Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 53-63, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1139 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Pelin Yilmaz Sancar, Sem- settin Civelek, Murat Kursat (2021) The morphological, karyological and phylo- genetic analyses of three Artemisia L. (Asteraceae) species that around the Van Lake in Turkey. Caryologia 74(3): 53-63. doi: 10.36253/caryologia-1139 Received: November 23, 2020 Accepted: May 26, 2021 Published: December 21, 2021 Copyright: © 2021 Pelin Yilmaz Sancar, Semsettin Civelek, Murat Kursat. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID PYS: 0000-0002-6134-622X SC: 0000-0003-1398-585X MK: 0000-0002-0861-4213 The morphological, karyological and phylogenetic analyses of three Artemisia L. (Asteraceae) species that around the Van Lake in Turkey Pelin Yilmaz Sancar1,*, Semsettin Civelek1, Murat Kursat2 1Fırat University, Faculty of Sciences, Department of Biology, 23119 Elazıg, Turkey 2Bitlis Eren University, Faculty of Arts and Sciences, Department of Biology, 13000 Bitlis, Turkey *Corresponding author. E-mail: peyilmaz@firat.edu.tr Abstract. Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in five subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classi- fication and phylogeny but its natural classification still has not been achieved. The aim of this study is to try to solve the problematic systematic relationship between three dif- ferent Artemisia species growing in close proximity to each other in the light of mor- phological, karyological and molecular data. The roots, stems, leaves, flowers structures of the plant samples collected from different populations belong to these species were investigated within the framework of morphological studies. Additionally, the chromo- some counts and karyotype analysises of these species were made and idiograms were drawn in the karyological studies. In the context of phylogenetic studies, ITS (ITS1- 5.8S-ITS2) and trnT - trnL3’ regions of 22 individuals belonging to 3 taxa were stud- ied. According to results of phylogenetic anlysis, it has been found that there is com- pleted speciation genetic isolation mechanism between the species Artemisia spicigera, Artemisia taurica and Artemisia fragrans that inhibit gene flow. Also Artemisia fragrans and Artemisia spicigera species are very similar to each other in terms of morphological characteristics. However, since populations of the species Artemisia fragrans are autopol- yploid, the dimensional values of their morphological squares are larger than those of the species Artemisia spicigera. This study is important as it is the first molecular based study relating with some species of Artemisia growing naturally in Turkey. Keywords: Artemisia, karyology, morphology, phylogeny, cpDNA trnT-trnL3’, r-DNA ITS. INTRODUCTION Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in five subgenera (Vallès et al., 2011). The majority of the members of this genus have a high economic value (Chehregani et al., 2010; Hayat et al., 2010). Due 54 Pelin Yilmaz Sancar, Semsettin Civelek, Murat Kursat to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still has not been achieved (Mcarthur et al., 1981; Torrel et al., 1999; Torrell and Vallès 2001; Vallès et al., 2003; Kurşat, 2010; Kurşat et al., 2011). The genus is currently divided into five main groups [Artemisia, Absinthium (Mill.) Less., Dracuncu- lus (Besser) Rydb., Seriphidium Besser and Tridentatae (Rydb.) McArthur] but subgeneric classification is sub- ject to rearrangements in the light of recent molecular studies (Torrell et al., 1999; Vallès et al., 2003). Although a lot of investigation have been made of the genus Artemisia, enhancing the available morpho- logic and karyological data (Kawatani and Ohno 1964; Vallès 1987a; Torrell et al., 1999; Torrell and Vallès 2001; Torrell et al., 2001; Vallès and Mcarthur 2001; Vallès et al., 2001; Kreitschitz and Vallès 2003; Inceer and Hay- irlioglu-Ayaz 2007; Pellicer et al., 2007; Chehregani and Hajisadeghian 2009; Nazirzadeh et al., 2009; Chehrega- ni et al., 2010), still the chromosome numbers of some species of the genus remain unknown or doubtful. The genus has two basic chromosome numbers, the largely predominating x=9 and the less extended x=8. x = 9 is not only the most common basic number in the genus Artemisia, but in the tribe Anthemideae and the family Asteraceae as well (Mcarthur and Sanderson 1999; Oliva and Vallès 1994; Schweizer and Ehrendorfer 1983; Sol- brig 1977; Vallès and Siljak-Yakovlev 1997). A high per- centage of Artemisia species are polyploid. This phenom- enon is present in all of the major groups into which the genus is divided. Both basic chromosome numbers (x=8 and x=9) show polyploidy, with levels up to 12x for x=9 and 6x for x=8 (Vallès and Mcarthur 2001). The gene regions that have been used for phylogeo- graphic and phylogenetic inferences in plants come from the single copy portions (LSC and SSC) of the chloroplast genome, and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA). Several molecular methods have been used to determine the genetic diver- sity and relationships among different Artemisia spe- cies, including karyotyping (Mcarthur and Pope 1979), cpDNA restriction site variation analysis (Kornkven et al., 1999), polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) analysis of several genes (Lee et al., 2009; Mahmood et al., 2011), microsat- ellite (SSR) polymorphism analysis (Tripathi et al., 2009; Shafie et al., 2011) and Random amplified polymorphic DNA (RAPD) analysis (Mcarthur et al., 1998a; Mcarthur et al., 1998b; Sangwan et al., 1999). Nevertheless, very few of Artemisia species have been verified with molec- ular phylogenetic studies based on nucleotide sequence data in Turkey, so far (Koloren et al., 2016). So, the aim of this study is to try to solve the prob- lematic systematic relationship between three different Artemisia species growing in close proximity to each other in the light of morphological, karyological and molecular data (using of rDNA ITS and trnT-trnL3’ regions sequence data). Additionally, the first molecular data for Artemisia spicigera, Artemisia taurica, and Arte- misia fragrans from Turkey has been submitted to the GenBank databases. MATERIAL AND METHODS Morphological evaluation Plant specimens were collected from around the Van Lake during the vegetative, flower and seed season in 2010, collected by M. Kurşat, Ş. Civelek and P.Y. Sancar. Morphological examinations consist of instant observa- tions on the samples in the field and macroscopic and microscopic examinations on the samples that have been converted into herbarium material in the laboratory. In order to determine the minimum and maximum val- ues of the examined characters, 10 samples were taken from each locality and measurements were made. Meas- urements of small structures were made with a ruler under a stereo microscope. Measurements of macro- scopic structures were made using the ruler again with the naked eye. The herbarium materials of the collected samples are stored in Firat University Herbarium (FUH). The list of the examined specimens, localities, collected date and voucher numbers were given in Table 1. Karyology Meristematic cells of root tips are used in the car- yological studies. The seeds (about 15-20 seeds for each type) were germinated on moist filter paper in petri dishes between 20-25°C. The actively growing root tips, 1 cm in length, were excised from the germinating seeds and pretreated with aqueous colchicine (0.05%) for 3-3.5 h at room temperature. Then, the root tips were fixed with Farmer (1:3 glacial acetic acid–absolute etha- nol) for at least 24 h at 4°C, hydrolysed in 0.1 N HCl at room temperature for 1 min, and subsequently rinsed in tap water for 3–5 min. Then they were stained in Feul- gen for 1 h and mounted in 45% acetic acid (Gedik et al., 2014). Digital photographs from at least five well-spread metaphase plates from each species were taken using an Olympus BX51 microscope (Olympus Optical Co. Ltd, Tokyo, Japan), and were recorded with an Olym- pus Camedia C-4000 digital camera (Olympus Optical 55The morphological, karyological and phylogenetic analyses of three Artemisia species Co. Ltd) (Figure 1). The short arm, long arm and total lengths of each chromosome were measured and the relative lengths (RL), arm ratios (AR), and centromeric indices (CI) were determined from images of selected cells. Levan et al. (1964) was used for the classification of chromosomes. The number of somatic chromosomes, ploidy level, karyotype formula, morphometric parameters, A1 and A2 values (The intrachromosomal asymmetry index - A1 and the interchromosomal asymmetry index - A2) were determined for each taxa (Romero Zarco 1986) (Table 2). Idiograms of haploid chromosomes were drawn (Figure 2). The examined taxa and characteristics of somatic chromosomes are given in the results section. Genomic DNA isolation, PCR, and Sequencing Genomic DNA isolation was performed manually as described CTAB method by Doyle and Doyle (1987). In PCR studies conducted by using trna- trnd primers and ITS4-ITS5 primers, trnT - trnL3’ and ITS (ITS1- Table 1. Information of Artemisia populations location in field. Taxa Locality Altitude Lat - Lon Date Voucher number A . s pi ci ge ra Ant valley slope 5.5 km after passing Aktuzla 1555m 39°.21’-42°.15’ 10.10.2010 Yilmaz Sancar, Kurşat and Civelek 5007,5008,5022 Hınıs - Varto highway, 24 km before Varto roadside, slopes 1780m 39°.13’-41°.42’ 10.10.2010 Yilmaz Sancar, Kurşat and Civelek 5009,5024 Van - Hakkâri road, after 1 km of Gürpınar road separation, Çavuştepe locality roadside 1799m 38°.20’-43°.25’ 25.11.2010 Yilmaz Sancar, Kurşat and Civelek 5013 A . f ra gr an s Kuzgun Koran Pass hills 2142m 38°.23’-42°.47’ 09.10.2010 Yilmaz Sancar, Kurşat and Civelek 5001,5002,5010,5011 Between Edremit and Gürpınar, 15 km before Gürpınar 1714m 38°.19’-43°.14’ 09.10.2010 Yilmaz Sancar, Kurşat and Civelek 5003,5012 Muradiye - Şelale location 1788m 39°.03’-43°.25’ 10.10.2010 Yilmaz Sancar, Kurşat and Civelek 5005,5016 Between Malazgirt - Aktuzla, around Nurettin village roadside slopes 1728m 38°.50’-43°.25’ 26.11.2010 Yilmaz Sancar, Kurşat and Civelek 5017 After passing Aktuzla 5.5 km, Ant Valley slopes (roadside slopes) 1555m 39°.21’-42°.15’ 26.11.2010 Yilmaz Sancar, Kurşat and Civelek 5006,5019,5020,5021,5023 A . t au ri ca Van-hakkari highway, after 1 km of Gürpınar crossroads, Çavuştepe area, roadsides 1799m 40°.25’-43°.20’ 25.11.2010 Yilmaz Sancar, Kurşat and Civelek 5004,5027,5028, Figure 1. Somatic metaphase in A. spicigera (2n=18), and haploid idiogram (Scale bars: 1 μm). 56 Pelin Yilmaz Sancar, Semsettin Civelek, Murat Kursat 5.8S-ITS2) region for 22 samples has been multiplied (Taberlet et al., 1991). The sequence of primers that were used to amplified both trnT - trnL3’ region and ITS (ITS1-5.8S-ITS2) region were given in Table 3 (Taberlet et al., 1991). The following protocol on a BıoRad Ther- mal Cycler : 2 min 95 °C initial denaturation, 35 cycles of 1 min 95 °C denaturation, 40 s 60 °C (for trn region) and 55 °C (for ITS region) annealing and 1 min 72 °C extension, followed by a 5 min final extension at 72 °C. PCR products were monitored in agarose gel with a 1 % ratio. Two-way reading was applied to the amplifica- tion products. PCR purification process was realized before sequence analysis. The purification and sequenc- ing process was realized by the Macrogen Company. Table 2. Somatic chromosome numbers (2n), ploidy level, karyotype formula, ranges of chromosome length, total karyotype length (TKL), and asymmetry indexes (A1, A2) of the studied taxa. Taxon 2n Ploidy level Karyotype formula Chromosome length range (μm) TKL (μm) A1 A2 A. spicigera 1 18 2x 1M+5m+3sm 4,94-5,56 46,72 0,27 0,04 A. spicigera 2 18 2 x 2M+5m+2sm 4,26-4,81 40,79 0,23 0,02 A. spicigera 3 18 2 x 1M+6m+2sm 4,64-5,14 43,02 0,24 0,04 A. spicigera 4 18 2 x 1M+6m+2sm 4,69-4,97 43,40 0,25 0,02 A. spicigera 5 18 2 x 2M+6m+1sm 4,55-5,50 42,28 0.26 0,03 A. spicigera 6 18 2 x 3M+4m+2sm 4,42-5,25 41,40 0,22 0,03 A. spicigera 7 18 2 x 1M+4m+4sm 4,34-5,33 44,50 0,28 0,04 A. taurica 1 36 4 x 3M+13m+2sm 3,48-4,07 67,31 0,22 0,05 A. taurica 2 36 4 x 4M+12m+2sm 4,57-5,51 92,47 0,25 0,05 A. taurica 3 36 4 x 4M+11m+3sm 4,44-5,50 89,70 0,23 0,06 A. fragrans 1 36 4 x 3M+10m+5sm 4,10-5,35 85,51 0,35 0,08 A. fragrans 2 36 4 x 3M+11m+4sm 3,13-3,53 59,52 0,24 0,01 A. fragrans 3 36 4 x 2M+14m+2sm 3,40-3,94 66,85 0,25 0,05 A. fragrans 4 36 4 x 2M+10m+6sm 4,81-6,29 100,08 0,26 0,07 A. fragrans 5 36 4 x 1M+14m+3sm 3,97-4,65 77,57 0,27 0,05 A. fragrans 6 34 4 x 3M+12m+2sm 3,98-4,89 75,78 0,20 0,07 A. fragrans 7 36 4 x 3M+13m+2sm 3,67-4,14 70,40 0,24 0,04 A. fragrans 8 36 4 x 1M+13m+4sm 3,43-4,75 70,15 0,29 0,08 A. fragrans 9 36 4 x 3M+12m+3sm 3,98-4,99 82,53 0,23 0,07 A. fragrans 10 36 4 x 2M+11m+5sm 3,83-4,79 76,09 0,28 0,07 A. fragrans 11 36 4 x 2M+10m+6sm 3,65-4,51 71,96 0,27 0,06 A. fragrans 12 36 4 x 3M+12m+3sm 3,40-4,49 70,52 0,27 0,08 Figure 2. Somatic metaphase in A. taurica (2n=36), and haploid idiogram (Scale bars: 1 μm). 57The morphological, karyological and phylogenetic analyses of three Artemisia species The obtained data was uploaded to NCBI and GenBank accession numbers were taken. The GenBank accession numbers were given in Table 4. Phylogenetic Analysis Phylogenetic analysis was conducted using the pro- gram Molecular Evolutionary Genetics Analysis software (MEGA X) (Kumar et al., 2019). In order to evaluate the data of chromatograms (sequencing), Finch TV 1.4 ver- sion is used. DNA sequence alignments of 22 individuals, Variable sites, number of parsimony informative sites, genetic distance, nucleotide diversity, and divergence within species were computed by MEGA X version. DNA sequence alignment of all the individuals is made subject to statistical analysis within the scope of this program. Ultimately, phylogenetic trees were constructed by Maxi- mum Parsimony Method with 100 bootstrap replicates (Nei and Kumar, 2000; Kumar et al., 2019). RESULTS Morphological results It was observed that A. taurica and A. fragrans spe- cies had a larger size compared to A. spicigera species in accordance with the environmental conditions it grows and the number of chromosomes and ploidy levels. Detailed morphological measurements of the studied species are as in Table 5. Karyological results A. spicigera K.Koch This taxa general spread is the Eastern Anatolia Region in Turkey. Samples were collected from three populations and 6 individuals of three different locali- ties (Table 1). The samples were labelled “P.Y. 5007-5008- 5009-5013-5022-5024”. The number of chromosomes in all the samples examined was 2n=2x=18 and it con- sists of 4M, 10m and 4sm chromosomes. The metaphase chromosome length is 1.46–3.06 μm and longest to shortest chromosome ratio is 2.0:1. Chromosome arm ratios are 1.28–2.27 μm, the centromeric index is 30.55– 43.83 μm, and relative lengths are 4.20–8.78 μm (Table 2, Figure 1,). Secondary structures and satellite chromo- somes (sat-chromosome) were not observed in this spec- imens. A. taurica Willd. The species A. taurica shows the wide distribution in the steppes of Central, Eastern and Southeastern Anato- lia in Turkey. Samples were collected from 6 individuals of three different localities (Table 1). The samples were labelled “P.Y. 5004-5027-5028”. The number of chromo- somes in all the samples examined was 2n=4x=36 and it consists of 2M, 14m and 2sm chromosomes. The meta- phase chromosome length is 3.40–3.94 μm. Chromo- some arm ratios are 1.27–2.57 μm, the centromeric index is 33.09–49.20 μm, and relative lengths are 5.08–5.89 μm (Table 2, Figure 2). Secondary structures and satellite chromosomes (sat-chromosome) were not observed in this specimens. Table 3. The base sequences of the primers used (Taberlet et al., 1991). Primers Base sequences (5’ – 3’) ITS 5 (F): 5’ GAA AGT AAA AGT CGT AAC AAG G 3’ ITS 4 (R): 5’ TCC TCC GCT TAT TGA TAT GC 3’ trn a (F): 5’ CAT TAC AAA TGC GAT GCT CT 3’ trn d (R): 5’ GGG GAT AGA GGA CTT GAA C 3’ Table 4. GenBank accession numbers for the rDNA ITS (ITS1-5.8S- ITS2) and trnT-trnL3’ regions of the studied samples Specimens GenBank Accesion Numbers ITS region trnT-trnL3’ region Artemisia fragrans 1 MT159779 MT648006 Artemisia fragrans 2 MT159780 MT648007 Artemisia fragrans 3 MT159781 MT648008 Artemisia fragrans 4 MT159782 MT648009 Artemisia fragrans 5 MT159783 MT648010 Artemisia fragrans 6 MT159784 MT648011 Artemisia fragrans 7 MT159785 MT648012 Artemisia fragrans 8 MT159786 MT648013 Artemisia fragrans 9 MT159787 MT648014 Artemisia fragrans 10 MT159788 MT648015 Artemisia fragrans 11 MT159789 MT648016 Artemisia fragrans 12 MT159790 MT648017 Artemisia spicigera 1 MT159791 MT648018 Artemisia spicigera 2 MT159792 MT648019 Artemisia spicigera 3 MT159793 MT648020 Artemisia spicigera 4 MT159794 MT648021 Artemisia spicigera 5 MT159795 MT648022 Artemisia spicigera 6 MT159796 MT648023 Artemisia spicigera 7 MT159797 MT648024 Artemisia taurica 1 MT159798 MT648025 Artemisia taurica 2 MT159799 MT648026 Artemisia taurica 3 MT159800 MT648027 58 Pelin Yilmaz Sancar, Semsettin Civelek, Murat Kursat A. fragrans Willd. This taxa only spread is the Eastern Anatolia Region in Turkey and this species a new record for Turkey (Kur- sat et al., 2014). Samples were collected from five popula- tions and 14 individuals of four different localities (Table 1). The samples were labelled “P.Y. 5001-5002-5003-5005- 5006-5010-5011-5012-5016-5017-5019-5020-5021-5023”. The number of chromosomes in all the samples exam- ined was 2n=4x=36 and it consists of 4M, 10m and 4sm chromosomes. The metaphase chromosome length is 1.46–3.06 μm and longest to shortest chromosome ratio is 2.0:1. Chromosome arm ratios are 1.28–2.27 μm, the centromeric index is 30.55–43.83 μm, and relative lengths are 4.20–8.78 μm (Table 2, Figure 3). Secondary structures and satellite chromosomes (sat-chromosome) were not observed in this specimens. Phylogenetic results In this part of the study, a phylogenetic tree display- ing the phylogenetic position of three Artemisia species Table 5. Comparison in terms of key features that distinguish of the species of A. spicigera, A. taurica and A. fragrans Characters A. spicigera A. fragrans A. taurica Stem length (cm) 20–50 20-75 20–45(–60) Dimensions lower leaves (cm) 0.5–2.5 × 0.3–1.5 2.5-4 × 1–2 1–2.5 × 0.5–1.2 Dimensions of cauline leaves (cm) 0.5–1.5 × 0.3–1.5 1-2.5 × 0.5–1.5 0.5–2.5 × 0.3–1 Dimensions of floral leaves (cm) 0.1–1 × 0.1–0.2 0.1–1.2 × 0.1–0.8 0.1–1 × 0.1–0.4 Orientation of synflorescence branches usually horizontal usually ascendant usually horizontal Capitula length (mm) 1–3 mm long 1–5 mm long (1–) 3–5 mm long, Outer phyllaries dimensions (mm) 0.2–0.4 × 0.2–0.4 0.5–0.8 × 0.3–0.5 0.6–0.9 × 0.5–0.8 Middle phyllaries dimensions (mm) 1.2–2.2 × 0.5–1 1–1.2 × 0.8–1.5 1–2.2 × 1.3–1.7 Inner phyllaries dimensions (mm) 3–3.4 × 0.5–1 3.3–3.8 × 1–1.2 4–4.2 × 1.2–1.5 Corolla colour yellow or red yellow or red yellow or pinkish red or purplish red Corolla dimensions (mm) 2.5–3.2 × 0.5–0.8 1.7–3.5 × 0.2–0.6 2.8–3.3 × 0.5–1 Pistil length (mm) 1.8–3 2.1–3.2 3.1–3.9 Ovarium dimensions (mm) 0.4–0.6 × 0.2–0.4 0.5–0.8 × 0.3–0.6 0.7–1 × 0.2–0.7 Style length (mm) 1.2–1.6 1.5–1.9 1.5–2.2 Forks length of bifid stigma (mm) 0.2–0.5 0.3–0.7 0.4–0.7 Stamens length (mm) 2.2–3.2 3–3.5 3–4.2 Filaments length (mm) 0.8–1.3 1.3–1.6 1–1.5 Anhters dimensions (mm) 1.4–1.7 × 0.1–0.3 2–2.5 × 0.2–0.5 2–2.7 × 0.1–0.3 Number of flowers in capitula 3-5 5-8(-10) Achenes (cypselas) dimensions (mm) 1.2–2.2 × 0.5–1.2 2–2.5 × 0.8–1.4 1.8–2.7 × 0.8–1.4 Somatic chromosome number 2n=2x=18 2n=4x=36 2n=4x=36 , 2n=6x=54 Figure 3. Somatic metaphase in A. fragrans (2n=36), and haploid idiogram (Scale bars: 1 μm). 59The morphological, karyological and phylogenetic analyses of three Artemisia species with respect to each other was constructed (Figure 4). Populations of these species were collected from 9 differ- ent regions cultivated in Van Lake around and 22 indi- viduals were included in the analyzes. İn addition to, the reference base sequences of two individuals belong to species A. sieberi (KJ004347.1) and A. maritima (NC045093.1) were also included in our analysis to dem- onstrate the accuracy of the study (Shahzadi et.al., 2020.) Haplocarpa scaposa (EU846325.1 and DQ444824) was used as an outgroup (McKenzie et.al. 2006; McKenzie and Barker 2008). Sequence data of plants used as out- group and sister group were taken from NCBI. Within the scope of the studies, DNA isolation of 22 individuals from leaf tissue was made by CTAB method, then rDNA ITS (ITS1-5.8S-ITS2) region and non-coding trnT-trnL3’ region of cpDNA was ampli- fied in PCR using specific primers (Table 2). The base sequences of the obtained regions were analyzed and their genetic characteristics were compared and infor- mation was obtained about the proximity and distance of taxa to each other. For a more accurate visualization of the results of the alignment, about 50-100 base from the head and the end were not evaluated by us. As a result of the research done from NCBI for Artemisia genus, the base length of the ITS (ITS1-5.8S-ITS2) region was found to be 700-750 bp, trnT-trnL3’ in total and the base length was 900-1000 bp in total, and in our study, it was found to be of similar length in accordance with the literature. The analyses were performed with the X version of the MEGA program and the method that would give the best result for us was selected from the “Find Best DNA Models” step of the program. As a result, it was decided that Maximum Parsimony method would give the most accurate result of the tree drawn with Tamura 3-param- eter step. In addition, two different DNA regions were evaluated at the same time and a complex tree has been obtained to achieve a more accurate result (Figure 4). As a result of the calculations made with the Maxi- mum Parsimony (MP) method, in both the separate and co-evaluations of the sequences of the ITS and trn regions of the examined individuals, in total ~1745 base pairs were taken into consideration and the number of variable regions (V) 204, The number of conserved regions (C) was 1507 parsimony number (PI) 34, and GC ratio was 41.6%. These calculated values are given in Table 6. DISCUSSION Artemisia is one of the most complex genera and it is represented by the large number of species, diverse morphological types, ploidy and complicated genetic relationships (Winward and Mcarthur 1995). Because of this, the clarification of the genus’s taxonomy using classical botanical tools and morphological characteris- tics has many difficulties (Torrel et al., 1999). Therefore, Table 6. PCR amplified region length and summary statistics from the rDNA ITS (ITS1-5.8S-ITS2) and the cpDNA (trnT-trnL3’) data- set of genus Artemisia. Molecular Diversity Parameters ITS (ITS1-5.8S- ITS2) Region trnT-trnL3’ Region Co-evaluated of ITS (ITS1-5.8S- ITS2) and trnT- trnL3’ Regions Total Sample Count 22 22 22 Total Characters ~725 ~1020 ~1745 GC Ratio (%) 52.7 34.3 41.6 Protected Regions (C) 577 930 1507 Regions with Variation (V) 142 62 204 Parsimony Informative Regions (Pi) 14 20 34 Figure 4. Maximum Parsimony tree obtained from the co-eval- uation of sequences of the ITS (ITS1-5.8S-ITS2) and trnT-trnL3’ regions of individuals. 60 Pelin Yilmaz Sancar, Semsettin Civelek, Murat Kursat usage of molecular markers and caryological data is a valuable and promising addition to the traditional mor- phology-based classification (Turuspekov et al., 2018). In this study a phylogenetic systematic study is conducted by using the morphological, karyological and phyloge- netic data of three species in Artemisia that grow around the Van Lake in Turkey. 22 individuals of taken from 8 different populations belong to taxa of the A. spicigera, A. taurica and A. fragrans were examined morphologi- cal measurements, karyotype analysis and analysing the base slice of the regions being obtained, it was tried to get information about the closeness and distance of taxa with each other. This research is important as it is the first molecular based study relating with some Artemisia species growing naturally that around the Van Lake in Turkey. Additionally, the first molecular data about these species from Turkey has been submitted to the GenBank international databases. A lot of research has been carried out to better understand the morphological, karyological, anatomical, and phylogenetic analysis of the genus Artemisia and its relationships to the other (four) subgenera, Absinthium, Dracunculus (Besser) Rydb, Seriphidium Besser ex Less. and Tridentatae (Rydb), in different parts of the world. Polyploidy is currently considered a prominent force in plant evolution and represents the most common mode of sympatric speciation in plants (Wendel and Doyle 2005). Polyploids, moreover, may have superior levels of adaptability and higher probabilities of survival than their diploid relatives (Thompson and Lumaret 1992; Soltis and Soltis 2000). Most of the Artemisia that colo- nize extreme and arid habitats are polyploids. This fact supports the hypothesis that polyploids have more tol- erance of extreme environmental conditions (Pellicer et al., 2007). Chromosome data currently available show poly- ploidy to be the most significant evolutionary trend in chromosome number within Asteraceae (Chehregani et al., 2010). Accordig to Chehregani et al. (2010), the high- est variation in chromosome number was observed in A. spicigera. In this species; different chromosome numbers (2n=2x=18, 2n=3x=27, 2n=4x=36, 2n=5x=45, 2n=6x=54 and 2n= 8x= 72) were identified in different populations that collected from different parts of Iran. However, in our study, the number of chromosomes in all studied populations was found to be 2n=2x=18. But Tabur et al. (2014), the number of chromosomes in all A. spicig- era populations they work from Turkey have recorded as 2n=2x=18. Accordingly, the ploidy level of A. spicigera kind in Turkey, we can say that 2x. The phylogenetic relationship among the differ- ent Artemisia species collected from different regions of Pakistan based on the chloroplast gene RPS11 was investigated by Mahmood et al. (2011). The molecular phylogenetic analyses of the Hawaiian Artemisia and its worldwide divergence based on nuclear and chloroplast DNA markers were reported by Hobbs and Baldwin. (2013). As discussed by Haghighi et al. (2014), the phy- logenetic relationships among Artemisia species based on nuclear ITS and chloroplast psbA-trnH DNA mark- ers using three sections of Artemisia, Dracunculus and Serphidium propose that the ITS and cpDNA psbA-trnH markers are practicable in the systematic revision of troubled taxa at the intra-genus level in plants. Further- more, Pellicer et al. (2014) performed phylogenetic anal- ysis of the annual Artemisia within its major lineages and suggested that annual Artemisia have been specially misidentified at a subgeneric level and verified that they are phylogenetically restricted to basal grades. However, to date, very few Artemisia species have been verified with molecular phylogenetic studies based on the nucle- otide sequence data in Turkey (Koloren et al., 2016). Civelek et al. (2010) have carried out a revisionary study of the genus Artemisia in Turkey. According to results of the revisionary study based on the morpho- logical features, it was observed that growing around the Lake Van that in the populations thought to belong to the A. spicigera species, there are some groups show- ing significant morphological differences from this spe- cies. These groups were found to be similar to A. spicig- era and A. taurica in terms of morphological, but it has been accepted that they were closer to A. spicigera. In these populations, a new variety (A. spicigera var. van- ensis) belonging to the species A. spicigera was made, but the variety was not certain as it was not published (nomen nudum). The researchers stated that they are not sure about the accuracy of this systematic arrange- ment and stated that these populations should be stud- ied in detail. To solve this systematic problems, in Flora of Turkey specified to be very close to each other A. spi- cigera and A. taurica species of, planned to investigate detailed morphological and cytogenetic aspects and the research was conducted. While these studies continue, after literature search and cytogenetic observations in these populations, have been identified as belonging to the species A. fragrans case a new record for the flora of Turkey and published (Kursat et al., 2014). However, it has been stated that a molecular study is needed to confirm these results. For this purpose, it was decided to phylogenetically evaluate the populations of A. spicigera, A. taurica and A. fragrans species around the Van Lake with various molecular markers. gramer In molecular studies, ITS (ITS1-5.8S-ITS2) in rDNA and trn regions in cpDNA were amplified with specific primers and ana- 61The morphological, karyological and phylogenetic analyses of three Artemisia species lyzed with MEGA program. In the phylogenetic family tree created after the analysis, it was observed that the examined individuals of the species in question were completely separated from each other, and the individ- uals of each species were grouped among themselves. According to these results, it has been determined that there is no gene flow between the populations of these species and they are completely independent from each other. According to the morphological and caryological data, it has been molecularly proven that the populations considered as A. spicigera var. vanensis (nomen nudum) are correct to be published as A. fragrans species. CONCLUSIONS According to this study results, it has been found that there is complete speciation genetic isolation mech- anism between the species A. spicigera, A. taurica and A. fragrans that inhibit gene flow. Also A.fragrans and A. spicigera species are very similar to each other in terms of morphological characteristics. However, since popu- lations of the species A. fragrans are autopolyploid, the dimensional values of their morphological squares are larger than those of the species A. spicigera. This study is so important as it is the first molecu- lar based study relating with some species of Artemisia growing naturally in Turkey. ACKNOWLEDGEMENTS This work was supported by the Firat University Sci- entific Research Projects Coordination Unit [grant num- ber: FF.2090]. 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