Evaluation and identification of morphological characters suitable for delimitation of Taraxacum species distributed in northeastern China

Abstract Taraxacum germplasm resources in northeastern China are not current and do not accurately reflect the actual distribution of the species. The objective of this study was to investigate the morphological traits of Taraxacum species distributed in northeastern China and identify those that will facilitate their classification in this region. Leaf, flower, and achene characteristics of 18 species were used for morphological classification. Scanning electron microscopy (SEM) was used to examine pollen morphology. Internal transcribed spacer (ITS) sequences were analyzed to determine sequence differences among the species and their utility in delimitation. Taxa were classified into groups based on their morphology. The ITS sequence analysis supported the taxon classification, but the genetic distances among the taxa did not reflect morphological differences. Phylogenetic analysis was used to divide the 18 species into three groups. Group I: T. coreanum (which has white flowers). Group Ⅱ: T. heterolepis, T. sinomongolicum, T. variegatum, T. asiaticum var. lonchophyllum, T. falcilobum, T. brassicaefolium, and T. erythropodium (outer involucre bracts, narrow membranous or nonmembranous). Group Ⅲ: T. formosanum, T. liaotungense, T. mongolicum, T. borealisinense, T. ohwianum, T. platypecidum, T. urbanum, T. antungense, T. asiaticum, and T. junpeianum (outer involucre bracts, broad membranous). The main taxonomic characteristics of Taraxacum floral organs and achene morphology.

Previous studies have employed morphological characteristics related to achene shape and color, overall achene length, achene beak length, leaf shape, leaf length, and leaf color to distinguish between Taraxacum species (Hidayat et al. (2016)). The taxonomic significance of seed (achene) structure has been highlighted by multiple researchers. For example, Ullah et al. (2021) investigated the ultrastructure of fruit and seed surface morphology among populations of the alpine Rosa sericea complex (Rosaceae) using scanning electron microscopy (SEM). Morphological and foliar characteristics are important tools for the identification of different plant groups, and they have been examined under light microscope (LM) and SEM to determine the taxonomic implications of the leaf epidermal anatomy of selected taxa of Scrophulariaceae from Pakistan (Ullah et al., 2020). Pollen morphology has assumed great significance in plant taxonomy. SEM has been used to evaluate the pollen diversity of the genus Sophora (Fabaceae) and its taxonomic significance (Liao et al., 2021).
The internal transcribed spacer (ITS) region of nuclear ribosomal DNA consists of internal transcribed spacer 1 (ITS-1), the 5.8S, and internal transcribed spacer 2 (ITS-2). This region has been widely used in the molecular phylogenetic analyses of many plant taxa, such as Asarum sieboldii Miq., Withania somnifera (L.) Dunal., Convolvulus prostrates Linn, and Evolvulus alsinoides (L.) L. var (Kim et al., 2014;Lee et al., 2013). In addition, a conserved 14 bp motif (5′-GAA TTG CAG AAT CC-3′) was found in the 5.8S gene, which is useful to differentiate between flowering plants and other plant groups (Martinez et al., 2015;Xiao et al., 2021). Kirschner revised the classification of dandelions in Central Asia by using color digital pictures to compare the characteristics seen in these with the known classification of dandelions, which is an important advancement in the classification of dandelions in Central Asia (Jan, 2016). In this study, we aimed to categorize 18 Taraxacum taxa from northeastern China based on DNA data and cluster analysis of morphological characteristics, and to evaluate whether the latter supports molecular-based phylogeny.

| Plant materials and growing conditions
The flora of northeastern China harbors 18 native Taraxacum taxa, which are defined based on the leaf margin, flower color, shape and texture of the outer bracts, shape of the inner bracts, achene length, and other characteristics. Their distribution in this region is shown in Figure 1. Eighteen taxa were collected from their natural habitats from April to October each year from 2011 to 2019; more than 30 individuals were collected for each taxon (Table 1 and

| Measurement of morphological characteristics
Vegetative and reproductive morphological traits that distinguish Taraxacum species were selected based on the treatment of the genus. The plant height was measured from the highest vertical point (stem or leaf apex) to the root tip. The largest leaves with a normal morphological appearance were selected for measurements.
The leaf length was measured from the lamina tip to its base, excluding the petiole. The leaf width was measured across the widest point.
The leaf thickness was measured across the thickest part. The shape, size, color, and ornamentation characteristics of achenes (e.g., shape and amount of ornamentation) were observed on the fruit wall. The shape and size of the coracoid base and the transition of the fruit body to the coracoid base were constricted suddenly or contracted gradually. The beak thickness and length, color of the crested hair, and beak length ratio (BL/AL: beak base length to achene length) were also measured.

| ITS sequence analysis
Genomic DNA from Taraxacum leaf tissue was extracted and purified using a modified cetyltrimethylammonium bromide (CTAB) method. The quality and quantity of the isolated DNA were verified using agarose gel electrophoresis. The universal forward and reverse primers, ITS-F (5′-AGG TGA ACC TGC GGA AGG ATC ATTG-3′) and ITS-R (5′-CTT CTC CTC CGC TTA TTG ATA TGCT-3′), were used to amplify the ITS region. A total of 25 ng genomic DNA and 5 pmol of each primer were used in the reaction that included 1 μmol·L −1 of primer, 2.0 mmol·L −1 MgCl 2 , 1.

| Data analysis
Data for 32 qualitative and quantitative morphological characteristics of the 18 taxa were subjected to analysis of variance (ANOVA) using SPSS v.21.0 at significance levels of p ≤ .01 and p ≤ .05, principal component analysis (PCA), and cluster analysis.
The obtained sequences of the ITS region were aligned using Clustal X and adjusted manually. Phylogenetic tree reconstruction based on the parsimony method was performed using PAUP* version 4.0b10. The insertions and deletions were treated as missing data. Clades' support was evaluated using the bootstrap analysis with 1000 replicates. The number of steps, consistency indices, and retention indices were calculated using the TREE SCORE command in PAUP*.
The plant height was divided into three categories: short (5-10 cm), medium (10-15 cm), and tall (15-20 cm). The leaf length and width ranged from 6 cm to 20 cm and from 1.2 cm to 2.9 cm, respectively (Table 3). The leaf thickness ranged from 0.61 mm to 1.26 mm. The leaf anatomy in cross-section and floral and achene traits differed among the 18 taxa, with pubescent leaves compared with thicker, glabrous leaves in other species (Figures 1-3). Three types of leaf margin were observed: deeply pinnatifid (pinnatisect), moderately pinnatifid (pinnatifid to pinnatisect), and shallowly pinnatifid. The apical lobe shape of the leaves was primarily triangular, but the shapes ranged from triangular, triangular-oblong, diamond-shaped triangle, to triangular-hastate ( The data characteristics of pollen size, spine width, spine density, germination pore size, and pore size can be used as an auxiliary basis for the classification of Taraxacum.

TA B L E 2 (Continued)
was 295 bp, and the 5.8S recombinant DNA (rDNA) sequence was 162 bp long. A BLAST search of all sequences showed 99% similarity to a partial sequence of the ITS region in T. coreanum (accession number JF837599.1). Of these, 162 (22%) were constant and 569 (78%) were potentially informative. The consistency index of the maximum parsimonious tree was 0.051 and the retention index was 0.007. The ITS-based topology of the 18 species was consistent with the morphological characteristics of plants. The total G + C content varied among the taxa, from 51.23% in T. ohwianum to 52.53% in T. coreanum.
Several base variation sites were identified in Taraxacum, and the information sites were approximately 3-4. The pairwise difference of the ITS base composition between the 18 taxa was within 1%.

| Evolution trend in Taraxacum species
The taxonomy of the Taraxacum complex group is controversial, owing to the lack of available criteria to evaluate the systematics of these species. The taxonomic and systematic ranking of these species is doubtful, owing to their complex morphological characteristics and molecular evidence (Li & Chen, 2013 consistent and similar to those found in previous studies (Yamaji et al., 2007). In the "Flora of China," the floral organs of dandelion were recorded as the main basis for morphological classification.
In the present study, we found that the achenes' characteristics of the dandelions can also be used as the basis of classification to identify species useful in traditional Chinese medicine. Achenes, as reproductive organs, are in a relatively enclosed environment, are less affected by external environmental factors than floral organs, and harbor stable genetic traits (Ning et al., 2012;Wu et al., 2011). Taraxacum are achenes with a short and thick beak that is not obvious, and an achene wall without strumae or small spines, or partially or completely covered with tumor or spines (Lee et al., 2011).
Therefore, the achene morphology indicates that Taraxacum is a well-evolved genus. It is worth mentioning that the BL/AL ratio, which is a fixed value in the same species of Taraxacum, indicates that beak base size and achene length are quality traits that are not easily affected by the environment and stable genetic characters, so they can be used as the basis for species classification.
The results of the ITS sequence analysis were supported by the morphological traits of the sampled taxa. Eighteen taxa from northeastern China were divided into three groups. The taxa within Group II were characterized by closely arranged outer bracts with hornlike protuberances, large achenes shaped like spindles or inverted cones, and covered with spines. Group III was characterized by outer involucres basally unrolled or rolled, outer bracts without hornlike projections, smaller achenes, and an achene lower half with tuberous projections. The results of this study support the taxonomic classification of this genus in the "Flora of China."

| The causes for the complicated nature of Taraxacum classification
The taxa examined during the eight years of the study under different environmental conditions differed in terms of their leaf, flower, inflorescence, pollen, and achene morphology (Wang et al., 2010).
Of the 32 analyzed morphological characteristics, 31 exhibited significant differences among the taxa. The PCA and cluster analyses of those characteristics arranged the taxa into three groups. In particular, the BL/AL ratio of the achene morphological characteristics was not used for species delimitation in the "Flora of China"; the results of our study suggest that this character is potentially useful in the identification of Taraxacum species.

| CON CLUS IONS
Dandelion plants, which belong to the Compositae family, constitute one of the most evolutionarily diverse subfamilies used in Chinese medicine and are widely distributed in China. In this study, we surveyed most regions in China. However, dandelion classification was difficult because species boundaries are often confused. In this study, we completed the dandelion germplasm resource classification and evaluation, which can supplement and perfect the "Flora of China" dandelion classification key points. Our results will clarify the distribution of species, facilitating the development of dandelion medicinal resources, determination of medicinal value, and identification of potential medicinal species for use in traditional Chinese medicine.

CO N FLI C T O F I NTE R E S T
There is no conflict to declare.

E TH I C A L A PPROVA L
This research does not involve any studies with human and animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.