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Cent. Eur. J. Biol. • 3(2) • 2008 • 169–176 DOI: 10.2478/s11535-007-0049-3

Central European Journal of Biology

Isozyme evidence on the specific distinctness and phylogenetic position of Vicia incisa (Fabaceae) Research article

Vello Jaaska

*

Department of Botany, Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, 51014 Tartu, Estonia

Received 1 October 2007; Accepted 19 November 2007

Abstract: V  icia incisa is a taxonomically controversial species that has been also treated as a subspecies of V. sativa because of a great morphological similarity. The phylogenetic position of V. incisa is uncertain because various DNA markers have provided contradictory results. Isozymes of V. incisa encoded by 15 loci and resolved with the use of polyacrylamide gel electrophoresis (PAGE) are described and compared with those of seven related species belonging to sections Vicia, Sepium, Lathyroides and Pseudolathyrus in order to get new evidence about its taxonomic rank and phylogenetic position. Phylogenetic relationships are analyzed with maximum parsimony and neighbour joining methods. Vicia incisa is shown to differ from all three subspecies of V. sativa including, sativa, cordata and nigra, by alternate variants of ten isozymes out of 15 analysed. Instead, V. incisa has much more similarity to V. grandiflora and V. sepium by sharing eight isozyme variants which differ from the subspecies of V. sativa. The most parsimony and neighbour joining analyses of the isozyme variation placed V. incisa as basally linked to the V. grandiflora and V. sepium species couple in the clade of section Sepium (= sect. Atossa), while the subspecies of V. sativa together with V. lathyroides formed a separate clade of section Vicia. The isozyme data provide further support to the species status of V. incisa. Keywords: V  icia • Isozymes • Phylogenetic relationships • Systematics

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

1. Introduction Vicia incisa Bieb. is a rare vetch species with sporadic and restricted distribution in the Mediterranean region, including Turkey, the Crimea of Ukraine, Bulgaria, Greece, Italy and south of France [1-5]. It has been attributed to section Vicia together with V. sativa L. sensu lato, V. barbazitae Ten. & Guss., V. grandiflora Scop. and V. pyrenaica Pourret [6,7]. By morphology, V. incisa resembles most closely V. sativa, being distinguishable from it mainly by incised leaflets. Taxonomically, V. incisa is a controversial species because it has also been treated either as a subspecies or variety of Vicia sativa, i.e. V. sativa subsp. incisa (Bieb.) Arcang. or V. sativa var. incisa (Bieb.) Boiss. [1,2,7,8]. In these cases, because of great morphological diversity, V. incisa has been split * E-mail: [email protected]

into a complex of up to seven subspecies. However, others have treated the subspecies of V. sativa along with V. incisa as separate species, i.e. V. sativa sensu stricto, V. angustifolia Reichard, V. segetalis Thuill., V. pilosa Bieb., V. cordata Wulf. ex Hoppe, V. macrocarpa (Moris) Berthol., and V. amphicarpa Dorthes [9-12]. Cytological studies have reported the somatic chromosome number 2n = 14 for all samples of V. incisa from Bulgaria [3], Italy [4] and Turkey [13] and revealed distinct differences from the V. sativa complex in the chromosome morphology and karyotype, thus providing evidence for the species status of V. incisa. Among the V. sativa complex, 2n = 14 is characteristic of V. amphicarpa, whereas V. sativa sensu stricto and most samples of V. angustifolia, including V. segetalis, have 2n = 12; V. cordata and some samples of V. segetalis have 2n = 10 [8,10,14,15]. Further evidence

169

Isozyme evidence on the specific distinctness and phylogenetic position of Vicia incisa (Fabaceae)

on the distinctness of V. incisa from the V. sativa complex has been presented by van de Wouw et al. [16] and Potokina et al. [17] on the basis of amplified fragment length polymorphism (AFLP) electrophoretic markers. The phylogenetic position of V. incisa in subgenus Vicia has been studied with two different types of DNA markers that gave contradictory results [18]. The maximum likelihood tree based on chloroplast restriction fragment length polymorphism (RFLP) data showed V. incisa in an unresolved polytomy with other taxa of the V. sativa group, whereas the parsimony tree of random amplified polymorphic DNA (RAPD) data resolved V. incisa from the V. sativa taxa into a separate clade together with V. lathyroides L. Previous isozyme studies of phylogenetic relationships between species of Vicia subgenus Vicia placed species of section Vicia in one major subgroup together with sections Sepium Radzhi. and Lathyroides (Buchenau) Tzvel., with V. bithynica (L.) L. of section Pseudolathyrus Tzvel. as basally linked to all of them [19,20]. However, V. incisa was not studied in these papers. The current work extends these previous studies by describing for the first time isozymes encoded by 15 loci in V. incisa. The isozyme characters of V. incisa are compared with those of seven related species of sections Vicia, Sepium, Lathyroides and Pseudolathyrus in order to provide new evidence about the taxonomic rank and phylogenetic position of V. incisa.

2. Experimental Procedures 2.1. Plant material

The list of species and accessions analysed for isozymes is given in Table 1. Taxonomic nomenclature is combined from several sources [6,7,11,21]. Notably, the priority rule of the International Code of Botanical Nomenclature is applied to the sectional names. The publication dates of the sectional names are given in Table 1. Taxonomic identifications of accessions were verified by the morphology of plants grown from seeds in a greenhouse, following the species descriptions in Ball [1], Davis and Plitmann [2], Tzvelev [5], Fedtschenko [9] and Potokina [12]. Vouchers of species are preserved in the herbarium of the Estonian Agricultural University (TAA).

2.2. Isozyme analysis and designation

Enzyme extracts for electrophoresis were made as described previously [20,22]. Preliminary analyses used two seeds per accession. The number of seeds 170

analysed was then doubled or tripled for isozymes that revealed polymorphism. Eight individuals of V. pyrenaica, ten individuals of V. lathyroides and twelve individuals of V. incisa and per accession were analysed. The following ten enzymes were assayed for isozymes: aspartate aminotransferase (AAT, EC 2.6.1.1), formate dehydrogenase (FDH, EC 1.2.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2), isocitrate dehydrogenase (IDH, EC 1.1.1.42), malate dehydrogenase (MDH, EC 1.1.1.37), 6-phosphogluconate dehydrogenase (PGD, EC 1.1.1.44), phosphoglucoisomerase (PGI, EC 5.3.1.9), phosphoglucomutase (PGM, EC 2.7.5.1), shikimate dehydrogenase (SKD, EC 1.1.1.25), superoxide dismutase (SOD, EC 1.15.1.1). The following four gel-buffer systems and three catholytes were combined for different enzymes to achieve better band resolution: Gel 1: 10% acrylamide, 0.2% N,N’-bisacrylamide (Bis), 0.25 M Tris, and 0.1 M HCl; applied for SOD with the glycine catholyte and for PGD and SKD with the 2-alanine catholyte. Gel 2: 7.5% acrylamide, 0.2% Bis, 0.25 M Tris, and 0.1 M HCl; applied for MDH and PGM with the glycine catholyte and for FDH and GDH with the 2-alanine catholyte. Gel 3: 7.5% acrylamide, 0.2% Bis, 0.125 M Tris, and 0.1 M HCl; applied for AAT with the glycine catholyte. Gel 4: 10% acrylamide, 0.2% Bis, 0.1% triethanolamine hydrochloride and 5 mM Trilon B (disodium EDTA); applied for IDH and PGI with the asparagine catholyte and with the 20 minutes overflow of the dye front. N,N,N’,N’‑Tetramethylethylenediamine (0.05 ml%), riboflavine (0.5 mg%) and ammonium persulfate (1 mg%) were added to the gel mixtures to initiate and catalyse their photopolymerization between two day‑light fluorescent bulbs during 1 h. The three catholytes used consisted of 80 mM glycine, 2‑ala­nine or asparagine with 10 mM Tris. The lower anode buffer for gel systems 1- 3 was 0.1 M Tris with 0.02 M acetic acid, and it was used re­peatedly while the pH remained over 7. The anolyte for the gel system 4 consisted of 0.1 M triethanolamine with 0.02 M acetic acid. Electrophoresis in the anodal direction was carried out in an ice‑refrigerated plexiglass apparatus for 120 x 800 x 2 mm vertical gel slabs by applying a pulsed cur­rent at 15 mA and 20‑30 V/cm until the marker dye, bromophenol blue, reached the gel end (about 2.5-3 hours). After electrophoresis, the gels were stained for isozymes by applying standard histochemical

V. Jaaska

Taxon name

Geographical origin and accession numbers with key letters (in parentheses) Genus Vicia L. section Vicia.

1. V. incisa Bieb.

Bulgaria (G696/74); Slovakia (G1001/84).

= V. sativa subsp. incisa (Bieb.) Arc. 2. V. sativa L. s. str.

France (BCA45/93); Greece, Crete (MHN48/02); Italy (BS41/03, BP53/03);

= V. sativa subsp. sativa

Syria VJ66/97); Turkey (IG 60668, TR57554, TR63181, TR63184, TR63199).

3. V. cordata Wulfen ex Hoppe

Greece (G459/78, G461/75, G465/75, G468/74, G469/74, G470/75); France

= V. sativa subsp. cordata

(BCA15/93); Italy (BP29/94); Portugal (BCO41/88, BCO35/99); Turkey

(Wulfen) Asch. & Graebn.

(G453/77, G454/77, G455/77, G457/77).

4. V. angustifolia Reichard

France (BCA130/88, MHN9/89, BD20/94, BCA30/96, BD3/97, BD4/97,

= V. sativa subsp. nigra (L:) Ehrh.

MHN47/02, MHN49/02, MHN50/02); Germany (BHU216/88, BHU219/88, BHU220/88, BHU114/96, BBD57/99, BH131/02, BH133/02); Italy (BGE51/93, BS28/94, BS77/96, BS78/96, BS50/97, BS42/03); Portugal (G431/75, BCO42/88, BCO129/96); Turkey (TAS7/93).

5. V. pyrenaica Pourret

France (BBD35/93, BJL148/00, G59/90, G69/89).

6. V. grandiflora Scop.

Armenia (EN2409, EN3215, EN3399); Germany (BBD172/88, BHU227/88,

subsp. grandiflora

BHU228/88) Hungary (IAB024931, IAB024932, IAB069107). Section Sepium Radzhi (1971), = sect. Atossa (Alef.) Aschers. & Graebn. ex Kupicha (1976).

7. Vicia sepium L.

Estonia (ML222/87, ML225/87, VJ27/89, VJ41/89); Finland (BHF96-97/88). France (BCA133/88, MHN51/02), Germany (BBD178/88, BHU94/99).

8. Vicia oroboides Wulfen

Bosnia-Herzegovina (BSA185/87, BSA164/88), Czech Republic (BPO24/99), Germany (BGU135/90, BLE114/99), Slovenia (BLJ105/02). Section Lathyroides (Buchenau) Tzvelev (1980), = sect. Wiggersia (Alef.) Maxted (1993).

9. V. lathyroides L.

Czech Republic (BPO38/97); Germany (BGU33/93, PI422500); Italy (BP141/02).

10. V. bithynica (L.) L.

France (BNA12/93, BNA48/99), Italy (BS57/87, BS253/88, BS8/92, BS45/94,

Section Pseudolathyrus Tzvelev (1980), = sect. Bithynicae (B. Fedtch. ex Radzhi) Maxted (1993). BP58/03); Ukraine, the Crimea (BNU106/87).

Table 1.

List of the taxa and accessions investigated. The accession numbers with key letters of seed sources are given in parentheses.

B: received from various botanical gardens (BG) coded by key letters following B: BBD, BG of Berlin-Dahlem (Germany); BCA, BG of Caen, France; BCO, BG of Coimbra (Portugal); BD, BG of Dijon, France; BF, BG of Firenze (Italy); BG of BGE, BG of Genova (Italy); BGU, BG of the Göttingen University; BH, BG of Hamburg (Germany); BHF, BG of the Helsinki University (Finland); BHU, BG of the Halle University (Germany); BJL, BG of the Justus-Liebig University in Giessen (Germany); BLE, BG of the Leipzig University (Germany); BLI, BG of the Lisboa University (Portugal); BLJ, BG of the Ljubljana University (Slovenia); BNA, BG of Nantes (France); BNU, BG of Nikita (Ukraine, the Crimea); BP, BG of Palermo (Italy); BPO, BG of Palacky University in Olomouc (Czech Republic); BRK, Royal BG of Kew (England); BS, BG of the University of Siena (Italy); BSA, BG of Sarajevo (Bosnia); EN, collected by Dr. Estella Nazarova of the Armenian Institute of Botany in Yerevan (Armenia); IAB, the collection of the Institute for Agrobotany (Tapioszele, Hungary); IG, the collection of the International Center for Agricultural Research in the Dry Areas (ICARDA, Aleppo, Syria); MHN, Muséum d’Historie Naturelle in Paris (France); ML, collected by Dr. Malle Leht of the Estonian University of Life Sciences (Tartu, Estonia); PI, the collection of the USDA Regional Plant Introduction Station at the Washington State University (Pullman, Washington, USA); TAS, received form Dr. A. Sahin of Firat University in Elazig (Turkey); TR, the collection of the Aegean Agricultural Research Institute (Izmir, Turkey); G, the collection of the Institute of Plant Genetics and Crop Plant Research (Gatersleben, Germany); VJ, collected by the author. Original numbers are applied for the accessions received from seed banks and Dr. Nazarova, whereas accession from botanical gardens and other persons are labeled by numbers in the author’s seed collection, the number after a slash indicating the year of receipt. The accessions received from botanical gardens and persons were collected in wild from known localities.

methods [23]. Isozyme phenotypes were interpreted on the basis of existing knowledge of isoenzyme structure and genetic control [23], as described for Vicia species [19,22]. Isozymes of different genetic nature are specified following the nomenclature described previously [22]. Heterologous and paralogous isozymes (= heterozymes and parazymes, respectively) are designated by capital letters followed by numbers, indicating the electrophoretic mobility of their allozymic and orthozymic variants

in a scale 0-100. The mobility values of allozymes and orthozymes are unified for each electrophoretic system, using extracts of selected reference species on the same gel slab in different combinations.

2.3. Data analysis

Cladistic analysis of phylogenetic relationships was conducted using Fitch-Wagner parsimony by applying heuristic search with tree‑bisection reconnection (TBS) branch‑swapping, multiple parsimony (MULPARS), 171

Isozyme evidence on the specific distinctness and phylogenetic position of Vicia incisa (Fabaceae)

simple stepwise taxon application of 200 replications, using the program PAUP* 4.0b10 [24]. Phenetic analysis was also performed with the PAUP* program using the neighbour-joining clustering method, with a mean character difference by the Nei-Li distance measure. Reweighting of characters by maximum values of rescaled consistency indexes was applied in order to reduce the misleading effect of homoplasious characters [25]. Branch supports were estimated by bootstrapping with simple stepwise addition of 1000 replications and TBS branch-swapping, as implemented in PAUP*.

3. Results The data on the isozyme variants of V. incisa, in comparison with seven related vetch species belonging to sections Vicia, Sepium, Lathyroides and Pseudolathyrus, are given in Table 2. The zymogram variation patterns among the vetch species were described in detail previously and will not be repeated here [19,22]. The electrophoretic mobility values in Table 2 may differ from those previously reported because of differences in the electrophoresis gel composition used, in particular for AAT and GDA [19,22]. Table 2 also includes the results of numerous new accessions not analysed previously and thus supplements earlier studies [19,22]. Vicia bithynica of section Pseudolathyrus was chosen as outgroup because of its sister position to all species of sections Vicia, Lathyroides and Sepium on the isozyme trees of our previous works [19,22]. The data show that V. incisa differs from all three subspecies of the V. sativa sensu lato complex, nigra, cordata and sativa, in distinct orthozymes of ten heterozymes out of 15 analysed. In a sharp contrast, the subspecies of V. sativa have common orthozymes, differing mostly by the presence of additional allozymes of some heterozymes and in their relative occurrence. Importantly, no isozyme variation or differentiation was observed between the two accessions of V. incisa originating from different geographical regions, i.e. Bulgaria and Slovakia, at any of the 15 heterozymes studied. Only limited allozymic variation was found among the numerous accessions of V. sativa subsp. cordata and subsp. nigra of different geographical origin studied, while subsp. sativa variation between accessions with four allozymes of SKD-A, which was invariant in the two other subspecies. Unexpectedly, V. incisa revealed much more similarity to V. grandiflora and V. sepium by sharing common orthozymes GDH-B53, MDH172

A64, PGD-A82, SKD-A80, PGI-A71, SOD-B54, AATA78, and AAT-C30 by which they differ from the taxa of the V. sativa complex. In total, electrophoretic analyses revealed 39 orthozymes that are shared by two or more species and 22 taxon-specific orthozymes of 15 heterozymes among the eight species studied. The outgroup species V. bithynica was most divergent with six speciesspecific orthozymes. The isozyme data matrixes for cladistic and phenetic analyses of relationships among the species were compiled from Table 2. Rare variants detected in only some individuals of some accessions were not included. The cladistic most-parsimony analysis of the binary data matrix of 39 parsimony informative orthozymes against the outgroup species V. bithynica gave three most parsimonious trees of 73 steps length (not shown). The tree, however, had a low value of rescaled consistency index, RC = 0.288, reflecting high level of homoplasy in the isozyme data. This suggests a need for reweighting of characters in order to minimise the noise caused by homoplasy [25]. Reweighting of characters once by maximum value of RC gave a single stable tree of 17 steps length, with RC = 0.796 and retention index RI = 0.927 (Figure 1). The tree has the same topology as the unweighted one and resolves the species into two major clades. One clade includes all three subspecies of V. sativa studied, with subsp. cordata and nigra as most closely related sister taxa, followed by subsp. sativa as basally linked to them. Vicia lathyroides is placed in the same clade as basally linked to the V. sativa taxa with a high bootstrap support (95%). The second clade includes V. grandiflora and V. sepium as closely related sister species followed by V. incisa, V. oroboides and V. pyrenaica as successively linked to them. The phenetic analysis of the same data matrix of 39 shared orthozymes with the neighbour-joining method based on the Nei-Li distance yielded a tree of 74 steps length (RC = 0.278, not shown) that has essentially the same topology as the most parsimony tree, except linking V. pyrenaica basally to V. lathyroides and the three V. sativa subspecies. Reweighting of characters once by maximum values of RC gave a tree of 17 steps length (not shown) that has the same topology as the cladogram in Figure 1.

V. Jaaska

V. grandiflora

V. pyrenaica

subsp. sativa

subsp. cordata

subsp. nigra

6

10

9

4

10

14

26

2

N

82

56

56

44;56;54

56

61/82

61/82

61/82

56

FDH-A

75

80

75

80

75

75

75

75

75

68

GDH-A

42

42;45

42

53;42

53

42

42

42

42

53

GDH-B

63

65

57

65

65

65

65

65

65

65

IDH-A

59

60

64

64;59

64;59

59

59

59

59

64

MDH-A

36

37

42;54

42;48r

42

42;48r

42

42

36;42

42

MDH-B

75

72

72

77;82

77;72

72;66

72

72

72

82

PGD-A

67

72;67;76

67;72;76

72;80;84;70

80;84;72;78

72;84;65;78

65;67;72;76

72

72

80

SKD-A

65;60

69;57

69

64;71;49

57;64;71

64

64;57

64

64;57

71

PGI-B

77

80;73

80;77

77;80

73

77

77

77

77

77

PGM-A

70

58

70

58

58

58

48

58

58;67

58

SOD-A

54

42;30

54

54

54

54

42

42

42

54

SOD-B

39

39

39;32

39;32

39;32

39;32

39

39

39

39

SOD-C

71

71

78

71;64

64;78

71

71

71;55

71

78

AAT-A

45;36

45

45

38;30

30

45

45

45

45;52

30

AAT-C

Taxon

V. sepium

4

71

Section Pseudolathyrus Tzvel., outgroup

V. lathyroides

Section Lathyroides

Section Sepium

V. sativa sensu lato

V. incisa

Section Vicia

V. oroboides

9

V. bithynica

Table 2.

Unified electrophoretic mobilities (scale 0-100) of formate (FDH), glutamate (GDH), isocitrate (IDH), malate (MDH), 6-phosphogluconate (PGD), and shikimate dehydrogenase (SKD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), superoxide dismutase (SOD), and aspartate aminotransferase (AAT) orthozymes in Vicia species: N - the number of accessions analysed, r - rare variant, bold - taxon-specific variant.

173

Isozyme evidence on the specific distinctness and phylogenetic position of Vicia incisa (Fabaceae)

Figure 1.

A single most parsimonious tree produced by the cladistic analysis of 39 parsimony informative isozyme characters after reweighting once on the basis of maximum value of RC: length = 17 steps, CI = 0.859, RI = 0.927, RC = 0.796. Bootstrap supports are given above nodes.

4. Discussion The isozyme data show that V. incisa has distinct orthozymes of most heterozymes studied in comparison with the subspecies of V. sativa that differ from each other mostly by the occurrence of additional allozymes of some heterozymes. This result provides a new convincing support to a species distinctness and status of V. incisa from the V. sativa complex, in agreement with the evidence from the recent AFLP studies [16,17]. Among the taxa of the V. sativa complex, V. incisa is morphologically most similar to V. sativa subsp. cordata. Because of this great resemblance the latter is treated in some works [2,13] even as a variety of former, V. sativa subsp. incisa (Bieb.) Arc. var. cordata (Wulfen ex Hoppe) Arc. The data presented in Table 2 show that the two, morphologically largely similar taxa are clearly divergent and distinguishable by alternate orthozymes of ten heterozymes out of 15 analysed. An important result of the isozyme study is that V. incisa is not closely related to the V. sativa complex despite of great morphological similarity, but instead reveals much more isozyme similarity to both V. grandiflora and V. sepium that are attributed to different sections Vicia and Atossa (synonym of sect. Sepium) in traditional taxonomic treatments [6,7]. Our isozyme data are consistent with the AFLP data [17] that tied V. incisa with V. grandiflora on the UPGMA phenogram and showed all taxa of the V. sativa aggregate in a separate cluster [17]. However, an earlier study [18] showed V. incisa together with V. lathyroides were basally linked to the taxa of the V. sativa group on the parsimony tree based on RAPD whereas, V. grandiflora was linked with V. sepium in a separate clade. The maximum likelihood tree based on RFLP retained V. incisa in an unresolved polytomy 174

with other taxa of the V. sativa group, but placed V. grandiflora and V. sepium together in a separate, basally linked clade [18]. In addition, the two studies agree that V. grandiflora and V. sepium are closely related sister species, but disagree about linking V. incisa in the same clade with the taxa of the V. sativa group. The isozyme data agree with the RAPD data in resolving V. incisa from the V. sativa group, but disagree about linking V. incisa with V. lathyroides in the same clade as sister species and also about placing V. grandiflora and V. sepium separately from V. incisa on the RAPD tree [18]. The existence of conflicting evidence about the phylogenetic position of V. incisa based on isozymes and three different DNA markers indicates a need of further study with the use of more molecular markers. Our isozyme cladogram ties V. incisa as basally linked to the V. grandiflora and V. sepium couple in the same subclade, whereas the subspecies of V. sativa appear together with V. lathyroides in a separate clade. The isozyme data and cladogram thus support the inclusion of V. lathyroides in the type section together with the V. sativa aggregate [6], instead of treating it in a separate section Lathyroides [11] or Wiggersia [7]. Remarkably, the cladistic analysis of the isozyme variation reveal that sections Vicia and Sepium (= Atossa) of the morphology-based traditional taxonomy [5,6,7,11] are not monophyletic groups because V. incisa and V. grandiflora of section Vicia are linked as sister species in the same subclade with V. sepium, the type species of section Sepium, whereas V. sativa, the type species of section Vicia, appears in a separate clade. The isozyme similarity between V. grandiflora and V. sepium is in agreement with the evidence about their biochemical similarity in sharing poisonous 2-cyanoalanin amino acids in seeds [25] and karyological similarity [26], thus supporting the treatment of V. grandiflora together with V. sepium in section Sepium rather than in the type section. Also, V. grandiflora and V. sepium have seeds with linear hilum extending over 50% of the seed circumference. On the grounds of this similarity in the seed hilum length, the two species were grouped together with V. oroboides in the section Atossa [27]. However, others preferred to keep V. grandiflora in the type section because of similarity to V. sativa in other morphological features [6,7]. The seeds of V. incisa differ distinctly from V. grandiflora in having spherical seeds 3-4 mm thick with linear hilum about 25-30% of seed circumference (personal observation), resembling more seeds of V. sativa. The plants with incised leaflets were described to occur rarely within V. grandiflora (= var. dissecta

V. Jaaska

Boiss.) and V. barbazitae Ten. & Guss. (= var. incisa (Orph.) Boiss.) [2,7]. These taxa were not available for the present study; all nine accessions of V. grandiflora analysed belonged to the widespread type variety. A thorough study of phylogenetic relationships within and between sections Vicia and Sepium of current taxonomy was not possible at the present time because of difficulties in obtaining seeds of the rare Near East and Transcaucasian endemics V. barbazitae, V. balansae and V. truncatula. This would be a task to pursue in the future.

Acknowledgement The author thanks all botanical gardens, seed banks and colleagues listed in Table 1 for kindly sending seeds for the study. This work was supported by grant ETF 5739 from the Estonian Science Foundation and by grant TF 0362481s03 from the Estonian Ministry of Education and Science.

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