Molecular phylogeny based on its sequences of nrDNA ITS of Adonis (Linnaeus, 1753) (Ranunculaceae) from various ecological sites of Turkey


 Background Genus Adonis (Linnaeus, 1753) contains approximately 40 annual and perennial species, which are widely distributed in the temperate zones of Asia and Europe, and less frequently in southwestern Asia, northern Africa and the Mediterranean region. In the present study, molecular phylogeny of genus Adonis L. from Turkey was evaluated with the aid of nrDNA ITS. Methods Samples of 10 natural Adonis taxa belonging to 21 different populations including 64 Adonis ecotypes were collected from different regions of the country during vegetation period between 2014 and 2018. ITS1, ITS4, P16 and P25 primers within Internal Transcribed Spacer (ITS) technique were used to estimate infraspecific variation in different populations of Adonis species. Results We identified about 600 bp DNA sequences were obtained from 21 different populations including 64 Adonis ecotypes. The dendrogram obtained from Adonis species and out-group sequences had two large main groups. While the out-group species were placed in the first large main group, the sect. Consiligo (perennial) and sect. Adonis (annuals) were placed in different sub-groups of the second large main group. Genetic similarity among Adonis species varied between A. microcarpa and A. dentata (98.46%). Conclusions Present analyses revealed that phylogenetic classification (grouping) of Adonis taxa largely depended on morphological structure and present ITS primers were quite efficient in putting forth the genetic diversity of such species. The results obtained from molecular data can be used to explore the genetic variation pattern, population structure, and the evolutionary history of natural Adonis species in the future.


Introduction
Ranunculaceae family, distributed almost all over the world, is considered as one of the essential groups of ancient angiosperms. It is a monophyletic family and estimated to have a history of approximately 75 million years according to the fossil records [1][2][3]. This family with about 43 genera and 2346 species is represented by 20 genera and 204 species in Turkey and 51 of these species are endemic [4,5].
Within the family, Adonis L. genus contains approximately 40 annual and perennial species, which are widely distributed in the temperate zones of Asia and Europe, and less frequently in southwestern Asia, northern Africa and the Mediterranean region [6][7][8][9][10][11]. Adonis species are distributed in habitats such as fallow elds, cultivated elds, industrial areas, airports, railway, highway sides, mountain steppes, forest, meadows, rocky slopes, shrubs, ruins, pastures and altitudes between 0-3500 m. [12]. In the latest taxonomic studies, the genus was divided into 2 sections, annual species in sect. Adonis and perennials in sect. Consiligo DC, and is represented by 11 taxa in Turkey [4,[12][13][14][15]. Previous studies on Adonis were mostly restricted to morphological [16,17], ecological [18], palynological [19] and cytological analyses [20]. Large variations in morphological characteristics and hybridizations between some species are the main problems in identi cation of Adonis species. Many previous studies reported that molecular techniques had great contributions to understanding phylogeny, evolution and taxonomy of Adonis and the other species [21][22][23]. Recently, Ro et al. [24] investigated phylogenetic relationships among 31 species belonging to Ranunculaceae family distributed in America using ITS regions (26S DNA) and reported that Adonis vernalis and Trollius laxus species were closely related. Johansson [21] studied phylogenetic relationships of Adonis amurensis, A. annua, A. brevistyla, A. cyllenea, A. pyrenaica and A. vernalis species with rps16 regions and reported that these species were close relatives and total size of genome was 151.3 kb in A. annua and 156.5 kb in Adonis vernalis. Suh et al. [25] studied phylogenetic relationships among 60 genotypes and 12 populations belonging to A. amurensis, Adonis pseudoamurensis, A. multi ora, and A. vernalis in Korea based on ITS and 5.8S regions in nuclear DNA. In that study, systematic status of A. pseudoamurensis taxon, which was previously introduced as a new species based only on morphological data, was supported with the molecular data. Cai et al. [26] studied phylogeny of 82 species including A. amurensis, A. multi ora, A. ramosa and A. shikokuensis of Ranunculaceae family widespread in China with the aid of ITS and 5.8S regions. With that study, previously classi ed sub-families and tribes based on morphological data were revised according to the phylogenetic tree obtained from molecular data. Despite several previous studies, molecular studies on Adonis generally focused on perennial species and the information about phylogeny and genetic diversity of this genus is quite limited. The main objective of this study was to analyze genetic diversity and phylogenetic relationships among and within Adonis genus naturally growing in Turkey based on nrDNA ITS. To the best of our knowledge, our ndings re ect the great genetic diversity of Adonis populations and molecular data obtained from this study can be used to explore the genetic variation pattern, population structure, and the evolutionary history of natural Adonis species in the future.  Table 1). Botanical identi cation of all samples was carried out based on the related literature [4,8,9,13,16,[27][28][29][30][31][32][33][34][35]. Adonis species should be collected during the growing season since the morphological characteristics such as achene and ower are very important easy to identify (Fig. 2). In our study, morphological analysis was performed based on Flora of Turkey using the following features: ower diameter, sepal shape, sepal width and length, feather in sepal, number of petals, petal shape, petal color, petal width and length, blackish in petal base, aggregate width and length, surface type of achene, achene width and length, hump position, hump shape, hump width and length, beak width and length, beak shape, beak surface and beak color.

Materials And Methods
DNA extraction, PCR ampli cation, sequencing Total genomic DNA from each accession was extracted as previously described by [36]. The quality of DNA was con rmed by electrophoresis in 0.8% agarose gel, and the DNA concentration was measured using The NanoDrop® ND-1000 UV/Vis spectrophotometer. The nal DNA concentration was adjusted to 50 ng/µL for ITS analysis, and the diluted DNA was stored at -20°C. PCR reactions were prepared using ITS1

Results And Discussion
In this study, about 600 bp DNA regions were obtained from 64 genotypes of 10 Adonis taxa natural distributed in Turkey. The data for investigated Adonis species and out-groups were subjected to genetic distance clustering analysis and resultant dendrogram is presented in Fig. 3.
According to resultant dendrogram, outgroups are clearly different from the examined Adonis species. In dendogram, Adonis members were separated into two large groups based on their genetic distance to each other. In the rst large group, Consiligo section including perennial A. volgensis and A. paryadrica species was close to perennial out-group taxa A. vernalis, Adonis section composed of annual species was clearly separated from them. In the second large group, annual Adonis species were placed together close to each other. Such a case proved that genotypes of Turkish ora and present Adonis taxa were separated at section-level based on their length of life and general morphology. Considering the annual Adonis section, it was observed that A. aleppica, A. microcarpa, A. dentata, and A. annua) species (from A. aleppica FK117-1 to A. annua FK143-2) were placed in the 1st main group of large main groups and the other species (from A. ammea FK135-2 to A. aestivalis subsp. aestivalis FK144-3) were placed in the 2nd main group. A. microcarpa generally has red owers and rarely has yellow owers. Red-ower samples were largely close to A. annua species). Yellow-ower A. microcarpa FK122-4 was very similar morphologically to A. dentata, thus was placed in the same group with it. Although yellow-ower forms of this species are weakly monophyletic, such a case should be elucidated with further studies (Fig. 3).
Genetic similarity data were given in supplementary le. There were quite high genetic similarity coe cients between yellow-ower A. microcarpa and A. dentata (98.46%) and they had very close relationships with each other. Morphological characteristics facilitating phylogenetic separation of the taxa are presented in Fig. 3. Genetic distances among Adonis species varied between 1.16-26.43%. Based on only primers used in this study, the lowest genetic distance ratios were respectively observed as 1.16% (between A. microcarpa-102 and A. annua-123) and 1.21% (between A. microcarpa-122 and A. dentata-108); the greatest genetic distance ratios were respectively observed as 26.43% (between A. volgensis-167 and A. aestivalis subsp. aestivalis-144), 26.215 (between A. paryadrica-179 and A. aestivalis subsp. aestivalis-144) and 22.975 (between A. aestivalis subsp. parvi ora-129 and A. volgensis-167). With regard to intraspeci c genetic distances, the lowest values were observed in A. annua (0.42%), A. dentata (0.43%) and A. eriocalycina (1.15%); the greatest values were observed in A. aestivalis subsp. aestivalis (19.01%) and A. volgensis (5.02%). The dendrogram revealed that grouping of the species of the genus largely depended on morphological structure. The A. volgensis and A. paryadrica species of Consiligo section were perennial and they were quite similar in rhizome structure, ower diameter and color, number of petals, aggregate and achene structures. Since A. aestivalis sub-species were morphologically similar, they were also in similar phylogenetic grouping. While A. aestivalis subsp. aestivalis and A. aestivalis subsp. parvi ora had similar general morphology, aggregate structure, achene shapes, ower colors and petal stains, they were different in ower diameter, achene sizes and surfaces. A. annua and A. microcarpa species closely positioned in the dendrogram were similar in hairs, spread zones, owering periods, non-hairy stems, sepal hairs, ower color, petal stains, achene sequence, size and surface, but different in achene shape, dorsal hump, ower diameter and sepal shape (Fig. 2).
In this study, intra and inter-species genetic relations of Adonis species were determined with the aid of molecular data gathered from 10 Adonis taxa widespread in Turkey, an out-group Ranunculus asiaticus taxa morphologically close to Adonis and Delphinium polycladon taxa morphologically far from Adonis. In Flora of Turkey [13], based on morphological characteristics, perennial A. volgensis and A. paryadrica species were separated from annual species in number of petals, ower colors and aggregate structures and placed under sect. Consiligo. The present phylogenetic dendrogram also supported such a separation. Heyn and Pazy [20] reported number of chromosomes as 2n=16 for A. annua and A. dentata, as 2n=32 for A. microcarpa and as 2n=48 for A. aestivalis. According to the present ndings, A. annua and A. dentata species with the same number of chromosomes were genetically quite close to each other (98.44% similarity coe cients). In the study of Cai et al. [26] and Wang et al. [42], Adonis species were genetically placed together with Ranunculus species in the same group and separated from Delphinium species. In the present dendrogram, Adonis species were placed at close positions to morphologically close Ranunculus asiaticus species (Fig. 3). In Kandemir et al. [15], the status of the Adonis cyllenea var. paryadrica was re-evaluated and it was decided to change its taxonomic status to species level as A. paryadrica according to leafy stems, young carpels more densely pubescent, sepals ½ to petals, and tall rhizomatous. In our results, taxonomic status of A. paryadrica was supported with molecular data. Fischer et al. [45] [47] indicated the most signi cant factors threatening endemic and endangered plant species as changing ecological conditions and potential genetic risks resulting from reduced genetic variation [22]. In the present study, genetic similarity between endemic A. paryadrica populations close species was quite high. Right at this point, it was proved once again that calculation of genetic variation played a signi cant role in decisions to be made for the preservation of endemic and endangered species.

Conclusions
Our molecular ndings on genetic diversity of different Adonis's species can be informative and used to explore the genetic variation pattern, population structure, and the evolutionary history of natural plant species in the future. Molecular data obtained by ITS primers can provide useful information to deal with various aspects of taxonomic classi cation of Adonis and the natural plant species. Along with the evolutionary process, it is known that there are some variations between species and species in plants necessitating revision studies. This paper is the rst report the employing ITS primers on Adonis L. (Ranunculaceae) for study genetic variation of various ecological sites of Turkey. In the present study, it was determined that the taxonomic classi cation of Adonis of the ITS sequences is highly conserved among the species in this study, supporting successfully reconstructing the phylogenies at the species level. It has been concluded that the ITS sequences of nrDNA of Adonis provide enough data to identify and classify the economically relevant species along with the distinctive structural characters.