Genetic Resources and Crop Evolution47: 171–183, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Population diversity of the Vicia sativaagg. (Fabaceae) in the flora of theformer USSR deduced from RAPD and seed protein analyses

Elena Potokina1,∗, Duncan A. Vaughan2, E.E. Eggi3 & N. Tomooka21Department of Plant Introduction and Taxonomy, N.I. Vavilov Institute of Plant Industry (VIR), Bolshaya Mor-skaya Street, 42, 190000 St. Petersburg, Russia2Crop Evolutionary Dynamics Laboratory, Genetic Resources 2, National Institute of Agrobiological Resources(NIAR), Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan3Laboratory of Crop Cultivars Identification, Seed Production Company ‘Gossortsemovoch’, 1st Verkhnyi proezd8, Promzona Parnas, 194292 St. Petersburg, Russia(∗Author for correspondence; present address: Department of Taxonomy, Institute for Plant Genetics (IPK),D-06466 Gatersleben, Germany)

Received 26 January 1999; accepted in revised form 7 June 1999

Key words:evolution, genetic resources, RAPDs, seed protein electrophoresis, taxonomy,Vicia

Abstract

The genetic diversity of 58 wild and weedy populations representing taxa within theV. sativaaggregate fromthe former USSR, 4 cultivars ofV. sativa, 2 accessionsof V. cordataand 3 accessions ofV. macrocarpafromMediterranean countries were analysed using randomly amplified DNA fragments (RAPDs) and seed protein elec-trophoresis (SDS-PAGE). Interspecific variation between taxa in theV. sativaaggregate could readily be detectedusing both techniques. RAPDs and seed protein patterns were found to be an effective means of identifyingaccessions that cannot be identified clearly by morphological criteria alone. RAPD and seed protein analysisrevealed a clear relationship between observed genetic variation of populations and their geographical distribution.Populations from each region had their own gene pools. Geographical variation was detected inV. segetalis. Thedegree of genetic divergence between local populations was usually related to proximity. In several locationswhere wild and weedy populations of differentV. sativaagg. taxa grow sympatrically, intermediate forms couldbe detected at the DNA and protein levels. Both RAPD and seed protein analysis support the view that theV.sativaaggregate consists of 8 taxa warranting recognition at the species level. Several species in this aggregate areevolving intra-specific groups which can readily be detected at the molecular level.

Abbreviations:PCR – polymerase chain reaction; RAPD – randomly amplified polymorphic DNA; SDS-PAGE –sodium dodecylsulfate electrophoresis in polyacrylamide gel.

Introduction

The Vicia sativa aggregate comprises several poly-morphic annual autogamous taxa. It is considered tobe a rather recently evolved branch of the genusVicia,and this aggregate appears to be currently in a pro-cess of dynamic evolution (Hanelt & Mettin, 1989).Taxonomists differ in their views regarding speciationin this aggregate. Some authors recognise 8 speciesin this aggregate (Fedchenko, 1948; Mettin & Hanelt,

1964; Hanelt & Mettin, 1966; Tzvelev, 1987; Potok-ina, 1997). Others consider this aggregate to be onevariable species,V. sativasensu lato, with subspeciesand varieties (Ball, 1968; Plitmann, 1970; Stankevich,1978; Zohary & Plitmann, 1979; Maxted, 1995).

The speciation process in this group could havebeen initiated by chromosomal rearrangements causedby mutations (Mettin & Hanelt, 1973; Hanelt & Met-tin, 1989) or by hybridization between well definedtaxa (Zohary & Plitmann, 1979; Ladizinsky, 1981).

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Most of the species recognised in theVicia sativaaggregate are sympatric in their distribution and oc-cupy similar habitats. Sympatric populations oftenhave plants of intermediate morphology, which maybe natural hybrids between taxa. Consequently keycharacters to distinguish taxa are sometimes lackingin populations, and the taxonomy of this group iscomplicated.

Since a population is the basic unit of evolution,population analysis may effectively describe intra andinter taxon diversity. Phenotypic analysis of 58 pop-ulations of wild and weedyV. sativaagg. taxa fromdifferent regions of the former USSR has been con-ducted using methods and approaches of populationphenetics (Potokina, 1997). It identified centers ofmorphological diversity forV. sativaL. sensu stricto,V. angustifoliaReichard andV. segetalisThuill. How-ever, due to genotype X environment interaction mor-phological characters alone provide insufficient data todescribe genetic diversity.

We used a combination of both molecular (RAPDanalysis) and biochemical (seed proteins) markers tobetter understand genetic diversity in local populationsof V. sativaagg. from different regions of the formerUSSR. The polymerase chain reaction (PCR) of ran-domly amplified polymorphic DNA (RAPD) markersis an effective method to detect genetic diversity andrelationships among closely related taxa inVicia L.subgen.Vicia (Potokina et al., 1999). RAPD markersprovide a high level of discrimination at the intra-specific level (Jarret & Austin, 1994). Electrophoreticanalysis of seed globulins has detected genetic dif-ferences at the species level and also has revealedintraspecific variation in leguminous species (Przybyl-ska, 1995; Salgado et al., 1995). Electrophoresis ofseed storage proteins has successfully been used foridentification of taxa withinV. sativaagg. (Potokina &Eggi, 1991). Consequently these techniques were usedto analyse the populations that were previously de-scribed in a morphological study by Potokina (1997).The objectives of this paper are:

1) to analyse the genetic diversity among local weedyand wild populations of theV. sativa aggregateusing RAPD markers and seed protein variability;

2) to determine the relationship, if any, between theobserved genetic variation at the DNA level, seedprotein patterns and geographical distribution andtaxonomical interpretation of populations.

Materials and methods

Plant material

The samples analysed in this study comprise 55 wildpopulations ofV. sativaL. s. str.,V. segetalisThuill.,V. angustifoliaReichard,V. amphicarpaDorth. andV.incisaBieb., collected during the expeditions in 1987–1990 in the former USSR: Caucasus (Azerbaijan,Nagornyi Karabakh, Krasnodarskyi area); Crimea andKerch peninsula (Ukraina); Central Asia (Turkmenia,Kopetdag Gershi); the North European part of Rus-sia (Arkhangelsk, Vologda, St. Petersburg, Novgorodprovinces) (Figure 1). Each of these populations wascollected as a bulk of seeds with one pod from eachplant and collected from as many plants in the popula-tion as possible. The samples are now deposited in theVIR (Vavilov All-Russian Institute of Plant Industry,St. Petersburg, Russia) germplasm collections. Theidentification and morphological diversity of popula-tions has been discussed previously (Potokina, 1997).Seeds included in the analysis were the original onesand had not undergone any cycles of multiplication.To estimate the genetic variability ofV. sativaagg. atboth the inter- and intra-specific levels we included inthe RAPD analysis 2 accessions ofV. cordataWulf.ex Hoppe, 3 accessions ofV. macrocarpa(Moris)Bertol., 1 accession ofV. incisa Bieb. and 1 acces-sion of each ofV. pilosa Bieb. andV. amphicarpaDorth. which were received from VIR and Gatersleben(Germany) germplasm collections. Four cultivars ofcommon vetch (V. sativaL. sensu stricto) were alsoanalysed for RAPD. The species and accessions usedfor RAPD and seed protein analysis are shown inTable 1.

RAPD analysis

Materials were grown in pots at NIAR, Tsukuba, Ja-pan (36◦05′ N; 140◦05′ E). Young leaves from 8–12individuals were used to extract DNA for each pop-ulation, using the standard CTAB method for small-scale extraction of DNA (Williams et al., 1993). Theconcentration of DNA was adjusted to 5 ng/µl.

Twenty primers, which gave clear polymorphicbands with six test samples were chosen from 70primers tested. The 10 base sequences of each primerused in this study were as follows (5′-3′): (P1) GTCT-GACGGT; (P2) CAGCTCAAGT; (P3) ACGCTG-ATCA; (P4) CTTGCCTCCC; (P5) GAGTGCGCAG;(P6) cgCGGACGAT; (P7) TGGTCGCACG; (P8)GAAGGCGCGT; (P9) GTCACTCCCC; (P10) GTCC

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Figure 1. Map showing the regions the material studied came from. Regions: Arkhangelsk, Vologda, St. Petersburg provinces (A); Novgorodprovince (B); Crimea and Kerch peninsula (C); Krasnodarskyi territory (D); Azerbaijan & Nagornyi Karabakh (E); Turkmenia, Kopet-DagGershi (F).

ACACGG; (P11) TGGGGGACTC; (P12) AAGACT-GTCC; (P13) CAGTTCTGGC; (P14) AAGACT-GTCC; (P15)GAAGGCTCTG; (P16) TCCATGC-CGT; (P17) CTGGCGGCTG; (P18) GCGGAAATAG; (P19) CAGGCGGCGT; (P20) GCTGGACATC.

Amplification of DNA was performed on a thermalcycler (Techne Inc., Princeton, NJ) by mixing 2µl of5 ng/µl DNA with a premix of 5.4µl sterile distilledwater, 1µl of 10X buffer (Perkin Elmer, Norwalk,CT), 0.8µl 25 mM MgCl2 (Perkin Elmer), 0.2µl10 mM mixture of dNTPs and 0.5µl of primer, 0.2µlof Taqpolymerase (1 U/µl) (Amplitaq, Perkin Elmer).Amplification was carried out for 1 min at 94◦C, fol-lowed by 48 cycles of 30 s at 94◦C, 1 min at 36◦Cand 2 min at 72◦C. A further cycle at 72◦C for 10 minwas performed before cooling and storing in a refriger-ator. The amplified products were visualised on 0.8%agarose gels containing ethidium bromide in 1XTAE(40 mM Tris base, 20 mM glacial acetic acid, 2 mM

Na2EDTA-2H2O) at 60 V for 4 h and photographedunder UV light. The molecular marker was lambdaDNA digested withPst I.

RAPDs were scored as dominant markers (pres-ence versus absence of specific bands) and trans-formed into a 1 (present)/0 (absent) matrix over allpopulations and all accessions studied. The geneticsimilarity coefficient was calculated between a pair ofpopulations using the formula of Nei & Li (1979). Thegenetic similarity matrix was then used in hierarch-ical cluster analysis using UPGMA (NTSYS statisticalpackage, Rohlf, 1992).

Seed protein electrophoresis

Cotyledons of mature seeds were examined in at leastten individuals for each population. Bulk analysis ofseed protein extracts from 8–10 individuals of eachpopulation or accession was also conducted.

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Table 1. Vicia sativaaggregate species, populations and accessions included in the study

Taxon Number of Locality Latitude Longitude Country, area Indication on

populations the map

V. sativa 1 Shingadulan 38◦47′ N 48◦25′ E Azerbaijan E

2 Gosmalany 38◦40′ N 48◦22′ E Azerbaijan E

3 Martuni 39◦47′ N 46◦54′ E Nagornyi Karabakh E

4 Nngi 39◦47′ N 46◦54′ E Nagornyi Karabakh E

5 Shushikend 39◦46′ N 46◦46′ E Nagornyi Karabakh E

6 Ponezhukay 44◦56′ N 39◦12′ E Russia, Krasnodarskyi area D

7 Dzerokay 45◦0′ N 40◦23′ E Russia, Krasnodarskyi area D

8 Ay-dere 38◦24′ N 56◦45′ E Turkmenia, Kopetdag Gershi F

9 Iol-dere 38◦17′ N 56◦45′ E Turkmenia, Kopetdag Gershi F

10 Shekhim-dere 38◦34′ N 56◦43′ E Turkmenia, Kopetdag Gershi F

11 Tutly-kala 38◦24′ N 56◦43′ E Turkmenia, Kopetdag Gershi F

121 cv. ‘Sivushskaya’ Daghestan

131 cv. ‘Anafa’ Portugal

141 cv. ‘Precoce 332’ Algeria

151 cv. ‘MorocDII 6235’ Morocco

V. segetalis 16 Gosmalany 38◦40′ N 48◦22′ E Azerbaijan E

17 Nngi 39◦47′ N 46◦54′ E Nagornyi Karabakh E

18 Susha 39◦45′ N 46◦44′ E Nagornyi Karabakh E

19 Levashi 42◦26′ N 47◦19′ E Daghestan

20 Labinsk 44◦38′ N 40◦43′ E Russia, Krasnodarskyi area D

21 Shuntuk 44◦27′ N 40◦10′ E Russia, Krasnodarskyi area D

22 Khadyzhensk 44◦26′ N 39◦32′ E Russia, Krasnodarskyi area D

23 Shaumjan 44◦55′ N 40◦20′ E Russia, Krasnodarskyi area D

24 Dzugba 44◦19′ N 38◦18′ E Russia, Krasnodarskyi area D

25 Ponezhukay 44◦56′ N 39◦12′ E Russia, Krasnodarskyi area D

26 Uchkuevo 44◦38′ N 33◦32′ E Ukraine, Crimea C

27 Sevastopol 44◦37′ N 33◦32′ E Ukraine, Crimea C

28 Schastlivoye 44◦34′ N 34◦4′ E Ukraine, Crimea C

29 Nikita 44◦31′ N 34◦14′ E Ukraine, Crimea C

30 Frunzenskoye 44◦35′ N 34◦20′ E Ukraine, Crimea C

31 Aju-Dag 44◦34′ N 34◦18′ E Ukraine, Crimea C

32 Malorechenskaya 44◦46′ N 34◦33′ E Ukraine, Crimea C

33 Rybach’ye 44◦47′ N 34◦36′ E Ukraine, Crimea C

34 Meganon 44◦48′ N 35◦4′ E Ukraine, Crimea C

35 Sudak 44◦52′ N 34◦58′ E Ukraine, Crimea C

36 Planerskoye 44◦58′ N 35◦14′ E Ukraine, Crimea C

37 Nasypnoye 45◦3′ N 35◦17′ E Ukraine, Crimea C

38 Marfovka 45◦13′ N 36◦5′ E Ukraine, Kerch peninsula C

39 Lenino 45◦19′ N 35◦39′ E Ukraine, Kerch peninsula C

40 Tarkhan 45◦26′ N 36◦26′ E Ukraine, Kerch peninsula C

41 Kamensk 45◦19′ N 35◦39′ E Ukraine, Kerch peninsula C

42 Zybino 45◦14′ N 34◦29′ E Ukraine, Kerch peninsula C

43 Kara-Kala 38◦34′ N 56◦44′ E Turkmenia, Kopetdag Gershi F

44 Chandyr 38◦24′ N 56◦43′ E Turkmenia, Kopetdag Gershi F

45 Ay-dere 38◦24′ N 56◦45′ E Turkmenia, Kopetdag Gershi F

46 Iol-dere 38◦17′ N 56◦45′ E Turkmenia, Kopetdag Gershi F

47 Shekhim-dere 38◦34′ N 56◦43′ E Turkmenia, Kopetdag Gershi F

V. angustifolia 48 Avdeevo 59◦5′ N 33◦32′ E Russia, Novgorod province B

49 Pochep 59◦5′ N 33◦33′ E Russia, Novgorod province B

50 Gory 59◦5′ N 33◦34′ E Russia, Novgorod province B

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Table 1. Continued

Taxon Number of Locality Latitude Longitude Country, area Indication on

populations the map

51 Podlitov’e 58◦22′ N 32◦1′ E Russia, Novgorod province B

52 Vekshino 57◦38′ N 31◦13′ E Russia, Novgorod province B

53 Kholmy 58◦59′ N 35◦24′ E Russia, Novgorod province B

54 Demjansk 57◦39′ N 32◦28′ E Russia, Novgorod province B

55 Zabel’ye 59◦17′ N 34◦53′ E Russia, Leningrad province A

56 Vashino 59◦39′ N 36◦15′ E Russia, Vologda province A

57 Mitino 61◦13′ N 44◦42′ E Russia, Arkhangelsk province A

V. macrocarpa 58 35812/VIR Portugal

591 36064/VIR Portugal

601 35484/VIR Portugal

V. cordata 61 35800/VIR Portugal

621 35807/VIR Portugal

V. pilosa 631 665195/Gatersleben Ukraine, Crimea

V. amphicarpa 642 Kamensk 45◦19′ N 35◦39′ E Ukraine, Kerch peninsula C

651 724184/Gatersleben Unknown

V. incisa 66 Aju-Dag 44◦34′ N 34◦18′ E Ukraine, Crimea C

671 1001184/Gatersleben Ukraine, Crimea

1Accessions used for RAPD analysis only.2Accessions used for seed protein electrophoresis only.

Total seed proteins were analysed by one-dimensional sodium dodecylsulfate polyacrylamidegel electrophoresis (SDS/PAGE). Seed protein ex-tracts were prepared by removing the seed coat, thensoaking 4 mg of homogenising cotyledon meal in 1 MTris-HCl pH 6.8 buffer at room temperature. Samplesof supernatant obtained after centrifugation at 12 000gfor 10 min were mixed with an equal volume of thebuffer containing 0.625 M Tris-HCl 6.8, 2% SDS, 5%mercaptoethanol and 0.01% bromophenol blue. Poly-acrylamide gels were prepared according to Laemmli(1970). The loading gel was 5% acrylamide in a2.5 M Tris-HCl pH 6.8 buffer with 10% SDS. Theresolving gel was prepared with 10% acrylamide ina 3.5 M Tris-HCl pH 8.8 buffer with 10% SDS. Theelectrode buffer was tris-glycine (6.0 g of Tris base,28.8 g of glycine, 20 ml of 10% SDS in 2 l of wa-ter, pH 8.3). Samples were run for 3.5 h at 30 mAin a Hiy Kalur apparatus. Polyacrylamide gels werestained with 0.1% Coomassie Blue for 12 h and thenwashed with 7% acetic acid (Methodical Instructions,1990). The approximate Mrs of polypeptide bandswere estimated using the following standard proteins:bovine serum albumin (67 kDa), egg albumin (45kDa), chymotrypsinogenA (25 kDa), myoglobin (17.8kDa) and cytochrome C (12.4 kDa).

Gels were scored for the presence and absence ofspecific bands and the values were used to compile adata matrix. A similarity index for all possible com-parisons between accessions was calculated by Dicecoefficient. An UPGMA phenogram was constructedbased on the program NTSYS-pc (Rholf, 1992).

Results

Differentiation between taxa

The number of amplified DNA fragments varied from1 to 5 per accession for each primer, and the fragmentsranged from 200 to 1700 bp in length. Each primergenerated from 6 to 19 distinctive bands across all 65accessions of 8 taxa of theVicia sativaaggregate. Us-ing 20 primers, a total of 229 bands were visualised;among these 216 were polymorphic in at least onepairwise comparison between populations.

Phenetic analysis of RAPD results, presented byUPGMA dendrogram (Figure 2A), clearly differenti-ated each of the 8 species of theV. sativaaggregate:V. sativa, V. angustifolia, V. segetalis, V. cordata, V.macrocarpa, V. amphicarpa, V. pilosaand V. incisa.There are considerable differences between the ge-netic similarity value for inter- versus intra-specific

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A

B

Figure 2. A: UPGMA phenogram generated from RAPD markers for 66 local populations and accessions of 8 species of theV. sativaagg;B:UPGMA phenogram based on similarity matrix of seed storage proteins of 54 local populations and accessions of 7 species ofV. sativaagg.Clusters:V. sativas. str. (b),V. segetalis(d), V. angustifolia(e).

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relationships. The similarity value between species isalways less than 0.5, while the similarity coefficientsfor intraspecific relations vary between 0.75 and 1.Of the species examinedV. incisa is most distantlyseparated from other species of theV. sativaagg. (Fig-ure 2A). Our results support Stankevich’s (1978) viewthat this species should be removed from theV. sativacomplex. RAPD analysis suggests thatV. segetalisandV. angustifoliaare less closely related than previouslyassumed based on morphological studies. Some au-thors do not separate these taxa, joining them in onegroup –V. sativasubsp.nigra (L.) Ehrl. (Ball, 1968;Plitmann, 1970; Stankevich, 1978; Maxted, 1995).However, the results of ecogeographical study (Po-tokina, 1997) and the RAPD results presented hereshow that these two taxa can be clearly distinguished,supporting their separate specific ranking.

The UPGMA dendrogram obtained from seed pro-tein electrophoretic analysis of 55 local populationsof V. sativaagg. and one accession ofV. cordataandV. macrocarpafor comparison is presented in Fig-ure 2B. All species of theV. sativaagg. can be clearlydelineated by their electrophoretic patterns. Each spe-cies has its own ‘protein portrait’, the most specificbanding region was the banding pattern of the vicilinzone of the spectra (approx. 20 kDa) as in the caseof Potokina & Eggi (1991). The closely related spe-ciesV. angustifoliaandV. segetaliscan also be easilyseparated by electrophoresis (Figure 2B). Since theelectrophoretic patterns have the complex molecularbasis that includes nucleotide substitutions, insertionsand deletions, and co- and post-translational modi-fications, it is difficult to relate phenotypic changesin the electrophoretic banding pattern to changes atthe molecular level (Gepts, 1995). Hence, one isusually limited to phenetic analyses with this cat-egory of markers. However, close agreement is foundbetween UPGMA dendrograms, derived from RAPDand seed protein banding. Both dendrograms showedclear inter- and intraspecific differences (Figure 2Aand B; clustersb, d, e), and similar topology, ex-cept for V. angustifoliabranch. It is now clear thatthere is a close relationship between 10-chromosome(V. cordata)and 12-chromosome (V. macrocarpa, V.sativa, V. segetalis, V. angustifolia)species and theirdivergence from 14-chromosome speciesV. amphi-carpa and V. pilosa. Furthermore, both sets of dataseparatedV. incisafrom the other species ofV. sativaagg.

Table 2. Frequency of two types of protein electrophoretic patternsoccurring inV. segetalispopulations from Central Asia

Populations, locality Number of plants Number of

examined type 1 type 2

seg 43, Kara-Kala 12 8 4

seg 44, Chandyr 10 10 0

seg 45, Ay-Dere 18 4 14

seg 46, Iol-Dere 8 3 5

seg 47, Shekhim-Dere 15 2 13

Differentiation within taxa

V. sativa sensu strictoRAPD analysis revealed two groups of popula-tions amongV. sativa from the former USSR (Fig-ure 2A).The first group includes local populations ofCaucasus. The second clade unites the populationsfrom Central Asia. A previous study of populationpolymorphism at the phenotypic level did not revealany clear distinctions betweenV. sativapopulationsfrom Caucasus and Central Asia, but indicated that theCaucasus was a region of higher variability. RAPDpolymorphisms showed higher variation in Caucasusthan in Central Asia. Protein electrophoresis also de-tected the differences between Caucasus and CentralAsia (Figure 2B).

The results of RAPD analysis revealed that com-mon vetch cultivars of the Mediterranean are genetic-ally more closely related to theV. sativapopulationsfrom Caucasus than those from Central Asia.

V. segetalisRAPD analysis and seed protein electrophoresis onV. segetalis revealed two eco-geographic groups:Crimea-Caucasus and Central Asia (Figure 2). Suchclear differences between the regions were not de-tected at the phenotypic level (Potokina, 1997). Thecentre of morphological diversity forV. segetalisap-peared to be the Crimea and Caucasus rather thanCentral Asia (Potokina, 1997). RAPD analysis re-vealed considerable variability within both of thesepopulation clusters. RAPD analysis revealed that bothCrimea-Caucasus and Central Asia represent distinctcenters of diversity forV. segetalis.

Analysis of bulk seed samples (8–10 individuals)enables average electrophoretic patterns to be visu-alized. To improve the resolution of intrapopulationvariation for V. segetalisfrom Central Asia proteinelectrophoretic analyses of 8–15 individuals from each

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Figure 3. SDS-PAGE protein patterns illustrating intraspecific variation detected in populations ofV. segetalis.(A) Patterns produced byindividual seeds from population N25 (Crimea) against the corresponding pattern of population N46 (Central Asia). Two types of proteinpattern detected in population N46 are marked with∗ – type 1, ∗∗ – type 2. (B) Comparison of protein patterns produced by bulks of seeds frompopulations of the Caucasus-Crimea region (17, 25, 26) and Central Asia (46, 47).

population were conducted (Figure 3A). Two proteinprofile types which could not be distinguished in bulksamples were revealed. One was the same type asfound in the populations from the Caucasus-Crimeagroup (Figure 3A,type 1). A second type was foundonly in Central Asian populations (Figure 3A,type2). The frequency oftype 2varied within populationsfrom 0 to 0.87 (Table 2). We found one population(seg 44), that lacked thetype 2. As a result, this pop-ulation falls outside the Central Asia cluster and isjoined to the Caucasus-Crimea cluster (Figure 2B,∗).No differences at the phenotypic level have been de-tected between plants with different electrophoreticpatterns. Thus, ecogeographical divergence ofV. se-getalis between Caucasus-Crimea and Central Asiaregions can be explained by a change of the geneticcomposition of Central Asian populations, due to theappearance of a new genotype (type 2).

RAPD analysis of the populations collected alongthe South Crimea coast and in the neighboringKrasnodarskyi area revealed that populations locatedin close proximity generally clustered together (Fig-ure 4B, dendrogram C1,C2,C3,D). In the steppe regionof the Crimea peninsula and the Krasnodarskyi area(Figure 4B, dendrogram C3,D) populations have rathersimilar genetic composition over a large area (100–200 km). In the mountainous part of the Crimea popu-lations located in close proximity as a rule have similar

profile based in RAPD (Figure 4, graphs C1,C2). How-ever, genetic differences between populations were notalways directly related to proximity. Some populations(N23, Krasnodarskyi area; N30 Crimea) had an uniquegenetic composition found only at that location. Thepossible reason for this may be related to the localisolation in mountain valleys.

V. angustifoliaRAPD analyses showed thatV. angustifoliaconsists oftwo groups (Figure 2A): populations from the north-west (Novgorod province) and the north-east of theEuropean part of Russia (Arkhangelsk, Vologda, St.Petersburg provinces). No polymorphism was detectedby seed protein analysis. LikeV. segetalis, genetic dif-ferences between geographically distant populationsof V. angustifoliausually reflect proximity.

Possible gene flow in populations of V. sativa agg.

Different taxa ofV. sativaaggregate are not fully re-productively isolated from one another. Various taxawithin V. sativa agg. are cross compatible and theF1 hybrids are partially fertile (Sveshnikova, 1936;Yamamoto, 1966, 1973; Mettin & Hanelt, 1973; Lad-izinsky, 1978, 1981). The fertility of F2 populationsderived from crosses betweenV. sativa taxa differ-ing in chromosome numbers and karyotypes indicatesthat gene flow between plants with different chromo-

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Figure 4. (A) Geographical variation ofV. segetalispopulation in the Crimea and Krasnodarskyi territory reflected by RAPD clusters on theUPGMA tree;(B) A part of the UPGMA dendrogram which containsV. segetalispopulations.∗ – population which differed from its adjacentpopulations.

some numbers and karyotypes is possible (Ladizinsky,1981). It has been suggested that this may occur innatural populations where mixed stands of plants be-longing to differentV. sativa agg. taxa are found(Ladizinsky, 1981).

We have observed five localities, whereV. sativaand V. segetalisor V. segetalisand V. amphicarpahave colonized the same habitats and populations arepolymorphic (Table 3). Although plants with inter-mediate morphological characters have been found,it was possible to identify plants at the species levelby the pod and seed characteristics (see Potokina,1997). Wherever identity was doubtful, each plantfrom such a mixed population was easily identified byelectrophoretic protein pattern.

Nevertheless, some evidence to support the hypo-thesis that gene exchange may occur between popu-lations of related taxa growing together was found.The RAPD electrophoregram (Figure 5) shows theresults of fragment amplification, using the primer15, for populations N4 and N17 ofV. sativaand V.segetalis, respectively. The amplification pattern ofboth these species shows a clear interspecific differ-ence for one band using this primer. However, onepopulation ofV. sativa(N4) had the same band as allV. segetalissamples have. This band distinguishedV.segetalisfrom all otherV. sativasamples. The pop-ulations N4 (V. sativa) and N17 (V. segetalis) werecollected separately from the same habitat (Nngi set-tlement, Nagornyi Karabakh, Table 3). For other

primers the sample N4 (V. sativa) had similar poly-morphisms to otherV. sativasamples and thus it fellwithin the cluster ofV. sativafrom the Caucasus inthe UPGMA dendrogram. Co-habitation is often con-sidered to be closely associated with the occurrenceof intermediate plants and forms bridging the mainmorphological types due to gene exchange (Zohary &Plitmann, 1979; Ladizinsky, 1981). We suppose thatamong seeds analyzed as a bulk forV. sativa, thereare several intermediate forms. It seems probable thatgene flow may have occurred betweenV. sativaandV. segetalisin this habitat, and intermediate formscould be detected at the molecular level, but not themorphological level.

Results of seed protein analyses also providedevidence for a natural hybridization between sym-patric taxa. The seed protein electrophoregram for 9plants ofV. amphicarpa(N64) andV. segetalis(N41)from the same habitats in the Crimea region is shownin Figure 6 (see also Table 3). Interspecific differencesof protein pattern are evident. On the electrophore-gram each plant is presented by a pair of seeds (lanes).Lane 3 belongs to a plant, which has been identifiedmorphologically asV. amphicarpa. However, its ‘pro-tein portrait’ has an intermediate profile between twospecies (Figure 6, marked by pointers).

Four plants of unusual protein pattern, unlike thewidely distributedtype 1or type 2, were found in apolymorphic population ofV. segetalis,growing to-gether withV. sativanear Ay-Dere settl., Turkmenia

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Table 3. Populations ofV. sativaagg. taxa collected from the same location

Taxa (accession number) Locality Habitat

V. sativa(4) & V. segetalis(17) Nngi settl., alt. 1200 m, weedy in wheat

Nagornyi crops, mountain slope (30◦),Karabakh, together withVicia narbonensis,

Transcaucasus V. hybrida, V. michauxii

V. sativa(6) & V. segetalis(25) Ponezhukay settl., alt. 80 m, weedy in wheat crops,

Krasnodarsky area along the road, together with

Russia, Northern V. tetrasperma, V. hirsuta,

Caucasus V. villosa, Lathyrus aphaca,

L. hirsuta

V. sativa(8) & V. segetalis(45) Ay-dere settl., alt. 900 m, fallow, along the

Turkmenia, road, together withV. michauxii,

Kopetdag Gershi, V. hybrida, L. aphaca

Central Asia

V. sativa(9) & V. segetalis(46) Iol-dere canyon, alt.1100 m, on the mountain

Turkmenia, slope, along the stream, together

Kopetdag Gershi, withV. narbonensis,

Central Asia V. hyrcanica, L. aphaca,

L. inconspicuus

V. segetalis(41) & Kamensk settl., alt. 2 m, grass steppe, sandy soil,

V. amphicarpa(64) Kerch peninsula, in gramineous phytocoenose

Ukraine

Figure 5. RAPD profile for 11 populations ofV. sativaand 6 populations ofV. segetaliswith primer N15. The same RAPD banding patternwas found for populations N4 (V. sativa) and N17 (V. segetalis) which were collected in the same locality.

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Figure 6. SDS-PAGE seed protein patterns ofV. amphicarpa(1–5) andV. segetalis(6–9) collected in a common phytocoenose. Each plant waspresented in analysis by two seeds (pair of lanes on the electrophoregram). Polypeptide bands considered as a result of hybridization betweentwo species are marked with pointers.

(seg 45, Table 3). At the phenotypic level those plantshave been identified asV. segetalis, and were distin-guished by denser pubescence on pods, stems andleaves. A comparison of these four plants with all pos-sible protein patterns ofV. sativaandV. segetalisfromCentral Asia did not reveal any analogies. This typeseems to be a combination of the bands ofV. sativaandV. segetalis, and has been found nowhere else.

Discussion

Although the wild and weedy populations of theV.sativa aggregate consist of well defined taxa, somemorphological intermediate forms have been detec-ted (Potokina, 1997). DNA and protein level analysescould also reveal intermediate forms. All the plantshaving intermediate protein patterns or RAPD profileswere found in the populations where different taxagrow sympatrically. This could support the view thatthese intermediate forms can be a result of gene flow.

Genetic divergence inV. sativaagg. may be facil-itated by incomplete autogamy in two ways. Firstly,a rather high probability of cross-pollination (up to

10%) (Hanelt & Mettin, 1970, 1989) allows geneflow between neighbouring populations of the samespecies. This diffusion of genes may lead to allelicsimilarity on a local scale, while geographically dis-tant populations differ in their gene array (Yablokov,1986). This pathway of genetic divergence was ob-served in the populations ofV. sativafrom the Cau-casus and Central Asia,V. segetalisfrom the Crimeaand Krasnodarsky territory, in the populations ofV.angustifoliain the north of European Russia.

The second means of genetic divergence couldinvolve rapid fixation of mutations in a few genera-tions and it occurs mainly within annual, autogamousplants. By this process even in the absence of welldeveloped isolation barriers incipient speciation canoccur. This process has previously been described inVicia as sympatric speciation where two lines ofV.cordatahave been reported (Mettin & Hanelt, 1973).The plants were very similar in morphological respect,their karyotypes however were clearly differentiated.The differences between them were on the same orderas between good species of theV. sativacomplex. Inour study we found similar examples of such ‘new

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Figure 7. Visual interpretation reflecting diversity at three levels of analysis: RAPD, seed protein electrophoresis and morphological for threespecies of theV. sativaagg. Branchesa,b,c,d,e,f correspond to clusters on UPGMA trees derived from RAPD and seed protein analysis(Figure 3).

lines’ in the populations ofV. segetalisfrom CentralAsia, identified by an distinct electrophoretic pattern.As in the case ofV. cordata, plants with distinct elec-trophoretic patterns could not be distinguished mor-phologically. Some of them could be detected, usingbiochemical markers, as unique samples, others oc-curred with high frequency in almost all populations(proteintype 2of V. segetalis).

Based on our results, the genetic diversity ofV.sativaagg. in the wild and weedy populations couldbe described as a hierarchical model, a tree. The ma-jor clusters of the tree corresponded to eight taxa ofV. sativa agg.: V. sativa sensu stricto, V. segetalis,

V. angustifolia, V. cordata, V. macrocarpa, V. amphi-carpa, V. pilosa, V. incisa, sufficiently differentiatingto support their separate specific ranking.

We now present the structure of genetic diversityin three species ofV. sativa agg. (V. sativa sensustricto, V. segetalisand V. angustifolia)in the areaof the former USSR, based on diversity observed atthe phenotypic, biochemical and DNA levels in Fig-ure 7. RAPD polymorphisms divided each of thesethree species into two intraspecific groups. Based onseed protein analysis two intraspecific groups were de-tected inV. sativaandV. segetalis,but no intraspecificgroups were found inV. angustifolia. At the pheno-

183

typic level this intraspecific differentiation cannot bedetected, and interspecific differences are debatable.These results support the opinion that genetic diversitycan best be quantified on the basis of data obtained asclose to the DNA level as possible ( Hintum, 1995).

The V. sativaaggregate comprises, at present, acomplex of well separated taxa and derivated forms,representing various degrees of phylogenetic diver-gence. Although results of RAPD analysis and seedprotein electrophoresis showed considerable geneticdivergence between and within taxa ofV. sativaag-gregate, these clear-cut differences are not alwaysreflected at the phenotypic level.

Acknowledgements

This research was undertaken while the first authorwas a Science and Technology fellow of the Japan-ese Government. We thank Dr Ru-Quang Xu for helpin DNA extraction manipulations and technique. Wethank Professor P. Hanelt for providing, from theGatersleben genebank, some of the accessions used inthis experiment.

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