Our taxon sampling strategy was designed to include Aster and relatives genera based on the recent classification (Li et al., 2012; Fu et al., 2016). We downloaded nrDNA ITS sequence matrix of 71 species from other molecular phylogenetic studies (Li et al., 2012; Zhang et al., 2015a; Zhang et al., 2019b), representing 17 genera and major clades of Aster and its relatives. Meanwhile, we downloaded the complete plastome sequence matrix of 17 species, representing the genus Aster and its relatives.

One fresh leave for each specimen was dried in silica gel (the list of samples is shown in Table 1 ). Total genomic DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the CTAB DNA extraction protocol (Doyle and Doyle, 1987) and the manufacturer’s instructions. In this study, to determine the systematic position of the new species, we used nuclear ribosomal DNA ITS sequences for phylogenetic inference. The spacer region of the nuclear ribosomal repeat (nrDNA ITS) was separately amplified with standard PCR following the method described in Zhang et al. (2015, 2019). The new species, A. yaoshanensis was sequenced, and the GenBank accession numbers and sample information of the ITS sequences used in this study are shown in Table 2 (accession number of A. yaoshanensis: OL461705.1). Voucher specimens were deposited at the herbarium of the Sichuan Normal University (SCNU).

In this study, we also used the complete plastome sequence of A. yaoshanensis for phylogenetic inference. We followed the CTAB DNA extraction protocol (Doyle and Doyle, 1987) and the manufacturer’s instructions, to extract total genomic DNA of A. yaoshanensis, using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China). Then, the NanoDrop 2000 Spectrophotometer and Qubit 4 Fluorometer (Thermo Fisher Scientific, Wilmington, DE, USA) were used to test quality and quantity of the total genomic DNA. We constructed DNA libraries using Illumina Paired-End DNA Library Kit (Illumina Inc., San Diego, CA, USA) in the NovaSeq 6000 platform with a paired-end reading length of 150 bp (NovoGene Inc., Beijing, China).

The complete plastome of A. yaoshanensis was assembled by SPAdes3.15.1 (Bankevich et al., 2012) with default parameters. The circular plastome was obtained by using Bandage (Wick et al., 2015). Subsequently, the results were annotated using PGA (Qu et al., 2019). The annotation results were checked using Geneious R11 (Kearse et al., 2012). The plastome map was drawn on the OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html). The tRNA sequences were also confirmed by tRNAscan-SE v2.0.7 (Lowe and Eddy, 1997), on the platform Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html). The genome sequence of A. yaoshanensis has been deposited in GenBank (accession number: ON120847.1). We performed nucleobase content and genes analysis using the platform JSHYClound (www.jshycloud.net).

Phylogenetic analyses of the ITS data set ( Table 2 ) were performed using maximum likelihood (ML) and Bayesian inference (BI) analysis using RAxML (Stamatakis et al., 2008) and MrBayes (Ronquist et al., 2012), respectively, on the CIPRES science gateway portal (https://www.phylo.org/portal2/, Miller et al., 2010). Prior to ML and BI analyses, the best fit model selected was GTR+T+G according to the Akaike information criterion (AIC) implemented in ModelTest (Posada and Crandall, 1998). RAxML was conducted with the fast bootstrap option, using 1,000 replicates, bootstrap value (BS) of 80%–100% were interpreted as strong support, 60%–80% as moderate. In BI analysis, four Markov chain Monte Carlo chains were run for 7,000,000 generations each, and were sampled every 1,000 generations, starting with a random tree, and Bayesian posterior probabilities (PP) were calculated for the majority consensus tree of all sampled trees after discarding 25% of trees sampled. Bayesian PP more than 0.95 were considered to be strong support, from 0.9 to 0.95 to be moderate. Finally, the trees were visualized using FigTree (Rambaut, 2012). The species Chrysanthemum indicum L. and Dendranthema indicum (L.) Des Moul. were selected as the outgroup following previous works (Panero and Funk, 2002, 2008; Panero et al., 2014).

To investigate the phylogenetic positions of A. yaoshanensis within the Aster genus, 17 complete plastome sequences were downloaded from the NCBI database ( Table 3 ). Phylogenetic analyses of the complete plastome data set were performed using ML in RAxML on the CIPRES platform (https://www.phylo.org/portal2/). We used ModelTest to predict the best fit model. The fast bootstrap option and 1,000 replicates were used in RAxML. Finally, the trees were visualized using FigTree.

Based on the topology of the ITS sequence for ML analysis, in Mesquite v3.81 (Maddison, 2008), we used the “Trace character history” option to analyze the ancestral morphological characters in the genus Aster. An ML approach using Markov k-state 1 parameter model (Mk1; Lewis, 2001) was used to reconstruct character evolution. The data employed for the reconstruction of the evolution of morphological characters were obtained from literatures (Li et al., 2012; Norbert et al., 2017), and our own observations of morphological character variation using herbarium specimens held at SCNU and PE (Thiers, 2024), and CVH (https://www.cvh.ac.cn/). The codes associated with these morphological features include (A) Inflorescence type: listed in order of 0–7, (B) the length of pappus: (0) none pappus, (1) short pappus, (2) long pappus, (3) longer pappus; (C) habit (0) dwarf herb, (1) tall herb, (2) subshrub, (3) shrub. A list of morphological characters and their character state coding used for the ancestral state reconstruction is detailed in Table 4 . The differences of eight types of inflorescence can be seen in the statement at the end of Table 4 . Based on the codes, we reconstructed the evolution of three morphological characters for genera of Aster and its relatives.

With the information collected through fieldwork, the conservation status of the new species was evaluated according to the IUCN, 2024 categories and criteria.

In this study, the structure of plastome of A. yaoshanensis is highly conserved, and it has standard quadripartite structure, including a large single-copy (LSC), a small single-copy (SSC), and a pair of IRs (IRa and IRb). The size o f A. yaoshanensis is 152,384bp, and GC content is 37.37%. Overall, the plastome have 133 genes, including 87 protein-coding genes, 38 tRNA, and 8 rRNA, of which 115 were unique and 18 were duplicated in the IR regions ( Table 5 ). The arrangements of these 133 genes were totally collinear. There are two introns of three genes (rps12, ycf3, clpP), and single intron of 16 genes (ndhA, ndhB, petB, petD, atpF, rbcL, rpl16, rpl2, rps16, rpoC1, trnA-UGC, trnG, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC). The gene rps12 was trans-spliced and the genes ndhD, psbL were RNA editing ( Table 5 ). These results were performed in the plastome map ( Figure 1 ).

The total length of ITS sequence alignment with gaps was 650 bp. Analysis of the data using ML and BI methods obtained similar trees with high ML Bootstrap (BS) and BI posterior probability (PP). Phylogeny analysis showed that A. yaoshanensis is positioned in the Aster clade, and it formed a clade with A. homochlamydeus Hand.-Mazz and A. handelii Onno (ML BS ≥ 90% and BI PP ≥ 0.90) ( Figure 2 ).

Based on phylogenetic analysis, A. yaoshanensis is positioned in the Aster clade, and it is the sister species to A. ageratoides var. scaberulus (Miq.) Y. Ling. (BS = 100) ( Figure 3 ).

Considering the similarity between new species and its related species in Aster, we obtained the morphological characteristics of the latter from specimens and literature, and made morphological comparisons between these species ( Table 6 ). The new species is most similar to Aster jishouensis W.P. Li & S.X. Liu ( Figure 4 ). It is mainly distinguished from the latter by its erect (versus oblique ascensional) stem, deep purple (vs. green) basal leaves, 4–5 seriate (vs. 5–7seriate) phyllaries, numerous (vs. 1–4) capitula. In addition, the results of ancestral trait reconstruction were shown in Figures 5 – 7 .

Aster yaoshanensis K. Qin, Z.X. Fu & P. Li, sp. nov. ( Figures 8 , 9 )

Type (Holotype). China, Guangxi, Laibin city, Jinxiu county, Dayaoshan Mountain National Nature Reserve, ca. 1100 m a.s.l., 24.1988°N, 110.2692°E, limestone, 12 November 2020, Zhixi Fu FZX5501 (SCNU).

Description. Perennial herbs, 90–130 cm tall, basally woody, rhizomatous. Stems erect, unbranched, finely striate, smooth and glabrous with many basal leaves and some cauline leaves. Basal leaves rather thick, ovate-lanceolate, apex acute to slightly obtuse, petiole narrowly winged, 3–3.5 cm × 1–1.5 cm, margin undulant serrate, abruptly mucronate, adaxially green, strigillose; abaxially amaranth, the vein strigillose. Cauline leaves, slightly reduced downward, adaxially green, densely strigose; abaxially subglabrous, midvein prominent abaxially, lowest leaves withered by anthesis, lower to upper leaves sessile or shortly petiole, the uppermost very small, blade narrowly lanceolate to oblanceolate, 2–14 cm × 1–4 cm, margin entire to undulant serrate (teeth mucronulate). Flowering branches attach to the leaf-axils, capitula numerous, in terminal or axillary radialized, 1.5–2 cm in diam; peduncles 2–8 mm, smooth and glabrous, bracts ovate, margin entire. Involucres campanulate, 6–7 mm × 2–3 mm, phyllaries 4-5 seriate, unequal, narrowly green apically, abaxially moderately to densely strigose, margin scarious, apex acute to obtuse, ciliate, outer phyllaries ovate, 1.5–2 × ca. 1 mm, middle phyllaries lanceolate-oblong, 2–2.5 × ca. 1 mm, inner phyllaries approximately oblong, 3–3.5 × 1–1.5 mm. Ray florets 8-18, white, lamina 6–7 mm × 1–1.5 mm, glabrous, eglandular; disk florets yellowish-green, 5–6 mm, tube base flared, lobes capreolary, tip recurved to cannular. Achenes long-obovoid, compressed, ca. 1.8–2.2 mm, 2-3-ribbed, sparsely strigillose. Pappus, bristles 2.5–3.5 mm. ( Figures 10 , 11 ).

Etymology. The specific epithet refers the famous Dayaoshan Mountain in Jinxiu county, Laibin city, Guangxi autonomous region, China.

Distribution and habitat. The new species, A. yaoshanensis is mainly distributed at an altitude of about 1000 m in Dayaoshan Mountain, Guangxi ( Figure 12 ), and mainly growing on limestone, slopes, forest margins, flooded lands, along streams, wet places.

Phenology. Flowering and fruiting from September to November.

Conservation status. According to the IUCN, 2024 categories and criteria, newly described species with a restricted distribution area are often considered in a threat category (CR, EN, VU) (Zhang et al., 2019; Blasco et al., 2021; Wagensommer and Venanzoni, 2021; Ibáñez et al., 2022), but more exploration in the occurring area of the new species is necessary for defining the distribution, population size, threats, etc., before being able to assess the conservation status of A. yaoshanensis. The new species is currently only found in rock crevices of mountain valleys in Dayaoshan National Nature Reserve. It is a narrowly distributed species with no more than 30 individuals found. It is very possible that the species is found in other localities with a more comprehensive collecting programme. Therefore, it would be best to assess the conservation status of the species as DD (Data Deficient).

Additional specimens examined (Paratypes). CHINA. Guangxi: Jinxiu county, Dayaoshan Mountain National Nature Reserve, alt. 1100m a.s.l., limestone, 12 November 2020, FZX5505 (SCNU), FZX5508 (SCNU), FZX5509 (SCNU) FZX5510 (SCNU), and FZX5511 (SCNU).

In our study, we successfully assembled the complete plastome of A. yaoshanensis collected from Guangxi for the first time. Based on the analysis of the plastome map, we found that the structure, gene location, size and gene content of A. yaoshanensis are highly conserved, which is very similar to the plastome structure of most reported Aster plants, such as A. ageratoides (Feng et al., 2021; Zhang et al., 2021; Wang and Liu, 2023). The plastomes of these Aster have a standard quadripartite structure, including two inverted repeat regions (IRs), an LSC region and an SSC region, and the general GC content was low in the whole sequence. This indicates the close relationship between A. yaoshanensis and Aster.

In order to further determine the evolutionary position of A. yaoshanensis in Aster, we conducted phylogenetic analysis of ITS sequences of Aster and its related genera ( Figure 2 ). Heteropappus, Kalimeris, Sheareria, Rhynchospermum, Rhinactinidia, Miyamayomena, Turczaninowia, Doellingeria, and other Aster taxa were deeply nested within the core Aster clade; this is consistent with previous studies (Li et al., 2012). In the Aster clade, A. yaoshanensis formed a compact clade with A. homochlamydeus and A. handelii (PP = 1.00, BS = 100), and the clade was sister to the clades comprising some Aster taxa, Heteropappus and Kalimeris. The result further proved that A. yaoshanensis is closely related to Aster. In addition, A. jishouensis and A. yaoshanensis were apparently embedded in different clades, indicating that the two species are not closely related.

Phylogenetic analysis of 17 plastomes within Aster and its related genera revealed that A. yaoshanensis is sister to A. ageratoides, and they form a powerful clade (BS = 100 in Figure 3 ), which was sister clade to the clade comprising with six Aster species (BS = 100). At the same time, Heteroplexis is not monophyletic and embedded within the Aster clade, which is sister clade to above eight Aster species (including A. yaoshanensis, BS = 100). This is consistent with Duan’s findings (Duan et al., 2022). Therefore, the Aster clade had strong support, and A. yaoshanensis should be merged into Aster.

Pappus length, inflorescence, and habit were often considered to be important diagnostic characters in previous taxonomic treatments of Aster and its related genera (Li et al., 2012; Norbert et al., 2017).

Pappus length. In Asteraceae, sepals usually degenerate to pappus to help the dispersal of fruits or seeds, and the pappus of Aster plants are varied in morphology (Chen et al., 2011; Norbert et al., 2017). Therefore, it is one of the important features in the classification of Aster and its related genera (Chen, 1986; Li et al., 2012). Based on the length of pappus, these plants are divide into four types (I–IV): none pappus, short pappus, long pappus, and longer pappus. The result indicated that type IV with a probability of 98% was the common ancestral trait ( Figure 5 ). A. yaoshanensis have longer pappus, belonging to the typical type IV.

Inflorescence. In genus Aster, the inflorescences belong to capitula (Chen et al., 2011), and the evolution of inflorescence is diverse (Li et al., 2012). Based on the number of capitulum and the way of inflorescences branching, Aster and its related genera could be divided into seven types (I–VII), and the legend can be seen in Figure 6 for details. The type of inflorescence is one of the important identification morphological characters of A. yaoshanensis as a new species, we define it as Type VII. Compared with A. handelii (I) and A. homochlamydeus (III) of the same subclade, A. yaoshanensis has more numbers of capitula, which mostly grows in the branch ends and leaf axils. A. jishouensis (VI) is very similar to A. yaoshanensis, but inflorescences have only 1–4 capitula. In addition, the ancestral reconstruction result showed that type I evolved independently at least 10 times in this taxa, type II originated from type III, and the evolutionary transformation may have occurred more than once.

Habit. In our analysis, we divided the habit of Aster and its relatives into dwarf herb, tall herb, subshrub, and shrub (Chen et al., 2011; Norbert et al., 2017; Jabbour et al., 2018). Phylogenetic analysis confirmed that tall herbs were the most probable ancestral feature of these plants (96%, Figure 7 ). Like A. jishouensis, A. yaoshanensis belongs to the tall herb, which may be the result of the evolution of the two plants to adapt to similar habitats. In addition, we found that dwarf herbs evolved independently at least nine times, subshrubs four times, and shrubs only twice.