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

Author's personal copy

(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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http://www.ncbi.nlm.nih.gov/

http://www.ncbi.nlm.nih.gov/

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

Genet Resour Crop Evol (2012) 59:683–699 685

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Table 1 continued


(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


(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


123


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)


123


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(*

)


123


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


123


(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


Fig. 7 Geographic occurrences of Tc(d)132 alleles among


Fig. 8 Geographic occurrences of Tf(s)225 alleles among ssp.

strangulata accessions


123


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


123


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).


123


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


123


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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