International Journal of Nature and Life Sciences (IJNLS) https://www.dergipark.gov.tr/ijnls e-ISSN: 2602-2397 Vol. 5 (1), June 2021, pp. 11-22 https://doi.org/10.47947/ijnls.840322 -------------------------------------------------------------------------------------------------Research article Molecular Phylogenetic Analyses of Vicia L. (Fabaceae) Taxa Growing in the Southeastern Anatolia Region Based on Chloroplast TrnL Sequences Alevcan Kaplan1*, Alaattin Selçuk Ertekin2, Esra Gündüzer3 1Batman University, Sason Vocational School, Department of Crop and Animal Production, Batman, Turkey,72060, orcid.org/0000-0001-6738-7527 2Ekinciler Street, Kalender Center, No: 9, Yenişehir, Diyarbakır, Turkey, 21100, orcid.org/0000-0003-2179-3778 3Gazi University, Life Sciences Application and Research Center, Ankara, Turkey, 06100, orcid.org/0000-00024474-7523 *Corresponding author: kaplanalevcan@gmail.com Received: 14 December 2020, Accept: 02 January 2021, Published Online: 01 June 2021 Abstract In the current study, some natural Vicia L. taxa growing naturally in the Southeast Anatolia Region were investigated from the point of view of molecular phylogenetic. For this purpose, transfer ribonucleic acid Leucine (trnL) was sequenced in order to study the phylogenetic relationships of the Vicia L. species. Lathyrus inconspicuous L. var. inconspicuous and Lathyrus cassius Boiss. were used as an outgroup. The length of the trnL area was determined approximately as 269-534 bp. The trnL sequences were submitted to the NCBI database and accession numbers received. We obtained information about the point mutations, as well as the protected and the changing characters. The taxa distinguished in the section level were separated and also grouped in terms of proximity on the phylogenetic tree (sect. Narbonensis, sect. Lathyroides, sect. Peregrinae, sect. Vicia, sect. Lentopsis). While, sect. Hypechusa, sect. Ervilia and sect. Cracca were not detailed to a great level of informativity. The data acquired were observed to be greatly reliable in terms of their ability to be used in solving taxonomical problems of the Vicia L. taxa. Key words: cpDNA, Fabaceae, molecular phylogeny, sequencing, Vicia L. 1. Introduction The legume family includes 720-730 genera and about 19.500 species. There are 69 genera in Turkey containing 1128 taxa and including 375 endemic taxa (Simpson, 2012). The genus Vicia is a member of the Vicieae legume tribe of Papilionoideae, and the genus itself has proven to be a popular group to study (Frediani et al., 2004). Vicia L. comprises about 210 species, is considered to have 22 divisions in two subgenus (Vicia and Vicilla), and is widely distributed throughout Europe and Asia (~110 species) North America (~ 20 species) and Africa (~15 species) (Kupicha, 1976; Weber and Schifino-Wittmann, 1999; Roze and Rurane, 2013). The genus 11 was originally localized to the Mediterranean and Irano-Turanian regions (Kupicha, 1981). The Mediterranean region is the main diversity center (Gunn, 1980; Naranjo et al., 1998). In Turkey, 64 species, 22 subspecies and 18 varieties of this genus have been recorded (Davis, 1970; Vural, 2000; Inceer and Ayaz, 2005; Başbağ et al., 2013). Vicia L. species are morphologically diverse and the morphological approach find it quite difficult to account for all the genetic variation that exist. Previous papers have reported morphological, anatomical, cytological, karyological and biochemical and molecular (although this has not been sufficient) data in order to elucidate the relationships within the Vicia genus (Frediani et al., 1992; Cremonini et al., 1998 a, b; Maggini et al., 1991; Venora et al., 2000; Tabur et al., 2002; Navratilova et al., 2003; Bryant and Hughes, 2011; Kahraman et al., 2013). Recently, plant organelle genomes (chloroplast and mitochondrial DNA) have been widely used for population genetics studies and in order to reveal phylogenetic relationships (Soltis et al., 1992). Non-coding regions in the chloroplast genome are often preferred for molecular systematic and plant-related population genetic studies (Shaw et al., 2006). DNA regions that are not coded show a high degree of mutation. For this reason, they are widely used to show evolutionary relationships (Taberlet et al., 1991). One of the non-coding regions in the chloroplast DNA is tRNA and this is used in determining evolutionary relationships. The most frequently used of these regions are trnT (UGU) - trnL (UAA) IGS, trnL5'-trnL3 'intron and trnL (UAA) - trnF (GAA) IGS regions (Taberlet et al., 1991; Kelchner, 2000). This study aimed to use transfer ribonucleic acid Leucine (trnL) as the differential region to examine the relationships within the Vicia genus growing in the Southeast Anatolian Region. 2. Materials and Methods 2.1. Plant material Plant material was collected from different localities of the study area from April to May, which is the vegetation periods of plants from natural habitats and identified according to the diagnostic morphological characteristics described in the Flora of Turkey and the East Aegean Islands (Davis, 1970). The study area is located in the Southeastern Anatolia Region. (Adıyaman, Batman, Diyarbakır, Gaziantep, Mardin, Siirt, Şanlıurfa) covers the area. Species were included in the eight different sections; Cracca S. F. Gray, Ervilia (DC) Link, Lentopsis Kupicha, Peregrinae Kupicha, Lathyroides (Buchenau) Tzvel, Hypechusa (Alef.) Aschers and Graebner, Vicia L. and Narbonensis (Radzhi) Maxted. To construct the phylogenetic relationships of the Vicia genus, we analyzed sequences of trnL regions from a total of 22 specimens and 25 taxa. Lathyrus cassius Boiss. and Lathyrus inconspicuous L. were used as out-group. DNA sequences of the studied taxa were entered into the NCBI database and an accession number was obtained for each species. Plant deposited under suitable conditions at the Herbarium of the Department of Biology (DUF), Faculty of Science and Art, Dicle University. 12 Table 1. The taxa of Vicia L. studied and their collecting sites, section, accession number and voucher name and herbarium number. No Taxon names Section Location Accession number MN862284 Voucher name and herbarium number A.Kaplan DUF 90 1 V. narbonensis L. var. narbonensis Narbonensis 2 V. hybrida L. Hypechusa 3 Cracca 4 V. villosa Roth subsp. varia (Host) Corb. V. mollis Boiss. & Hausskn. 5 V. galeata Boiss. Hypechusa 6 V. sativa L. subsp. sativa Vicia 7 Vicia Peregrinae Cracca 10 V. sativa L. subsp. nigra L. (Ehrh.) var. nigra L. (Ehrh.) V. michauxii Sprengel. var. stenophylla Boiss. V. monantha Retz subsp. monantha Retz. V. anatolica Turril. Batman, Batman University, central campus, 570 m. Batman, Batman University, central campus, 570 m. Adıyaman, Bozova to Adıyaman, 18.5 km, 589 m. Adıyaman, Hilvan to Bozova, Külünçe village, 5 km, 600 m Adıyaman, Hilvan to Bozova, Külünçe village, 5 km, 600 m. Diyarbakır, Dicle University campus, Bakbako stream around, 630-640 m. Diyarbakır, Çermik to Çüngüş, 6-7 km, 703 m. Şanlıurfa, Şanlıurfa to Suruç, 18km, 666 m. MN862285 A.Kaplan DUF 88 MN862286 A.Kaplan DUF 48 MN862287 A.Kaplan DUF 40 MN862288 A.Kaplan DUF 44 MN862289 A.Kaplan DUF 82 MN862290 MN862291 A.Kaplan & A.S.Ertekin DUF 35 A.Kaplan DUF 08 Şanlıurfa, Akçakale, 382 m. MN862292 A.Kaplan DUF 09 11 V. peregrina L. Hypechusa Mardin, Exit of Mardin 2 km, 1095m. MN862293 A.Kaplan DUF 12 Peregrinae Siirt, Çatılı village,1475 m. MN862294 A.Kaplan DUF 73 12 Hypechusa 15 V. ervilia (L.) Willd. Ervilia 16 V. galilaea Plitm. Et. Zoh. Narbonensis Diyarbakır, Ergani to Çermik, 13 km, 870875 m. Diyarbakır, Ergani to Çermik 13 km, 870875 m. Diyarbakır, Çermik to Çüngüş, 11.5-12 km, 905 m. Diyarbakır, Ergani to Çermik, 16-17 km, 880m Diyarbakır, Çermik to Siverek, 1 km, 732m MN862295 14 V. noeana Reuter ex Boiss var. noeana Reuter ex Boiss. V. sericocarpa Fenzl. var. sericocarpa Fenzl. V. lathyroides L. 17 V. cracca L. subsp. stenophylla Vel. Cracca Adıyaman, Ferryboat to Siverek, 4 km, 588m MN862300 A.Kaplan & A.S.Ertekin DUF 16 A.Kaplan & A.S.Ertekin DUF 18 A.Kaplan & A.S.Ertekin DUF 39 A.Kaplan & A.S.Ertekin DUF 21 A.Kaplan & A.S.Ertekin DUF 32 A.Kaplan DUF 56 18 V. palaestina Boiss. Cracca MN862301 19 V. lutea var. hirta (Balbis) Lois. Hypechusa Diyarbakır, Çermik to Çüngüş 11.5-12 km, 905 m. Diyarbakır, Devegeçidi picnic area, 732 m. 20 V. esdraelonensis Warb. & Eig Hypechusa MN862303 21 Cracca 23 V. villosa Roth subsp. eriocarpa (Hausskn) P.Ball. V. sativa L. subsp. amphicarpa (Dorth.) Aschers. & Graebn. V. assyriaca Boiss. 24 V. montbretii Fisch. & C.A.Mey. Lentopsis 25 V. caesarea Boiss. & Bal. Lentopsis 26 Lathyrus cassius Boiss. Lathyrus 27 Lathyrus inconspicuous L. Linearicarpus Diyarbakır, the way of Bilgekışla, 3.5 km, 740 m. Adıyaman, Hilvan to Bozova, Külünçe village, 5 km, 600 m. Diyarbakır, Dicle University campus, 630640 m. Şanlıurfa, Siverek Avurtepe village, Otlu avenue,1080 m. Şanlıurfa, Siverek Avurtepe village, Otlu avenue,1080 m. Siirt, Şirvan to Madenköy, 4-5 km, 1020 m. Diyarbakır, the way of Bilgekışla, 3.5 km, 740 m. Gaziantep, Islahiye Bahçe to Gaziantep, 22 km. 8 9 13 22 Hypechusa Hypechusa Lathyroides Vicia Hypechusa MN862296 MN862297 MN862298 MN862299 MN862304 A.Kaplan & A.S.Ertekin DUF 38 A.Kaplan & A.S.Ertekin DUF 59 A.Kaplan & A.S.Ertekin DUF 60 A.Kaplan DUF 41 MN862305 A.Kaplan DUF 80 MN862306 A.S.Ertekin DUF 79 MN862307 A.S.Ertekin DUF 78 MN862308 A.S.Ertekin DUF 184 MN862309 A.S.Ertekin DUF 4241 MN862310 A.S.Ertekin DUF 5249 MN862302 2.2. DNA extraction, PCR, sequencing, sequence alignment and phylogenetic analyses Total genomic DNA were isolated from dried and stored at room temperature leaf tissues using the Lefort et al. (1998) method. The purity and quantity determinations of extracted DNAs were determined by NanoDrop 1000c UV-Vis Spectrophotometer (Thermo Scientific) at A260 / A280 nm. The isolated DNA samples were checked in 1 % agarose gel electrophoresis (Figure 1). Specific primer pairs were used to amplify (amplification) the targeted region on genomic DNA. Amplification of trnL region was carried out by using primer 13 pairs trnc 5' CGA AAT CGG TAG ACG CTA CG 3' (Forward) / trnd 5' GGG GAT AGA GGG ACT TGA AC 3' (Reverse) (White et al., 1990). DNA amplification was performed in a 50 μl volume mixture containing genomic DNA (100 ng/μl), 10X PCR Buffer, MgCl2 (25 mM), dNTP mixture (25 mM), selected primer pair (50 μM), Taq polymerase (5u/μl) and sterile water (ddH2O). PCR reaction of each region consisted of almost same amount of Buffer (5 μl), DNA (1 μl), MgCl2 (1,5 μl), dNTP (1 μl), each primer (forward and reverse) (1,5 μl) and Taq polymerase (0.3 μl). PCR amplification was started with 2 min initial denaturation at 94 °C, and terminated with 5 min at 72 °C. Each reaction ended with a final 4°C hold step and consisted of 35 cycle numbers. Each reaction cycle consisted of denaturation step at 94 °C for 20 sec (1 min), annealing step at 57 °C for 30 sec and elongation step at 72 °C for 30 1 min. Amplicons were visualized by electrophoresis on 2 % agarose gels (Figure 2). After purification, products were sequenced in both directions using Applied 3130 Genetic Analyzer automated sequencer. All sequence chromatograms were opened using DNA dragon software. Ambiguous sites were checked manually and corrected by comparing the strands. Sequences of trnL region have been deposited in NCBI database for further studies. Molecular Evolutionary Genetics Analysis software (MEGA 5.1; [Beta 2] is used to measure total nucleotide length (base pair, bp) (Table 2). DNA sequences were aligned via the Clustal W program. The sequence data was analyzed by using the UPGMA method (Figure 3) based on the Kimura model (Kimura, 1980) and bootstrap analysis with 1000 replications (Felsenstein, 1985). The bootstrap value ranges from 0 % to 100 %. According to the Kress et al. (2005), bootstrap support values, it was defined as> 85% very strong, 70-85 % strong, 50-70 % weak and <50 % very weak. 70 % or greater bootstrap support is often associated with the identification of the correct phylogeny. If the bootstrap support for a certain branch is below 50 %; It is concluded that the relationship between the species is not fully clarified and the branching model is not determined and as a result, this branch will be determined in the tree as a fork (polytomy = uncertainty point) rather than a single node (Freeman and Herron, 1999). 3. Results 3.1. Observation of amplification products When PCR was performed on gDNA samples of targeted species, the presence of bands was visualized by 1% agarose gel electrophoresis (Figure 1). And then amplicons (trnL region) were visualized by electrophoresis on 2 % agarose gels (Figure 2). 14 Figure 1. 1 % gel image of Vicia species PCR products; 1) V. narbonensis var. narbonensis 2) V. hybrida 3) V. villosa subsp. varia 4) V. mollis 5) V. galeata 6) V.sativa subsp. sativa 7) V. sativa subsp. nigra var. nigra 8) V. michauxii var. stenophylla 9) V. monantha subsp. monantha. 10) V.anatolica 11) V. peregrina 12) V. noeana var. noeana 13) V. sericocarpa var. sericocarpa 14) V. lathyroides 15) V. ervilia 16) V. galilaea 17) V. cracca subsp. stenophylla 18) V. palaestina 19) V. lutea var. hirta 20) V. esdraelonensis 21) V. villosa subsp. eriocarpa 22) V. sativa subsp. amphicarpa 23) V. assyriaca 24) V. montbretii 25) V. caesarea 26) Lathyrus cassius 27) Lathyrus inconspicuous. Figure 2. Nuclear DNA of the Vicia species trnL region image of agarose gel (2%); 1) V. narbonensis var. narbonensis 2) V. hybrida 3) V. villosa subsp. varia 4) V. mollis 5) V. galeata 6) V.sativa subsp. sativa 7) V. sativa L. subsp. nigra var. nigra 8)V. michauxii var. stenophylla 9) V. monantha subsp. monantha 10) V. anatolica 11) V. peregrina 12) V. noeana var. noeana 13) V. sericocarpa var. sericocarpa 14) V. lathyroides 15) V. ervilia 16) V. galilaea 17) V. cracca subsp. stenophylla 18) V. palaestina 19) V. lutea var. hirta 20) V. esdraelonensis 21) V. villosa subsp. eriocarpa 22) V. sativa subsp. amphicarpa 23) V. assyriaca 24) V. montbretii 25) V. caesarea 26) Lathyrus cassius 27) Lathyrus inconspicuous. 3.2. Nucleotide sequences of the trnL region The base ratios obtained from approximately 408 bp of which trnL gene were analyzed in the series are given in Table 1. When Table 2 is examined trnL region sizes varied from 269 bp (Vicia narbonensis var. narbonensis and Vicia caesarea) to 534 bp (Vicia esdraelonensis). It appears to be A: 27,3 %, G: 16,4 %, C: 17,2 %, T (U):39,1 %. DNA sequences related to the samples are G - C ratio (33,6 %), A - T ratio (66,4 %). The DNA sequences of the samples are relatively rich in A - T, the G - C ratio is less. 15 Table 2. trnL region sizes in Vicia L. and Lathyrus L. (outgrup) taxa. Species name Vicia narbonensis var. narbonensis Vicia hybrida Vicia villosa subsp. varia Vicia mollis Vicia galeata Vicia sativa subsp. sativa Vicia sativa subsp. nigra var. nigra Vicia michauxii var. stenophylla Vicia monantha subsp. monantha Vicia anatolica Vicia peregrina Vicia noeana var. noeana Vicia sericocarpa var. sericocarpa Vicia lathyroides Vicia ervilia Vicia galilaea Vicia cracca subsp. stenophylla Vicia palaestina Vicia lutea var. hirta Vicia esdraelonensis Vicia villosa subsp. eriocarpa Vicia sativa subsp. amphicarpa Vicia assyriaca Vicia montbretii Vicia caesarea Lathyrus cassius Lathyrus inconspicuous Avg. T(U) 36,1 40,3 38,8 41,0 41,1 36,3 38,4 39,7 38,7 40,6 41,0 41,4 41,0 36,0 40,3 34,9 38,7 39,2 39,8 39,1 37,6 38,0 41,2 35,7 38,3 38,0 40,0 39,1 C 19,0 16,7 17,4 15,0 14,7 19,0 19,5 17,4 17,2 17,0 15,7 16,1 15,4 18,9 16,6 19,0 16,7 16,2 16,7 16,7 17,6 19,2 15,7 20,9 20,8 17,4 16,3 17,2 A 27,9 26,7 29,1 27,9 28,7 25,9 25,3 27,8 28,8 27,5 28,1 26,9 28,2 26,3 27,1 27,5 28,6 28,4 27,8 27,3 28,6 25,7 26,9 24,3 23,0 26,8 26,3 27,3 G 17,1 16,3 14,7 16,2 15,4 18,7 16,8 15,1 15,3 15,0 15,2 15,6 15,4 18,9 15,9 18,7 16,0 16,2 15,7 16,9 16,3 17,0 16,2 19,1 17,8 17,8 17,4 16,4 Total 269,0 461,0 443,0 427,0 428,0 347,0 292,0 478,0 431,0 488,0 402,0 435,0 415,0 350,0 409,0 327,0 426,0 444,0 497,0 534,0 490,0 276,0 413,0 325,0 269,0 511,0 448,0 408,7 3.3. Molecular phylogeny Phylogenetic associations and genetic diversity data were made with Mega (Molecular Evolutionary Genetics Analysis) 5. 1 Beta 2 software using sequence analysis results. Unweighted pair group method with arithmetic mean (UPGMA) method was used during the drawing of the phylogenetic tree (Figure 3). In order to increase the reliability of the obtained tree, bootstrap analysis with 1000 replications was preferred. The percentage of each branch shows how much that branch is supported. If the branch's reliability rate is below 50 %, the program qualifies this branch as very weak and collapses that branch, and the empty branch resulting from the collapsed branch is called a polytomy. Since the branching model cannot be determined in collapsed branches, no interpretation can be made about the relationship between taxa. It can be said that only this branch has created a polytomy. 16 Figure 3. Phylogenetic tree of Vicia species based on (UPGMA) analysis of the trnL region. Lathyrus cassius and Lathyrus insconspicuous were used as a outgroup. Bootstrap analysis of UPGMA was based on 1000 replicates. Percentage of each branch shows the support rate of that branch (Bootstrap value). 4. Discussion DNA sequencing technologies and recent advances in molecular biology enable us to characterize the genomes of organisms and many ongoing genome projects for various species are providing valuable insight into their biology and uses. In this context, many molecular studies of the Vicia genus have been carried out. Different coding and non-coding regions from both nuclear and chloroplast DNAs have been studied. For instance, phylogenetic relationships between Vicia faba (Fabaceae) and related species trnL sequence (Fennel et al., 1998), nuclear ribosomal DNA internal transcribed spacer (ITS) (Foladi et al., 2013) and matK (Steele and Wojciechowski, 2003) are, some of the molecular systems which have been extensively used for molecular systematic studies. In this study, we chose to use the transfer ribonucleic acid Leucine (trnL) region of nuclear DNA. The reasons for using chloroplast in plant phylogenetic studies are as follows: the structural stability of cpDNA, that it is haploid (n), that it generally has a uniperantal transfer, that it is non-recombinant (Small et al., 2004). In the unweighted pair group method with arithmetic mean (UPGMA) tree obtained in the present study (Figure 3), it was observed that Lathyrus species used as an outgroup were nested within other species and this branch was supported with a bootstrap value of 51 %. The ingroup consists of two main clades. Using the data from Figure 3, the branch with V. narbonensis var. narbonensis and V. galilaea (sect. Narbonensis) species was supported with a bootstrap value of 100 %. Molecular data is based on sequences of the trnL with high bootstrap values.In many 17 studies (Enneking and Maxted,1995; Fennel et al., 1998; Potokina, 1999; Schaefer et al., 2012), the species belonging to sect. narbonensis have been studied in the same branch. These results are in line with those of the current study. Schaefer et al. (2012), reported that the section was monophyletic. Our study supports the assertion that section is monophyletic. The species belonging to sect. Lentopsis (V. montbretii, V. caesarea) were supported with a bootstrap value of 81 %. Schaefer et al. (2012), in their study, sect. lentopsis examined the species in the tree of maximum likelihood in the same branch. Binzat (2012), Foladi, et al. (2003), Schaefer et al. (2012) also studied the molecular phylogeny of Vicia species in parallel with our work. The branch where V. sativa subsp. sativa, V. sativa subsp. nigra var. nigra, V. sativa. subsp. amphicarpa species are found is supported a bootstrap value with 88 % and this value shows us that we have achieved a meaningful result. Molecular data based on sequences of the trnL with high bootstrap values, support the placement of these species in section V. lathyroides, which was connected to this branch with a high bootstrap value, for example a 100 %. Van de Wouv et al. (2001), found that, V. sativa subsp. amphicarpa and V. sativa subsp. nigra var. nigra were located close to each other, while V. sativa subsp. sativa was located farther away in the UPGMA phylogenetic tree they obtained. Potokina, (1999) reported that the V. sativa complex coexists in the dendograms they obtained in their study. Leht, (2009), Potokina, (1999) and Schaefer et al. (2012) reported that this section is not monophyletic but polyphyletic. However, Potokina et al. (1999), examined V. lathyroides species as a separate branch in their study and supported the branch where this species was located with a value of 53 %. They also found it close to the Vicia section in all the trees they created. Jaaska, (2008) and Leht, (2009) reported that they found the species to be close to the Vicia section. Although the bootstrap value of the V. lathyroides species is low, it has established a close connection with the sect. Vicia as in the studies mentioned above. Schaefer et al. (2012) determined that the section was not monophyletic. The results we obtained in our study support the current studies. The branch with the highest bootstrap value in the group was the branch with V. michauxii var. stenophylla and V. peregrina species, which was supported with a bootstrap value of 78 %. Molecular data based on sequences of the trnL with high bootstrap values, supports the placement of these species in this section. In many studies (Fennel et al., 1998; Potokina et al., 1999; Leht, 2009; Emre, 2011; Schaefer et al., 2012), these species have been found in the same branch and examined in the Peregrinae section. Schaefer et al. (2012) in their study, they found that the section was monophyletic. This result is in line with our study. As is well known, unrooted trees are obtained with bootstrap method. The values on the branches of the trees are used to determine the most statistically reliable branches. The percentage ratio of each branch indicates how much that branch is supported. If the reliability rate of the branch is below 50 %, the program considers this branch very weak and collapses that branch, and the empty branch resulting from the collapsed branch is called polytomy. Since the branching model in collapsed branches cannot be determined, no comment can be made on the relationship between taxa. It can only be said that this branch has created a polytomy. In the phylogenetic tree that we have obtained, V. villosa subsp. varia, V. villosa subsp. eriocarpa, V. cracca subsp. stenophylla, V. monantha subsp. monantha, V. palaestina (sect. Cracca), V. ervilia (sect. Ervilia) V. lutea var. hirta, V. galeata, V. assyriaca, V. esdraelonensis, V. noeana var. noeana, V. hybrida, V. mollis, V. anatolica, V. sericocarpa var. 18 sericocarpa (sect. Hypechusa) were species branches where the species were found to have collapsed. Polytomy was observed since the reliability value was below 50 %. Therefore, the parser did not provide any information. In the current study, these taxa were found to be problematic, and showed a random distribution in phylogenetic trees, and were thus supported by a low bootstrap value. The tree that we created was not successful in separating these species. This may be because the taxa are actually differentiated from a single ancestral species or because of conflicting or missing data. The occurrence of polytomies in phylogenetic analysis may indicate an interesting evolutionary history that needs further research. In addition, molecular characters are more objective than morphological characters, but they are not accurate. Polymorphisms and uncertainties may arise in determining the basis for DNA sequence data. It may not be easily understood, especially if the sequences between taxa are very different when making sequence alignments. Also, the possibilities of paralogy arising from ancestral gene duplication or hybridization can cause confusion when comparing homologous sequences (Simpson, 2012). The genetic diversity of the species is already due to the variation in genes and their recombination (Lorenzetti, 1994, Lorenzetti, 1996). In the current study, such mutations occurring in species were indicative of genetic diversity and such differences manifested themselves in the dendogram we obtained. In the future, we propose that only studies on these taxa (various molecular marker systems such as biochemical, AFLP, RFLP, ISSR, SSR) should be conducted in detail. We also consider that such studies could study the MatK region in the chloroplast genome. It should not be forgotten that each method often leads to a different result. Expecting a specific and accurate result (a tree) is not appropriate anyway; there are no perfect trees, but some are useful. This study attempted to examine Vicia L. species morphologically and molecularly in a flexible way and to eliminate the deficiencies in the literature. It is our duty to carefully conserve the gene resources of the many Vicia L. species, which are almost as old as human history are and which are extensively cultivated, and pass them on to future generations. We hope that the economic integration studies found at the end of the inventory and the documentation studies conducted in basic sciences worldwide will now be carried out more effectively in Turkey and that the information presented will serve this purpose. Conflicts of Interests Authors declare that there is no conflict of interests References Başbağ, M., Hoşgören, H., & Aydın, A. (2013). Vicia Taxa in the Flora of Turkey. 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