Isozyme Evidence on the Specific Distinctness and Phylogenetic Position of Vicia Incisa (Fabaceae)

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: Vicia 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: Vicia • Isozymes • Phylogenetic relationships • Systematics

1. Introduction into a complex of up to seven subspecies. However, others have treated the subspecies of V. sativa along Vicia incisa Bieb. is a rare vetch species with sporadic with V. incisa as separate species, i.e. V. sativa sensu and restricted distribution in the Mediterranean stricto, V. angustifolia Reichard, V. segetalis Thuill., V. region, including Turkey, the Crimea of Ukraine, pilosa Bieb., V. cordata Wulf. ex Hoppe, V. macrocarpa Bulgaria, Greece, Italy and south of France [1-5]. (Moris) Berthol., and V. amphicarpa Dorthes [9-12]. It has been attributed to section Vicia together with Cytological studies have reported the somatic V. sativa L. sensu lato, V. barbazitae Ten. & Guss., chromosome number 2n = 14 for all samples of V. V. grandiflora Scop. and V. pyrenaica Pourret [6,7]. incisa from Bulgaria [3], Italy [4] and Turkey [13] and By morphology, V. incisa resembles most closely V. revealed distinct differences from the V. sativa complex sativa, being distinguishable from it mainly by incised in the chromosome morphology and karyotype, thus leaflets. Taxonomically, V. incisa is a controversial providing evidence for the species status of V. incisa. species because it has also been treated either as Among the V. sativa complex, 2n = 14 is characteristic a subspecies or variety of Vicia sativa, i.e. V. sativa of V. amphicarpa, whereas V. sativa sensu stricto and subsp. incisa (Bieb.) Arcang. or V. sativa var. incisa most samples of V. angustifolia, including V. segetalis, (Bieb.) Boiss. [1,2,7,8]. In these cases, because of have 2n = 12; V. cordata and some samples of V. great morphological diversity, V. incisa has been split segetalis have 2n = 10 [8,10,14,15]. Further evidence

* E-mail: [email protected] 169 Isozyme evidence on the specific distinctness and phylogenetic position of Vicia incisa (Fabaceae)

on the distinctness of V. incisa from the V. sativa analysed was then doubled or tripled for isozymes complex has been presented by van de Wouw et al. that revealed polymorphism. Eight individuals of [16] and Potokina et al. [17] on the basis of amplified V. pyrenaica, ten individuals of V. lathyroides and fragment length polymorphism (AFLP) electrophoretic twelve individuals of V. incisa and per accession markers. were analysed. The following ten enzymes were The phylogenetic position of V. incisa in subgenus assayed for isozymes: aspartate aminotransferase Vicia has been studied with two different types of (AAT, EC 2.6.1.1), formate dehydrogenase (FDH, DNA markers that gave contradictory results [18]. EC 1.2.1.2), glutamate dehydrogenase (GDH, The maximum likelihood tree based on chloroplast EC 1.4.1.2), isocitrate dehydrogenase (IDH, EC restriction fragment length polymorphism (RFLP) data 1.1.1.42), malate dehydrogenase (MDH, EC 1.1.1.37), showed V. incisa in an unresolved polytomy with other 6-phosphogluconate dehydrogenase (PGD, EC taxa of the V. sativa group, whereas the parsimony 1.1.1.44), phosphoglucoisomerase (PGI, EC 5.3.1.9), tree of random amplified polymorphic DNA (RAPD) phosphoglucomutase (PGM, EC 2.7.5.1), shikimate data resolved V. incisa from the V. sativa taxa into a dehydrogenase (SKD, EC 1.1.1.25), superoxide separate clade together with V. lathyroides L. dismutase (SOD, EC 1.15.1.1). Previous isozyme studies of phylogenetic The following four gel-buffer systems and three relationships between species of Vicia subgenus catholytes were combined for different enzymes to Vicia placed species of section Vicia in one major achieve better band resolution: subgroup together with sections Sepium Radzhi. and Gel 1: 10% acrylamide, 0.2% N,N’-bisacrylamide Lathyroides (Buchenau) Tzvel., with V. bithynica (L.) (Bis), 0.25 M Tris, and 0.1 M HCl; applied for SOD L. of section Pseudolathyrus Tzvel. as basally linked with the glycine catholyte and for PGD and SKD with to all of them [19,20]. However, V. incisa was not the 2-alanine catholyte. studied in these papers. The current work extends Gel 2: 7.5% acrylamide, 0.2% Bis, 0.25 M Tris, and these previous studies by describing for the first 0.1 M HCl; applied for MDH and PGM with the glycine time isozymes encoded by 15 loci in V. incisa. The catholyte and for FDH and GDH with the 2-alanine isozyme characters of V. incisa are compared with catholyte. those of seven related species of sections Vicia, Gel 3: 7.5% acrylamide, 0.2% Bis, 0.125 M Tris, Sepium, Lathyroides and Pseudolathyrus in order to and 0.1 M HCl; applied for AAT with the glycine provide new evidence about the taxonomic rank and catholyte. phylogenetic position of V. incisa. 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 2. Experimental Procedures asparagine catholyte and with the 20 minutes overflow of the dye front. 2.1. Plant material N,N,N’,N’‑Tetramethylethylenediamine (0.05 ml%), The list of species and accessions analysed for riboflavine (0.5 mg%) and ammonium persulfate (1 isozymes is given in Table 1. Taxonomic nomenclature mg%) were added to the gel mixtures to initiate and is combined from several sources [6,7,11,21]. catalyse their photopolymerization between two Notably, the priority rule of the International Code of day‑light fluorescent bulbs during 1 h. Botanical Nomenclature is applied to the sectional The three catholytes used consisted of 80 mM names. The publication dates of the sectional names glycine, 2‑alanine or asparagine with 10 mM Tris. The are given in Table 1. Taxonomic identifications of lower anode buffer for gel systems 1- 3 was 0.1 M Tris accessions were verified by the morphology of plants with 0.02 M acetic acid, and it was used repeatedly grown from seeds in a greenhouse, following the while the pH remained over 7. The anolyte for the gel species descriptions in Ball [1], Davis and Plitmann system 4 consisted of 0.1 M triethanolamine with 0.02 [2], Tzvelev [5], Fedtschenko [9] and Potokina [12]. M acetic acid. Vouchers of species are preserved in the herbarium Electrophoresis in the anodal direction was carried of the Estonian Agricultural University (TAA). out in an ice‑refrigerated plexiglass apparatus for 120 x 800 x 2 mm vertical gel slabs by applying a pulsed 2.2. Isozyme analysis and designation current at 15 mA and 20‑30 V/cm until the marker dye, Enzyme extracts for electrophoresis were made as bromophenol blue, reached the gel end (about 2.5-3 described previously [20,22]. Preliminary analyses hours). After electrophoresis, the gels were stained used two seeds per accession. The number of seeds for isozymes by applying standard histochemical

170 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).

Section Pseudolathyrus Tzvelev (1980), = sect. Bithynicae (B. Fedtch. ex Radzhi) Maxted (1993). 10. V. bithynica (L.) L. France (BNA12/93, BNA48/99), Italy (BS57/87, BS253/88, BS8/92, BS45/94, 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 in a scale 0-100. The mobility values of allozymes on the basis of existing knowledge of isoenzyme and orthozymes are unified for each electrophoretic structure and genetic control [23], as described for system, using extracts of selected reference species Vicia species [19,22]. Isozymes of different genetic on the same gel slab in different combinations. nature are specified following the nomenclature described previously [22]. Heterologous and 2.3. Data analysis paralogous isozymes (= heterozymes and parazymes, Cladistic analysis of phylogenetic relationships was respectively) are designated by capital letters conducted using Fitch-Wagner parsimony by applying followed by numbers, indicating the electrophoretic heuristic search with tree‑bisection reconnection (TBS) mobility of their allozymic and orthozymic variants 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, A64, PGD-A82, SKD-A80, PGI-A71, SOD-B54, AAT- using the program PAUP* 4.0b10 [24]. Phenetic A78, and AAT-C30 by which they differ from the taxa analysis was also performed with the PAUP* program of the V. sativa complex. using the neighbour-joining clustering method, with In total, electrophoretic analyses revealed 39 a mean character difference by the Nei-Li distance orthozymes that are shared by two or more species measure. Reweighting of characters by maximum and 22 taxon-specific orthozymes of 15 heterozymes values of rescaled consistency indexes was applied in among the eight species studied. The outgroup species order to reduce the misleading effect of homoplasious V. bithynica was most divergent with six species- characters [25]. Branch supports were estimated specific orthozymes. The isozyme data matrixes for by bootstrapping with simple stepwise addition of cladistic and phenetic analyses of relationships among 1000 replications and TBS branch-swapping, as the species were compiled from Table 2. Rare variants implemented in PAUP*. detected in only some individuals of some accessions were not included. The cladistic most-parsimony analysis of the binary data matrix of 39 parsimony 3. Results informative orthozymes against the outgroup species V. bithynica gave three most parsimonious trees The data on the isozyme variants of V. incisa, of 73 steps length (not shown). The tree, however, in comparison with seven related vetch species had a low value of rescaled consistency index, RC belonging to sections Vicia, Sepium, Lathyroides and = 0.288, reflecting high level of homoplasy in the Pseudolathyrus, are given in Table 2. The zymogram isozyme data. This suggests a need for reweighting variation patterns among the vetch species were of characters in order to minimise the noise caused by described in detail previously and will not be repeated homoplasy [25]. Reweighting of characters once by here [19,22]. The electrophoretic mobility values in maximum value of RC gave a single stable tree of 17 Table 2 may differ from those previously reported steps length, with RC = 0.796 and retention index RI because of differences in the electrophoresis gel = 0.927 (Figure 1). The tree has the same topology as composition used, in particular for AAT and GDA the unweighted one and resolves the species into two [19,22]. Table 2 also includes the results of numerous major clades. One clade includes all three subspecies new accessions not analysed previously and thus of V. sativa studied, with subsp. cordata and nigra as supplements earlier studies [19,22]. Vicia bithynica most closely related sister taxa, followed by subsp. of section Pseudolathyrus was chosen as outgroup sativa as basally linked to them. Vicia lathyroides is because of its sister position to all species of sections placed in the same clade as basally linked to the V. Vicia, Lathyroides and Sepium on the isozyme trees sativa taxa with a high bootstrap support (95%). The of our previous works [19,22]. second clade includes V. grandiflora and V. sepium The data show that V. incisa differs from all three as closely related sister species followed by V. incisa, subspecies of the V. sativa sensu lato complex, V. oroboides and V. pyrenaica as successively linked nigra, cordata and sativa, in distinct orthozymes to them. of ten heterozymes out of 15 analysed. In a sharp The phenetic analysis of the same data matrix of 39 contrast, the subspecies of V. sativa have common shared orthozymes with the neighbour-joining method orthozymes, differing mostly by the presence of based on the Nei-Li distance yielded a tree of 74 steps additional allozymes of some heterozymes and in length (RC = 0.278, not shown) that has essentially their relative occurrence. Importantly, no isozyme the same topology as the most parsimony tree, except variation or differentiation was observed between the linking V. pyrenaica basally to V. lathyroides and the two accessions of V. incisa originating from different three V. sativa subspecies. Reweighting of characters geographical regions, i.e. Bulgaria and Slovakia, once by maximum values of RC gave a tree of 17 at any of the 15 heterozymes studied. Only limited steps length (not shown) that has the same topology allozymic variation was found among the numerous as the cladogram in Figure 1. 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, MDH-

172 V. Jaaska Table 2. V. bithynica Tzvel., outgroup Pseudolathyrus Section V. lathyroides Section Lathyroides V. oroboides V. sepium Section Sepium V. grandiflora V. pyrenaica subsp. sativa subsp. cordata subsp. nigra V. sativa sensu lato V. incisa Section Vicia Taxon Unified electrophoretic mobilities (scale 0 - 100) of formate (FDH), number of accessions analysed, r - rare variant, bold taxon-specific variant. (SKD), phosphoglucoisomerase (PGI), phosphoglucomutase 9 280 82 4 67 42 75 56 6 05 05;26 45 42;48r 64;59 65 53;42 80 56 10 9 67 26 942;48r 59 65 42 75 56 4 06/27 26 942 59 65 42 75 61/82 10 46/27 26 942 59 65 42 75 61/82 14 66/27 26 936;42 59 65 42 75 61/82 26 56 2 D- D- D- D- MDH-A IDH-A GDH-B GDH-A FDH-A N 71 44 ;56; 54 542 75 55 56;94 77;72 42 64;59 65 53 75 68 42; 45 36 44 280 82 42 64 65 53 63 65 57 936 59 037 60 64 (PGM), superoxide dismutase (SOD), and aspartate aminotransferase glutamate (GDH), isocitrate (IDH), malate (MDH), 6-phosphogluconate 42; 54 D- G- K- G- G- SOD PGM-A PGI-B SKD-A PGD-A MDH-B 75 27;77 95 07 58 80;73 69;57 72;67;76 72 26;27 98;77 54 70 80;77 69 67;72;76 72 77;82 72; 66 26;77;66;777 64;57 65;67;72;76 72 272 72 272 72 67 72;80;84; 70 08;27 76;17 85 39;32 54 58 73 57;64;71 80;84;72;78 28;57 47 85 39;32 54 58 77 64 72;84;65;78 65;60 64;71; 49 47 84 39 42 58 77 64 45 77 64;57 17 85 978 39 54 58 77 71 77 43 71 39 54 70 77 78 85 93 71;64 39;32 54 58 77;80 48 58; 67 ASDBSDCATA AAT-C AAT-A SOD-C SOD-B -A (AAT) orthozymes in Vicia species: N - the (PGD), and shikimate dehydrogenase 42; 30 23 145 71 39 42 23 71 39 42 97 45 71 39 93 845 78 39;32 47 30 64;78 145 71 71; 55 45; 36 38 ;30 45 45; 52 30

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

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 Figure 1. A single most parsimonious tree produced by the cladistic analysis of 39 parsimony informative isozyme charac- V. incisa on the RAPD tree [18]. The existence of ters after reweighting once on the basis of maximum value conflicting evidence about the phylogenetic position of RC: length = 17 steps, CI = 0.859, RI = 0.927, RC = 0.796. Bootstrap supports are given above nodes. of V. incisa based on isozymes and three different DNA markers indicates a need of further study with the use of more molecular markers. 4. Discussion Our isozyme cladogram ties V. incisa as basally linked to the V. grandiflora and V. sepium couple in the The isozyme data show that V. incisa has distinct same subclade, whereas the subspecies of V. sativa orthozymes of most heterozymes studied in appear together with V. lathyroides in a separate clade. comparison with the subspecies of V. sativa that The isozyme data and cladogram thus support the differ from each other mostly by the occurrence of inclusion of V. lathyroides in the type section together additional allozymes of some heterozymes. This with the V. sativa aggregate [6], instead of treating it result provides a new convincing support to a species in a separate section Lathyroides [11] or Wiggersia distinctness and status of V. incisa from the V. sativa [7]. Remarkably, the cladistic analysis of the isozyme complex, in agreement with the evidence from the variation reveal that sections Vicia and Sepium (= recent AFLP studies [16,17]. Among the taxa of the Atossa) of the morphology-based traditional taxonomy V. sativa complex, V. incisa is morphologically most [5,6,7,11] are not monophyletic groups because V. similar to V. sativa subsp. cordata. Because of this incisa and V. grandiflora of section Vicia are linked as great resemblance the latter is treated in some works sister species in the same subclade with V. sepium, [2,13] even as a variety of former, V. sativa subsp. the type species of section Sepium, whereas V. incisa (Bieb.) Arc. var. cordata (Wulfen ex Hoppe) sativa, the type species of section Vicia, appears in Arc. The data presented in Table 2 show that the a separate clade. The isozyme similarity between V. two, morphologically largely similar taxa are clearly grandiflora and V. sepium is in agreement with the divergent and distinguishable by alternate orthozymes evidence about their biochemical similarity in sharing of ten heterozymes out of 15 analysed. poisonous 2-cyanoalanin amino acids in seeds [25] An important result of the isozyme study is and karyological similarity [26], thus supporting the that V. incisa is not closely related to the V. sativa treatment of V. grandiflora together with V. sepium in complex despite of great morphological similarity, section Sepium rather than in the type section. Also, V. but instead reveals much more isozyme similarity to grandiflora and V. sepium have seeds with linear hilum both V. grandiflora and V. sepium that are attributed extending over 50% of the seed circumference. On the to different sections Vicia and Atossa (synonym of grounds of this similarity in the seed hilum length, the sect. Sepium) in traditional taxonomic treatments two species were grouped together with V. oroboides [6,7]. Our isozyme data are consistent with the AFLP in the section Atossa [27]. However, others preferred data [17] that tied V. incisa with V. grandiflora on the to keep V. grandiflora in the type section because of UPGMA phenogram and showed all taxa of the V. similarity to V. sativa in other morphological features sativa aggregate in a separate cluster [17]. However, [6,7]. The seeds of V. incisa differ distinctly from V. an earlier study [18] showed V. incisa together with V. grandiflora in having spherical seeds 3-4 mm thick lathyroides were basally linked to the taxa of the V. with linear hilum about 25-30% of seed circumference sativa group on the parsimony tree based on RAPD (personal observation), resembling more seeds of V. whereas, V. grandiflora was linked with V. sepium in sativa. a separate clade. The maximum likelihood tree based The plants with incised leaflets were described on RFLP retained V. incisa in an unresolved polytomy to occur rarely within V. grandiflora (= var. dissecta

174 V. Jaaska

Boiss.) and V. barbazitae Ten. & Guss. (= var. incisa Acknowledgement (Orph.) Boiss.) [2,7]. These taxa were not available for the present study; all nine accessions of V. The author thanks all botanical gardens, seed banks grandiflora analysed belonged to the widespread type and colleagues listed in Table 1 for kindly sending variety. A thorough study of phylogenetic relationships seeds for the study. This work was supported by grant within and between sections Vicia and Sepium of ETF 5739 from the Estonian Science Foundation and current taxonomy was not possible at the present by grant TF 0362481s03 from the Estonian Ministry of time because of difficulties in obtaining seeds of the Education and Science. rare Near East and Transcaucasian endemics V. barbazitae, V. balansae and V. truncatula. This would be a task to pursue in the future.

References

[1] Ball P.W., Vicia L., In: Flora Europaea, Vol. 2. Cam- L. subsp. incisa (Bieb.) Arc. var. incisa, Turkish J. bridge University Press, Cambridge, 1968, 129-136 Bot., 1999, 23, 63-67 [2] Davis P.H., Plitmann U.P., Vicia. In: Flora of Turkey [14] Hanelt P., Mettin D., Cytosystematische Untersu- and East Aegean Islands, Vol. 3, Edinburgh Univer- chungen in der Artengruppe um Vicia sativa L. II, sity Press, Edinburgh, 1970, 274-325 Kulturpflanze, 1966, 14, 137-161 (in German) [3] Terziiski D., Karyological investigation of the vetch [15] Hollings E., Stace A., Karyotype variation and evo- Vicia incisa M.B. (Fabaceae Lindley), In: Scientific lution in the Vicia sativa aggregate, New Phytol., Works, “Vassil Kolarov” Higher Institute of Agricul- 1979, 73, 195-208 ture, Plovdiv, Vol. 32, Botany, 19-25 (in Bulgarian) [16] Van de Wouw M., Maxted N., Chabane K., Ford- [4] Roti Michelozzi G., Barberis G., Biosystematic notes Lloyd B.V., Molecular taxonomy of Vicia ser. Vicia on Vicia incisa M. Bieb. (Leguminosae), a rare spe- based on Amplified Fragment Length Polymor- cies from Italy, Atti Soc. Tosc. Sc. Nat. Mem., Serie phisms, Plant Syst. Evol., 2001, 229, 91-105 B, 1989, 96, 219-225 [17] Potokina E., Blattner R.F., Alexandrova T., Bach- [5] Tzvelev N., Vicia L., In: Flora Partis Europaeae mann K., AFLP diversity in the common vetch USSR, Vol. 6, Nauka, Leningrad, 1989, 127-147 (in (Vicia sativa L.) on the world scale, Theor. Appl. Russian) Genet., 2002, 105, 58-67 [6] Kupicha F.K., The infrageneric structure of Vicia, [18] Potokina E. , Tomooka N., Vaughan D.A., Alexran- Notes Roy. Bot. Gard. Edinburgh, 1976, 34, 287-326 drova T., Xu R.-Q., Phylogeny of Vicia subgenus [7] Maxted N., A phenetic investigation of Vicia L. subge- Vicia (Fabaceae) based on analysis of RAPDs and nus Vicia (Leguminosae, Vicieae), Bot. J. Linn. Soc., RFLP of PCR-amplified chloroplast genes, Genet. 1993, 111, 155-182 Res. Crop Evol., 1999, 46, 149-161 [8] Zohary D., Plitmann U., Chromosome polymorphism, [19] Jaaska V., Isoenzyme diversity and phylogenetic af- hybridization and colonization in the Vicia sativa finities in Vicia subgenu. Vicia (Fabaceae), Genet. group (Fabaceae), Plant Syst. Evol., 1979, 131, Res. Crop Evol., 1997, 44, 557-574 143-156 [20] Leht M., Jaaska V., Cladistic and phenetic analysis [9] Fedtschenko B., Genus Vicia L., In: Flora URSS Vol. of relationships in Vicia subgenus Vicia (Fabaceae) 13, Academy of Sciences of USSR, Moscow and by morphology and isozymes, Plant Syst. Evol., Leningrad, 1948, 406-475 (in Russian) 2002, 232, 237-260 [10] Mettin D., Hanelt P., Cytosystematische Untersu- [21] Radzhi A.D., Conspectus systematis specierum chungen in der Artengruppe um Vicia sativa L. I., Caucasicarum Generis Vicia L. Novitates System- Kulturpflanze, 1964, 7, 163-225 (in German) aticae Plantarum Vascularium, 7, 228-240 (in Rus- [11] Tzvelev N., Systema specierum generis Vicia L. in sian) parte Europaea URSS, Novitates Systematicae [22] Jaaska V., Isozyme variation and phylogenetic re- Plantarum Vascularium, 1980, 17, 200-208 (in lationships in Vicia subgenus Cracca (Fabaceae), Russian) Ann. Bot., 2005, 96, 1085-1096 [12] Potokina E.K., Vicia sativa L. aggregate (Fabaceae) [23] Wendel J.F., Weeden N.F., Visualization and interpre- in the flora of the former USSR, Genet. Res. Crop. tation of plant isozymes, In: Isozymes in plant biol- Evol., 1997, 44, 141-161 ogy, Dioscoroides Press, Portland, OR, 1989, 5-45 [13] Meric C., Dane F., Karyological studies on V. sativa [24] Swofford D.L., PAUP* 4.0b10. Phylogenetic analy-

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

sis using parsimony (* and other methods), Sinauer Associates, Sunderland, MA, 2002 [25] Farris J., A successive approximations approach to character weighting, Syst. Zool., 1969, 18, 374-385 [26] Tschierisch B., Hanelt P., Die freien Aminosauren der Samen von Vicia L. und die systematische Gliederung der Gattung, Flora, 1967, 157A, 389-406 (in German) [27] Ascherson P.F.A., Graebner C.O.R.P.P., Vicia, In: Synopsis der mitteleuropäischen Flora, Bd. 6, H. 2, Leipzig, 1909, 902-995 (in German)

176