleles among
ssp. strangulata accessions
Fig. 6 Geographic occurrences of Nf(d)237 alleles among
ssp. strangulata accessions
Fig. 7 Geographic occurrences of Tc(d)132 alleles among
ssp. strangulata accessions
Fig. 8 Geographic occurrences of Tf(s)225 alleles among ssp.
strangulata accessions
694 Genet Resour Crop Evol (2012) 59:683–699
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only by the accessions t9(1)s and AE929 from the west
of the area, Dagestan and Georgia, respectively
(Table 4).
In ssp. tauschii neither one of the 10 II/D&S
characteristic for the subspecies has a wide geo-
graphic occurrence (Fig. 4). Only among microsatel-
lites was found the mutation, Tc(m)917b, that has
wide geographic distribution in ssp. tauschii (Fig. 9).
Discussion
The few artefacts (outlined in Table 4) were found.
Obviously, existence of microsatellite allele
Tc(m)310b, characteristic for ssp. strangulata, in the
accessions k912 (India) and k1345 (Uzbekistan
(formally it is from Turkmenistan, but located on
the east, near Uzbekistan border)) belonging to ssp.
tauschii and geographically located far from each
other and ssp. strangulata range, is due to indepen-
dent mutations leading to homoplasy (Table 3).
It is known that recombination events in chloro-
plast could take place, at least in some species
(Marshall et al. 2001) and Ae. tauschii is thought to
be among them (Nakamura et al. 2001). It seems the
rare occurrence of Tf(s)225b and Nf(i)225b alleles
within ssp. strangulata on ‘‘wrong’’ branches of the
tree is due to recombination (Table 4). Geographic
location of ssp. strangulata accession KU2075 (east-
ern Precaspian Iran) having allele Tc(m)917b (other-
wise found in ssp. tauschii only) near ssp. tauschii
accession having this allele, indicates genetic
exchange between the subspecies and recombination
in cpDNA (Table 4; Fig. 9).
The last artefact is the occurrence of Ai(d)351b
allele in the two accessions on two distant branches
of the phylogeny tree (Table 4). As shown in Table 3,
in this cpDNA region several different deletions
could occur which will be aligned and presented by
computer program as the same sequence, probably,
this is a mispresentation.
Let us now consider the results obtained through
the study from the aspect of (1) basic principles of
population genetics, molecular evolution theory and
ecology, and of (2) what was previously known about
Ae. tauschii.
(A) According to the theory of molecular evolu-
tion (Kimura 1983), a neutral allele sooner or later
will be substituted by another in a species popula-
tion—that is the basis of molecular phylogeny. But in
the case of subspecies tauschii and strangulata which
are presented by numerous small fairly well isolated
populations (Dudnikov 1998) such a neutral allele
(e.g. cpDNA II/D&S) could exist practically for
infinitely long time: it will be substituted in some
local populations and fixed in the other—the poly-
morphism of the locus will retain in the subspecies in
general. Obviously, the mutation Tf(s)225b presents
an example of ‘‘early mutation event’’ (Barton et al.
2007, Fig. 27.28): according to the data obtained
(Table 4; Fig. 3), Tf(s)225b mutation originated in
Ae. tauschii before its subdivision into subspecies
tauschii and strangulata, and the polymorphism of
the Tf(s)225 locus was ‘‘inherited’’ by both the
subspecies (Fig. 10). It is the only one such mutation
among the 26 II/D&S considered, all the other 25
mutations are characteristic for either ssp. tauschii or
ssp. strangulata (Table 4; Fig. 3). Therefore it could
Fig. 9 Geographic occurrences of Tc(m)917b microsatellite
allele in Aegilops tauschii
Fig. 10 A presumed scheme of Ae. tauschii evolution. The
circles present local populations in the field of gene combina-
tions of Sewall Wright
Genet Resour Crop Evol (2012) 59:683–699 695
123
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be concluded that neither of the two Ae. tauschii
subspecies was an ancestor to one another.
Ae. tauschii divided into ssp. tauschii and ssp.
strangulata at the very beginning of its existence as
a species. It is completely in line with the putative
scheme of Ae. tauschii intraspecies divergence pro-
posed previously by Dudnikov (2000). According to
this scheme, subspecies tauschii and strangulata had
originated as a result of splitting of subdivided
population of an ancestor species into two parts in the
field of gene combination of Sewall Wright (Fig. 10).
It explains well how the mutation Tf(s)225b (which
appeared after the origin of Ae. tauschii but before its
subdivision into the subspecies) was retained in the
both subspecies; and why the mutations Tc(s)380 and
Tc(s)472, which existed even before the origin of
Ae. tauschii, could be found only in some local
populations of one of its subspecies—ssp. strangulata
(Fig. 10).
(B) Molecular genetic data outlined in §4 of
‘‘Results’’ confirm the conclusion previously made by
Zhukovsky (1928). Origin of Ae. tauschii (and each
of its two subspecies) has taken place in the west of
the species area.
(C) Peculiarities of evolutionary history of differ-
ent Ae. tauschii lineages have left traces on the
phylogeny tree presented on Fig. 3. As time passes
and mutations occur, the level of genetic variation is
growing in a ‘‘wealthy’’ big subdivided population.
On the contrary, a ‘‘poor’’ small isolated population is
incapable to retain genetic polymorphism. Newly
arisen mutations could be rapidly fixed in such
populations, replacing the previous allelic variant, but
the level of genetic variation does not increase
(Wright 1937; Kimura 1983). Such ‘‘unhappy’’
periods in a phylogenetic lineage history are dis-
played on ‘‘the tree’’ as consecutive mutations
without ‘‘branching out’’ (Fig. 3).
Both major phylogenetic lineages of ssp. tauschii,
Tf(s)225a and Tf(s)225b, are ‘‘branching out’’ well,
reflecting its successful, rapid geographic expansion
without any delay. The minor ssp. strangulata
lineage, Tf(s)225b, also seems to had no considerable
delay in geographic spread (Fig. 3). But the major
ssp. strangulata lineage, Tc(s)697b, had a marked
delay before its expansion started: in ssp. strangulata
evolutionary past this lineage existed as a small
isolated population for a long period of time. During
this time Tc(m)310b and Tc(s)697b alleles were fixed
in the population. Later this lineage divided in the
three lineages, Tc(s)492b, Nf(d)237b and Tc(d)132b,
again the ‘‘a’’ alleles were lost (Fig. 3), reflecting that
these lineages had originated not during the geo-
graphic expansion but before it started. At the
beginning of their existence, each of the Tc(s)492b,
Nf(d)237b and Tc(d)132b lineages was present for a
considerable time as a small isolated population.
The lineages that have been isolated for a long time
are rather common on ssp. tauschii secondary
‘‘branches’’ presenting the western part of the subspe-
cies range (Nf(s)567b, Nf(s)510b and Nf(d)199 lin-
eages (Fig. 3)). A really outstanding lineage, Tc(s)21b,
was pointed out in the western part of ssp. strangulata
area. This small lineage has existed as an isolated from
the beginning of existence of ssp. strangulata and
managed not to become extinct till now. Such history
has made local ssp. strangulata population t9(1)s the
most unique from all other local populations—at
cpDNA genetic markers and at enzyme-encoding
genes as well (Dudnikov 1998). Previously it was
noticed that local populations: t9(1)s from Dagestan
(ssp. strangulata lineage Tc(s)21b-Tf(d)53b) and
k1954 from north-western Iran (ssp. tauschii lineage
Nf(d)199b), have existed for a long time as small,
completely isolated populations. This ‘‘enabled’’ them
to fix extremely rare for Ae. tauschii slightly delete-
rious alleles (Dudnikov 1998). These alleles, that made
t9(1)s and k1954 so unique, belong to the loci which are
being maintained monomorphic in Ae. tauschii by
purifying selection, with the both subspecies having
the same alleles. At the same time, t9(1)s and k1954
have spike morphology typical for ssp. stangulata and
ssp. tauschii, respectively. They also have the sets of
alleles typical for their subspecies at genetic loci which
polymorphism was found to be adaptive and deter-
mining the subspecies attribution (Dudnikov 1998). In
Figs. 1, 2 and 3 local population t9(1)s stands far away
from other ssp. strangulata populations, since its
isolation led to fixation of many unique neutral cpDNA
genetic markers (Table 4, Fig. 3). At the same time,
natural selection prevented local population t9(1)s
from going down from the adaptive peak occupied by
ssp. strangulata in the field of gene combinations of
Sewall Wright (Fig. 10). As a result, the accession
t9(1)s retained characteristic for ssp. strangulata the
value of spike morphology index SI, and typical for
ssp. strangulata alleles Acph195, Got2105and Est5170 of
enzyme-encoding loci (Dudnikov 1998, 2003a).
696 Genet Resour Crop Evol (2012) 59:683–699
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The ‘‘fate’’ of unique genetic alleles, originated in
such small populations, completely isolated for a long
period of time, differs from the fate of cpDNA non-
coding sequences markers. Any contact of such
isolated local population with the other will lead to
the loss of unique genetic alleles: these slightly
deleterious alleles will be substituted by the common
alleles. Unique genetic markers, being neutral, have
much more chances ‘‘to survive’’ in this case (Kimura
1983). So, k1954 accession has unique alleles at
enzyme-encoding genes Nadhd1, Aco2 and vernali-
zation requirement gene Vrn-D2 (Dudnikov 2003a),
while accession KU2824, having the same with
k1954 unique markers Nf(d)199b and Ai(d)420b
(Table 3), possesses common alleles for Ae. tauschii
at all the three mentioned genes (Dudnikov unpub-
lished data).
The particular importance of the accession 9(1)s as
a unique genetic resource should be emphasized.
Previously, the accessions AE454, AE457, AE490,
KU-2829A, KU-2832 were reported to be similar
with AE929 in their genetic markers (Pestsova et al.
2000; Matsuoka et al. 2008; Mizuno et al. 2010) and
probably belong to the Tc(s)21b lineage, but there
were no reports yet that any of these accessions of
Georgian origin has rare alleles of functional genes.
9(1)s differentiates the most from all the other
Ae. tauschii accession not only at its genetic markers,
but also at the allelic constitution of functional genes
(Dudnikov 1998, 2003b).
(D) The following features of Ae. tauschii subspe-
cies should be emphasized. (1) According to enzyme-
encoding genes variation patterns (Dudnikov 2009)
and cpDNA sequences polymorphism (‘‘Results’’,
§1), tauschii and strangulata are similarly ancient
subspecies. (2) The level of cpDNA non-coding
sequences polymorphism is much higher in ssp.
strangulata then in ssp. tauschii (‘‘Results’’, §2); at
the same time (3) ssp. strangulata has much smaller
area than ssp. tauschii: ssp. strangulata has spread to
the east only up to central Kopet-Dag, while ssp.
tauschii—up to central Tien Shan and western
Himalayas (Jaaska 1981; Dudnikov 2000).
According to population genetics and molecular
evolution theory (Kimura 1983; Hedrick 2005), in
Ae. tauschii, which is presented by numerous small
fairly well isolated local populations, an allele could
had spread widely through the area only if (1) it was
‘‘supported’’ by natural selection or if (2) it was
involved in the species geographic expansion. For
cpDNA non-coding sequences loci, only the latter
variant was possible since their variations are thought
to be neutral. The phylogeny tree (Fig. 3) reveals that
there was no delay before ssp. tauschii had started its
geographic expansion (‘‘Results’’, §3); obviously, it
was the first of the two subspecies to do this.
Therefore, it was ssp. tauschii which ‘‘met’’ the vast
ecological niche which was free and could be rapidly
occupied. This rapid expansion resulted in the low
level of polymorphism at cpDNA II/D&S loci. The
rate of ssp. tauschii migration front moving eastward
was so high that at a time a chDNA II/D&S mutation
arose and attained high frequency in some eastern
boundary population, this local population already
‘‘found itself in the rear’’, far behind the migration
front. Therefore these alleles could not be involved
into ssp. tauschii geographic expansion and remained
geographically locally spread. Only microsatellites
due to their very high mutation rates had a chance to
be ‘‘captured and carried with the wave of ssp.
tauschii geographic expansion’’ and Tc(m)917b
appear to be such an example (Fig. 9). As expected
for a microsatellite, ‘‘DDBJ’’ data reveals that
mutation rate at Tc(m)917 locus among diploid
Aegilops/Triticum species, as well as at the other
mentioned above microsatellites, are much higher
than at II/D&S loci. (The rate of ssp. tauschii
geographic expansion is characterised here as rela-
tively high in comparison with cpDNA mutation rate.
Its absolute value in kilometres per year (generation)
was rather low due to the low migration capacity of
the species (Dudnikov 1998): it has taken ssp.
tauschii not less than 105 years (generations) to
spread through the area (Dudnikov 2009), and the
decrease of cpDNA II/D&S mutations occurrences in
ssp. tauschii from the west to the east (Fig. 4) reflects
this.).
Evolutionary success of ssp. strangulata was
concerned with Tc(s)697b lineage. According to the
phylogeny tree (Fig. 3, (‘‘Results’’, §3)), this major
lineage of ssp. strangulata had started its geographic
spread after a considerable delay. At this time all
Ae. tauschii range had been already occupied by ssp.
tauschii. The only opportunity for ssp. strangulata to
spread was to force out ssp. tauschii from those
natural habitats for which ssp. strangulata turned out
to be relatively better adapted. In contrast to ssp.
tauschii rapid geographic expansion through the vast
Genet Resour Crop Evol (2012) 59:683–699 697
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area, ssp. strangulata geographic spread was compli-
cated, multi-stage and slow process. Subspecies
tauschii was forced out from the relatively moister
and cooler natural habitats with several adaptive
forms of ssp. strangulata (presented as different
branches on the phylogeny tree (Fig. 3)). Adaptive
nature of ssp. strangulata geographic spread explains
the striking features of its cpDNA variation patterns
(Figs. 5, 6, 7). The narrow southern Precaspian area
and continental Iran to the south from it are separated
by watershed and belong to different phytogeograph-
ical regions, Euro-Siberian and Irano-Turanian,
respectively (Feldman 2001). Therefore, evolutionary
success of ssp. strangulata in these different natural
climatic zones was achieved by its different adaptive
forms, the lineages Nf(d)237b and Tc(d)132b,
respectively.
Ssp. strangulata, which ‘‘was late’’ with geo-
graphic expansion, had no chance to spread far to the
east, but it has almost completely forced out ssp.
tauschii from eastern Transcaucasia in Azerbaijan
and Precaspian area in Iran. A long time was
necessary for several adaptive forms of ssp. strangu-
lata, capable to force out ssp. tauschii from cool,
moist habitats, to originate and to spread geograph-
ically, and this time was enough for cpDNA II/D&S
could originate and to be incorporated in ssp.
strangulata geographic expansion. As a result, the
high level of cpDNA polymorphism was achieved in
ssp. strangulata (‘‘Results’’, §2).
(E) The previous study of enzyme-encoding genes
allelic variation revealed that in the western part of Ae.
tauschii area ssp. tauschii and ssp. strangulata exist as
the two neighboring-sympatric subspecies, and are in
contact with each other both ecologically and genet-
ically (Dudnikov 1998). In the three from 20 local
habitats studied in Transcaucasia, Ae. tauschii was
presented by mixed populations, with ssp. tauschii and
ssp. strangulata being presented approximately
equally (Dudnikov 1998). Also, local populations of
one of the subspecies with a minor admixture of plants
from another subspecies are common in the western
part of Ae. tauschii area (Dudnikov, unpublished data).
The genetic exchange between the subspecies is rather
common, but due to natural selection, eliminating
intermediate genotypes, ssp. tauschii and ssp. stran-
gulata exist for more than a million years (i.e.
generations, since it is an annual plant) as the two
distinctly genetically different, separate subspecies
(Dudnikov 1998). The adaptive process today main-
tains ssp. tauschii and ssp. strangulata geographic
distributions as a static status quo, and was revealed
through the previous allozyme variation studies
(Jaaska 1980, 1981; Dudnikov 1998, 2000, Dudnikov
and Kawahara 2006). The same adaptive process acted
dynamically at the beginning of Ae. tauschii evolu-
tionary history, when the two subspecies and different
adaptive forms had originated and competed with each
other in the course of geographic expansion. This
dynamic adaptive process, which had taken place in
the beginning of Ae. tauschii evolutionary history, is
reflected well in cpDNA non-coding sequences
polymorphism.
Acknowledgments I would like to express my sincere
gratitude to Miss. Rinata R. Husainova and Dr. Oleg V.
Vaulin for the help in the course of this study. I am also very
grateful to Prof. Sasa Stefanovic for useful discussion. And I
wish to thank Mrs. Holly Griesbach for refining the English of
the paper.
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