Phylogenetic relationship among taxa in the genus Adonis L. collected from Türkiye based on nrDNA internal transcribed spacer (ITS) markers

Genus Adonis L. contain 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. The aim of the study was to evaluate the phylogenetic relationship among Adonis taxa collected from Türkiye based on nrDNA Internal transcribed spacer (ITS) markers. Samples of 64 individual genotypes from 21 populations of 10 Adonis taxa were collected from different regions of the country during vegetation period between 2014 and 2018. ITS1, ITS4, P16 and P25 primers within ITS technique was used to genotype the plant materials. Then, genotypic data was used to estimate magnitude and organization of infraspecific variation in different populations of Adonis. About 600 bp DNA sequences were obtained from each 64 Adonis genotypes belonging to 21 different populations. The dendrogram obtained from Adonis taxa 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 taxa varied between A. microcarpa and A. dentata (98.46%). Principal component analysis indicated that two important components in Adonis taxa genotypes. The expected heterozygosity ranged from 0.0252 (sub-population A) to 0.3460 (sub-population C), with an average of 0.1154. In addition, population differentiation measurements (Fst) ranged from 0.0025 (sub-population C) to 0.9016 (sub-population A) with a relatively high average 0.6601. 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 of this study suggested that ITS markers could be used in the identification of genetic diversity among the Adonis taxa. The results obtained from molecular data can be used to explore the genetic variation pattern, population structure, and the evolutionary history of genus Adonis 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 Türkiye 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 fields, cultivated fields, industrial areas, airports, railway, highway sides, mountain steppes, forest, meadows, rocky slopes, shrubs, ruins, pastures and altitudes between 0 and 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 Türkiye [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 identification of Adonis taxa. 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].
The relationships among and within Adonis and closely related taxa belonging to the Ranunculaceae family have been inferred using various marker systems such as amplified fragment length polymorphisms (AFLP), inter-retrotransposon amplified polymorphism (IRAP), inter-primer binding site (iPBS), and random amplified polymorphic DNA (RAPD), etc. [11,[24][25][26][27]. However, the development of new scientific approaches based on nuclear DNA has been stimulated because of the potential problems due to cpDNA introgression among closely related taxa, as well as the lack of phylogenetic resolution, recently. Therefore, one of the most common alternatives is the sequencing of the internal transcribed spacer (ITS) of 18S-25S nuclear ribosomal DNA. In recent years, ITS analyses have been used to reconstruct classic taxonomy and resolve systematic problems that are difficult to resolve due to it having conserved lengths and a high degree of variability [3,28]. Phylogenetic relationships among 31 species belonging to Ranunculaceae family distributed in America were investigated using ITS regions (26S DNA) and reported that Adonis vernalis and Trollius laxus species were closely related [29]. In other study, phylogenetic relationships of Adonis amurensis, A. annua, A. brevistyla, A. cyllenea, A. pyrenaica and A. vernalis species were analyzed with rps16 regions and it was 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 [21]. In a study carried out in Korea, phylogenetic relationships among 60 genotypes and 12 populations belonging to A. amurensis, Adonis pseudoamurensis, A. multiflora, and A. vernalis were analyzed based on ITS and 5.8S regions in nuclear DNA [24]. In that study, systematic status of A. pseudoamurensis, which was previously introduced as a new species based only on morphological data, was supported with the molecular data. Phylogeny of 82 species including A. amurensis, A. multiflora, A. ramosa and A. shikokuensis of Ranunculaceae family widespread in China was analyzed using the aid of ITS and 5.8S regions [28]. With that study, previously classified 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. Therefore, the main objective of this study was to analyze genetic diversity and phylogenetic relationships among and within Adonis genus naturally growing in Türkiye based on nrDNA ITS.  Table 1). Botanical identification of all samples was carried out based on the related literature [4,8,9,13,16,[30][31][32][33][34][35][36][37][38].

Morphological analysis
In our study, morphological analysis was performed based on Flora of Turkey [13] using the following features: flower 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.

DNA extraction, PCR amplification, sequencing
Total genomic DNA from each accession was extracted as previously described by [39]. The quality of DNA was confirmed by electrophoresis in 0.8% agarose gel, and the DNA concentration was measured using The NanoDrop® ND-1000 UV/Vis spectrophotometer. The final 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 (5'-TCC GTA GGT GAA CCT GCG G-3'), ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'), P16 (5'-CCA YTG AAC CTT ATC ATT KAGA GGA -3') and P25 (5' GGG TAG TCC CGC CTG ACC TG-3') primers from previous reports [40][41][42]. PCR amplifications were performed in a thermal cycler (Labcycler). The PCR mixture consisted of 1X buffer, 2 mM MgCl 2 , 0.25 mM of each dNTP, 1 µM (20 pmol) primer, 0.5 U Taq polymerase, and 50 ng/µL DNA template in a 20 µL reaction mixture. The amplification conditions were as follows: an initial denaturation step of 3 min at 95 °C, 38 cycles of 60 s at 95 °C, 60 s at 67 °C, 120 s at 72 °C, and a final extension step of 10 min at 72 °C. The amplification products were resolved in 1.5% agarose gel in 1X SB buffer at 6 V/cm for 120 min, stained with ethidium bromide (0.5 μg/mL), and visualized under a UV-trans illuminator. The sizes of the amplicons were determined based on a DNA ladder between 50 and 1000 bp (Vivantis Product No: NM2421). PCR products were sequenced using an ABI 3500XL (Applied Biosystems, Foster City, CA, USA) automated sequencer.

Molecular cloning, sequencing and data analysis
The raw data obtained from the sequencing process were edited using ChromasPro Version 1.7.5 (Technelysium Pty. Ltd. 2003-2013). The sequence alignment was performed on ClustalW 2.1 program [43] and adjusted manually. The phylogenetic tree was constructed using the Neighbor Joining Tree-Jukes-Cantor model of Geneious V. 11.1.4 program. The phylogenetic tree was visualized using Interactive Tree of Life [44]. Branch support values were calculated using a full heuristic search using maximum number of trees 1000 and 1000 bootstrap replicates. ITS sequences of Ranunculus asiaticus L. (GU257963), Delphinium polycladon Eastw (AF258743), and Adonis vernalis (AJ347910) taxa used as out-group in the phylogenetic tree were retrieved from NCBI GenBank based on related studies [27,[45][46][47].
Molecular variances within and between the Adonis genotypes were estimated by the analysis of molecular variance (AMOVA) using GenAlEx 6.5 software [48]. The genetic structure of the ecotypes was determined using model-based cluster analysis (Structure v. 2.2) [49,50]. The number of populations (K) was expected every ten runs for every population, which varied from 2 to 10, characterized by a set of distinctive allele frequencies at each locus, and the individuals were sited in K clusters. Using this method, Markov chain Monte Carlo (MCMC) posterior probabilities were estimated. The MCMC chains were run with a 10,000-iteration burn-in period, followed by 100,000 iterations using a model allowing for admixture and correlated allele frequencies. The most expected value for K was predicted with Evanno's ∆K method [51] using Structure Harvester [52].

Phylogenetic relationship among taxa and principal component analyses (PCA)
In this study, about 600 bp DNA regions were obtained from 64 genotypes of 10 Adonis taxa distributed in Türkiye  1). The data for investigated Adonis taxa and out-groups were subjected to genetic distance clustering analysis and a dendrogram is presented in Fig. 3.
According to the dendrogram, outgroups are clearly different from the examined Adonis taxa. In dendogram, Adonis members were separated into two large groups based on their genetic distance. In the group I, Consiligo section including perennial A. volgensis and A. paryadrica species was close to perennial out-group taxa A. vernalis, Adonis section composed of annual taxa was clearly separated from them. In the group II, annual Adonis taxa were placed together close to each other. Such a case proved that genotypes of Turkish flora 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 group A under group II and the other taxa (from A. flammea FK135-2 to A. aestivalis subsp. aestivalis FK144-3) were placed in the group B. A. microcarpa generally has red flowers and rarely has yellow flowers. Red-flower samples were largely close to A. annua species). Yellow-flower A. microcarpa FK102 was very similar morphologically to A. dentata, thus was placed in the same group with it. Although yellow-flower forms of this species are weakly monophyletic, such a case should be elucidated with further studies (Fig. 3).
Genetic similarity coefficient data were given in Supplementary file 2. There was quite high genetic similarity correlation coefficient between yellow-flower 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 taxa varied between 1.16 and 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 -129 and A. volgensis-167). With regard to intraspecific 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 taxa 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, flower 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. parviflora had similar general morphology, aggregate structure, achene shapes, flower colors and petal stains, they were different in flower diameter, achene sizes and surfaces. A. annua and A. microcarpa species closely positioned in the dendrogram were similar in hairs, spread zones, flowering periods, non-hairy stems, sepal hairs, flower color, petal stains, achene sequence, size and surface, but different in achene shape, dorsal hump, flower diameter and sepal shape (Fig. 2).
The purpose of the principal components analysis is to obtain a small number of linear combinations, which account for most of the variability in used data. In our study, the relationships among parameters related to genotypes were investigated by principal component analysis (PCA). Individual clustering was performed according to principal components analysis with phenotypic data to visualize the population structure. Inference of population structure in the two grups based on PCA of the molecular data showed (Fig. 4).
Greatness of these variances influences good separation of genotypes. If there will be correlations between traits or similarities between genotypes, these components can provide appropriate grouping and separate the same genotypes into different groups [53]. Adonis accessions of molecular analysis of variance (AMOVA) results to assess variation in populations showed that within-populations (94.00%) were higher than between-populations (6.00%) ( Table 2).

Discussion
In this study, intra and inter-species genetic relations of genus Adonis were determined with the aid of molecular data gathered from 10 Adonis taxa widespread in Türkiye, an out-group Ranunculus asiaticus taxa morphologically   (Table 5). 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 findings, A. annua and A. dentata species with the same number of chromosomes were genetically quite close (98.44% similarity coefficients). In the study of Cai et al. [28] and Wang et al. [45], Adonis taxa were genetically placed together with Ranunculus species in the same group and separated from Delphinium species. In the present dendrogram, Adonis taxa were placed at close positions   [56] indicated the most significant 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 significant role in decisions to be made for the preservation of endemic and endangered species. ANOVA results to assess variation in populations showed it was 94% within the population but 6.00% between populations. The reason could be that the region has differences in altitudes and climatically, which tend to improve the level of diversity. Our results showed that the highest value was obtained at K = 4. Population stratification of the total genotype assembly indicated the existence of a distinct structure.

Conclusion
Our molecular findings on genetic diversity of different Adonis taxa 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 classification 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 first report the employing ITS primers on genus Adonis for study genetic variation of different phytogeographical regions of Türkiye.
To the best of our knowledge, a limited number of studies have been published on the phylogenetic relationships among Adonis populations in this region. In the present study, it was determined that the taxonomic classification 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. This work attempts to contribute to reflecting the genetic diversity of Adonis populations and our molecular data can be used to explore the genetic variation pattern, population structure, and evolutionary history of the genus Adonis. In addition, in the future, molecular genetic information along with morphological data will provide an intergraded characterization of existing diversity and will allow for its use in breeding efforts and in commercial Adonis.