Plastid phylogenomics contributes to the taxonomic revision of taxa within the genus Sanicula L. and acceptance of two new members of the genus

Introduction The genus Sanicula L. is a taxonomically complicated taxa within Apiaceae, as its high variability in morphology. Although taxonomists have performed several taxonomic revisions for this genus, the interspecific relationships and species boundaries have not been satisfactorily resolved, especially for those endemic to China. This study mainly focused on S. giraldii var. ovicalycina, S. tienmuensis var. pauciflora, and S. orthacantha var. stolonifera and also described two new members of the genus. Methods We newly sequenced sixteen plastomes from nine Sanicula species. Combined with eleven plastomes previously reported by us and one plastome downloaded, we performed a comprehensively plastid phylogenomics analysis of 21 Sanicula taxa. Results and Discussion The comparative results showed that 21 Sanicula plastomes in their structure and features were highly conserved and further justified that two new species were indeed members of Sanicula. Nevertheless, eleven mutation hotspot regions were still identified. Phylogenetic analyses based on plastome data and the ITS sequences strongly supported that these three varieties were clearly distant from three type varieties. The results implied that these three varieties should be considered as three independent species, which were further justified by their multiple morphological characters. Therefore, revising these three varieties into three independent species was reasonable and convincing. Moreover, we also identified and described two new Sanicula species (S. hanyuanensis and S. langaoensis) from Sichuan and Shanxi, China, respectively. Based on their distinct morphological characteristics and molecular phylogenetic analysis, two new species were included in Sanicula. In summary, our study impelled the revisions of Sanicula members and improved the taxonomic system of the genus.


Introduction
Sanicula L. is a distinctive genus of Apiaceae subfamily Saniculoideae with high medicinal value (Pryer and Phillippe, 1989).The genus comprises approximately 45 species that are widely distributed from East Asia to North America, with China and North America as two diversification centers (Van et al., 2013;Li et al., 2023).Among them, nineteen species and five varieties are distributed in China and eleven species and five varieties are endemic (Sheh and Phillippe, 2005;Pimenov, 2017;Xie et al., 2019;Li et al., 2023;Song et al., 2024).The most distinctive characteristic features of the genus are the fruits (mericarps) covered with scales, bristles, or hooked prickles, a rather prominent and persistent calyx, and two persistent styles that can easily distinguish it from other genera of Apiaceae (Pryer and Phillippe, 1989;Calviño and Downie, 2007).Published studies illustrated that Sanicula was closely related to the genus Eryngium L. However, Eryngium has its distinctive morphological features, such as capitate inflorescences and single bract per flower, which is easily distinguished from Sanicula (Vargas et al., 1998;Valiejo-Roman et al., 2002;Calviño and Downie, 2007;Calviño et al., 2008).Traditionally, plant taxonomists tended to study the genus based on morphological characteristics and to divide the genus into more smaller classification units, whereas many members of the genus always exhibited varied morphological features in rhizomes, foliage, flowers, and fruits (Shan and Constance, 1951), which have resulted in massive disagreements over classification system (De Candolle, 1830;Drude, 1898;Wolff, 1913;Shan and Constance, 1951).In addition, species relationships and species identification in the genus were also blurred, largely due to phenotypic plasticity or the lack of taxonomically robust morphological characters at the species level (Pryer and Phillippe, 1989;Vargas et al., 1999;Calviño and Downie, 2007;Pimenov, 2017;Li and Song, 2022;Li et al., 2022).For example, Li et al. (2022) found that S. pengshuiensis M. L. Sheh & Z. Y. Liu and S. lamelligera Hance were similar in overall morphology and thus treated the former as a synonymy of the latter.Furthermore, the misidentification of species and misuse of species names occurred frequently due to the various morphological features within species, such as S. chinensis Bunge and S. orthacantha S. Moore, as well as S. caerulescens Franch.and S. lamelligera Hance (Chen, 2019), which made it difficult to identify species accurately.Therefore, the revisions for species of this genus, traditionally recognized by morphological features, are necessary and urgent.
A robust phylogenetic framework could provide a valuable information to aid the taxonomic revision of Sanicula.In most angiosperms, plastids are usually considered to be inherited from the maternal parent and have low nucleotide substitution rates (Wicke et al., 2011;Wataru and Tsuneaki, 2023).Thus, the plastid genomes (plastomes) have been widely and successfully used for plant phylogenetic analyses (Duminil et al., 2012;Miller et al., 2014;Razafimandimbison et al., 2014;Zhang et al., 2018;Schneider et al., 2021;Xu and Hong., 2021;Ji et al., 2022;Scatigna et al., 2022;Xiang et al., 2022;Baldwin et al., 2023;Fu et al., 2023), especially for those taxonomically controversial taxa within the family Apiaceae (Gou et al., 2020;Ren et al., 2020Ren et al., , 2022;;Cai et al., 2022;Liu et al., 2022;Guo et al., 2023;Liu et al., 2023a;Lei et al., 2022;Gui et al., 2023;Peng et al., 2023;Qin et al., 2023;Song et al., 2023;Tian et al., 2023;Song et al., 2024).For example, Song et al. (2023) transferred Peucedanum franchetii C.Y.Wu & F.T.Pu under the genus Ligusticopsis Leute based on phylogenetic analysis of ten plastomes.Gui et al. (2023) investigated the divergence and morphological evolution of alpine Tongoloa H. Wolff using 27 plastomes and nuclear ribosomal DNA (nrDNA).Guo et al. (2023) reinterpreted the phylogenetic position and taxonomic revision of the genus Pterocyclus Klotzsch (Apiaceae, Apioideae) based on 105 complete plastomes, combined with nrITS and morphological evidence.Therefore, plastomes also provided a promising window for studying the genus Sanicula.In the previously published studies (Yang et al., 2022;Li et al., 2023;Song et al., 2024), researchers have used the plastomes data to investigate the phylogenetic positions of Sanicula members, which has significantly improved our understanding of this taxonomically confused group.However, sampling of this genus was limited and the interspecific relationships of some members were still unclear, such as S. giraldii H. Therefore, this study mainly focused on three Sanicula varieties: S. giraldii var.ovicalycina, S. tienmuensis var.pauciflora, and S. orthacantha var.stolonifera.These three varieties are endemic to China. Shan (1943) described a new species (S. subgiraldii R. H. Shan) of the genus.Later, Shan and Liou (1979) also described a new species in Nanchuan, Chongqing, China, which grows on shady woods or grassy places on mountain slopes at an altitude of 1,300 m-1,600 m.They observed that this species was very similar to S. giraldii, but the fertile flowers of the species were usually fewer than in S. giraldii (one to three per umbellule vs. three per umbellule), with characteristics of broadly ovate calyx teeth, larger size, and oblong fruit, hence they regarded this species as a variety of S. giraldii (Figure 1A) and named it as S. giraldii var.ovicalycina (Figure 1B).Pimenov (2017) treated S. subgiraldii as a synonym of S. giraldii var.ovicalycina based on reviews of the type specimens and morphological evidence.So far, this variety name was accepted and all authors agreed with this treatment by Shan and Liou (1979).The other variety is S. tienmuensis var.pauciflora described by Shan and Pu (1989).This variety was a narrowly circumscribed species, only occurring in Luding, Sichuan, China.It grows on the edge of ditches or under the forest with an altitude of 2,200 m.Sanicula tienmuensis var.pauciflora (Figure 1D) is considered to be a variety of S. tienmuensis (Figure 1C), mainly because it has fewer staminate flowers (two or three per umbellule), whereas S. tienmuensis has more staminate flowers (five or six per umbellule) (Shan and Pu, 1989).The remaining one variety is S. orthacantha var.stolonifera (Figure 1F), which was described as a variety of S. orthacantha S. Moore (Figure 1E) (Shan and Liou, 1979).This variety grows on mountain top with an altitude of 2,300 m-2,450 m in Emei Shan, Sichuan, China.It can be distinguished from S. orthacantha by its thin rhizome and elongate stoloniferous nodes (vs.thick, oblique rootstock bearing elongated, fibrous roots) and ovate calyx teeth, ca. 1 mm long and 0.5 mm wide (vs.narrowly lanceolate, acute, ca. 1 mm long, 0.1 mm wide) (Li and Song, 2022).Sheh and Phillippe (2005) also recognized this variety and stated that the rhizome with long and distinct nodes was its distinctive character.However, after critical examination of type specimen and careful observation in the field, we found that these three varieties were not similar to their type varieties, especially in fruit morphology (Figure 1).Therefore, we suggested that the taxonomic positions of S. giraldii var.ovicalycina, S. tienmuensis var.pauciflora, and S. orthacantha var.stolonifera need to be re-evaluated.
In addition, during two field botanical surveys of Apiaceae in July to September 2022 and March to June 2023, we (I and my colleagues Chang-Kun Liu, Ting Ren, Yu-Lin Xiao) collected two interesting Sanicula species: Sanicula sp.SBN2022073001 (Figure 2) and Sanicula sp.SBN2023041201 (Figure 3) in Hanyuan Country, Sichuan Province, and Langao County, Shanxi Province, respectively.Sanicula sp.SBN2022073001 grows under the mixed forest or roadsides at an altitude of 2,000 m-2,100 m.Sanicula sp.[1][2][3][4][5].Dorsal side views of fruits.8. Commissural side views of fruits.9. Transverse section.Song et al. 10.3389/fpls.2024.1351023Frontiers in Plant Science frontiersin.orgSBN2023041201 grows in stream banks in mixed forests with an altitude of 1,400-1,500 m.By consulting a large number of specimens and investigating the morphological and anatomical characters, we found that both exhibited distinctly different morphological characters with other species of this genus, including distinct differences in leaves, inflorescence, peduncle, bracts, bracteoles, fruit, and calyx teeth.Based on the combination of detailed morphological features and molecular evidence, we confirmed that these two new species actually represented two hitherto undescribed species of Sanicula.
In this study, we aim to (1) reveal the plastome features of three varieties and the two undescribed species of Sanicula; (2) uncover the phylogenetic positions of these three varieties and the two undescribed species; and (3) provide a taxonomic revision for these three varieties and accept two new members of the genus based on comparative plastome analyses, molecular phylogeny and morphological features.2 Materials and methods

Sample collection, DNA extraction, sequencing, assembly, and annotation
In this study, we collected sixteen individuals of nine Sanicula species in the wild and the fresh young basal leaves were immediately dried and stored with silica gel.All voucher specimens were deposited in the Sichuan University Herbarium (SZ) (Chengdu, China) (Supplementary Table 1).Herbarium codes are based on Index Herbariorum (https://sweetgum.nybg.org/science/ih/).We used the modified CTAB method (Pahlich and Gerlitz, 1980) to extract total genomic DNA from silica gel-dried leaves, which was then used for subsequent sequencing.
Before library preparation, we used agarose gel electrophoresis to test the quality and quantity of genomic DNA.Then, the DNA library with an average insert size of 400 bp was constructed using the TruSeq DNA Sample Preparation Kits (Illumina) referred to the manufacturer's protocol (Illumina, San Diego, CA, USA).The DNA library was sequenced using the Illumina NovaSeq platform at Shanghai Personal Biotechnology Co., Ltd.(Shanghai, China), with an average paired-end read length of 150 bp.At least 5 GB of raw data per species was generated.To obtain high-quality reads, the software fastP v0.15.0 (-n 10, -q 15) (Chen et al., 2018) was used to filter raw data.For the yielded high-quality reads, we employed two methods to assemble the complete plastomes.First, the GetOrganelle pipeline (Jin et al., 2020) was used to assemble the sixteen complete plastomes of nine Sanicula taxa, using the plastome sequence of S. giraldii (OQ612643) as a reference.To validate the accuracy of plastome assembly, we also assembled the sixteen plastomes using the NOVOPlasty v2.6.2 program (Dierckxsens et al., 2017), setting the rbcL sequence extracted from the plastome of S. giraldii (OQ612643) as the seed.The complete plastomes were initially annotated by Plastid Genome Annotator (PGA) (Qu et al., 2019) software, with S. giraldii (OQ612643) as a reference, and then manually checked and corrected the start and stop codons and intron positions in Geneious v9.0.2 (Kearse et al., 2012).Finally, the wellannotated plastomes were displayed by online program OrganellarGenomeDRAW (OGDRAW) (Lohse et al., 2007).

Comparative plastome analyses
We compared the IR length and gene location at the IR/SC boundaries among the 21 Sanicula plastomes in Geneious v9.0.2 (Kearse et al., 2012).Then, we detected the possible gene rearrangements using the whole genome alignment tool Mauve v1.1.3 plugin (Darling et al., 2004) in Geneious v9.0.2 (Kearse et al., 2012).In addition, we evaluated the degree of variation sequences of these 21 Sanicula plastomes using the LAGAN model implemented in the mVISTA (Frazer et al., 2004) tool with default parameters, setting S. astrantiifolia as the reference.Finally, to further investigate the hypervariable regions, the protein-coding genes, the non-coding regions, and the intergenic regions among the 21 Sanicula plastomes were extracted in Geneious v9.0.2 (Kearse et al., 2012) and aligned with MAFFT v7.221 (Katoh and Standley, 2013).The alignments with less than 200 bp in length were discarded, and then we calculated the nucleotide diversity (Pi) employing DnaSP v5.0 (Librado and Rozas, 2009).

Phylogenetic analyses
We performed the phylogenetic analyses using two datasets: dataset 1 was the 60 complete plastomes (16 newly sequenced) and dataset 2 included 73 ITS sequences (43 newly sequenced and assembled) (Supplementary Tables 1, 2).Among them, Hedera L. species were served as the outgroup referring to a previous study (Song et al., 2024).Sequences from the two datasets were respectively aligned using MAFFT v7.221 (Katoh and Standley, 2013) and adjusted manually when necessary.Both identified matrixes were subjected to Maximum-Likelihood (ML) analyses and Bayesian Inference (BI).For ML analyses, RAxML v8.2.8 (Stamatakis, 2014) was performed to reconstruct the phylogenetic trees and estimate the support value (BS) for each node with 1,000 rapid bootstrap replicates and the GTRGAMMA model referring to the RAxML manual.BI analyses were carried out using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2012), and the best-fitting evolutionary model (GTR+I+G) for plastome data and (GTR+G) for ITS sequences were determined by Modeltest 3.7 (Posada and Crandall, 1998) based on the Akaike information criterion (AIC).Two independent Markov chain Monte Carlo (MCMC) runs of 10 million generations were performed with sampling every 1,000 generations.When the average standard deviation of the splitting frequency fell below 0.01, the MCMC running finished.The initial 25% of trees was discarded as burn-in, and the remaining trees were used to generate the consensus tree and calculate posterior probabilities (PP).Finally, the phylogenetic trees were edited and displayed in FigTree v1.4.2 (Rambaut and Drummond, 2015).

Morphological observations
The fruit characteristics, as one of the most important morphological characters in the classification system of the Apiaceae, have been widely used in taxonomic studies of many genera of Apiaceae (Xiao et al., 2021;Xu et al., 2021;Cai et al., 2022;Lei et al., 2022;Qin et al., 2023).In the present study, we collected mature fruits from eight taxa (three varieties and three type varieties, and two new species) of Sanicula in the field and fixed them in formaldehyde-acetic acid-ethanol (FAA) solution.There were thirty representative fruit samples for each species (ten individuals from each species, each with three fruits) selected to observe their morphological characters, and then their overall structure and anatomy were photographed using a stereo microscope (SMZ25, Nikon Corp., Tokyo, Japan).The software MATO (Liu et al., 2023b) was used to measure the thirty representative fruit samples for each species, and then the average value was calculated.The terminology followed the reported study (Kljuykov et al., 2004).Moreover, we also observed other morphological characters based on extensive documentation, specimens information, and fieldwork.
We investigated the repeat sequences of the 21 Sanicula plastomes and detected a total of 977 repeats of four types, containing 482 forward repeats, 478 palindromic repeats, 15 reverse repeats, and two complementary repeats (Supplementary Figure 2A, Supplementary Table 5).All Sanicula plastomes possessed forward and palindromic repeats, twelve taxa had the reverse repeats, and the complementary repeats only occurred in Sanicula sp.SBN2023041201 and S. odorata (Supplementary Figure 2A).In addition, six types of SSRs (mono-, di-, tri-, tetra-, penta-, and hexanucleotide) were identified in the 21 Sanicula plastomes (Supplementary Figure 2B, Supplementary Table 5).The total number of SSRs was 1215, of which the most predominant SSR was mononucleotide (575) and the fewest SSRs were pentanucleotide (2).The number of SSRs also differed among the 21 Sanicula plastomes, with S. rugulosa Diels owing the fewest (54 SSRs) and S. odorata owing the most (64 SSRs) (Supplementary Figure 2B).It was noteworthy that all Sanicula species detected mononucleotide-to-tetranucleotide SSRs.Pentanucleotide SSRs were only found in S. flavovirens Z.H.Chen, D.D. Ma & W. Y. Xie, and hexanucleotide SSRs were only found in S. hacquetioides Franch., S. rubriflora F. Schmidt, and S. rugulosa Diels (Supplementary Figure 2B).Bases A and T occurred more frequently than bases G and C in all identified SSRs of the 21 Sanicula plastomes (Supplementary Table 5).
The 53 CDSs shared by the 21 Sanicula plastomes were extracted and connected to analyze the codon usage patterns.These sequences harbored 21,103-21,205 codons, and the codon usage bias was similar across all Sanicula plastomes (Supplementary Table 6).The highest number of codons were used to encode the Leucine, and the least number of codons were used to encode the Cysteine.We also found that the relative synonymous codon usage (RSCU) values of all codons varied from 0.34 to 1.92 in the 21 Sanicula plastomes.Specifically, thirty codons were used frequently with RSCU greater than 1.00 (Supplementary Figure 3).

Plastome comparison
The length of the IR region among the 21 Sanicula plastomes ranged from 26,176 bp (S. odorata) to 26,334 bp (S. rugulosa) (Supplementary Table 3), and the genes rps19, rpl2, trnH, trnN, ndhF, and ycf1 were located at the junctions of the IR/SC boundaries (Supplementary Figure 4).The results showed that 21 Sanicula plastomes were conserved in terms of the gene order and gene content at the IR/SC borders (Supplementary Figure 4).In detail, the rps19 gene, crossing the IRa/LSC boundaries, were located at the LSC and IRa regions with 221 bp and 58 bp.The IRa/ SSC borders were located between trnN gene and ndhF gene, with 2146 bp-2164 bp and 5 bp-11 bp away from the IRa/LSC borders.The borders of IRb/SSC were crossed by the ycf1 gene with 3,447 bp-3,479 bp in the SSC region and 1,819 bp-1,837 bp in the IRb region.In the IRb/LSC borders, all the junctions were within the genes between rpl2 and trnH with 115 bp-118 bp and 2 bp away from the IRb/LSC borders (Supplementary Figure 4).Mauve alignment results demonstrated that the gene order of 21 Sanicula plastomes were extremely conservative and no rearrangement occurred in gene organization (Supplementary Figure 5).The mVISTA program characterized genome divergence, and the result showed that the whole plastome sequences shared high similarity among the 21 Sanicula taxa (Supplementary Figure 6).
According to the sequence divergences, eleven mutation hotspot regions were selected as promising DNA barcodes, including five coding regions-cemA, rpl22, rbcL, matK, and ycf1 -which showed the Pi > 0.00458 (Figure 4A, Supplementary Table 7) and six non-coding regions-ycf4-cemA, trnH-psbA, trnE-trnT, rbcL-accD, ccsA-ndhD, and trnG-trnR-which showed the Pi >0.01225 (Figure 4B, Supplementary Table 7).Meanwhile, the average Pi in the SSC region was higher than that in the IR region (Figure 4C).We further found that the other genes (OGs) groups had a higher Pi median value among the functional groups of all protein-coding genes, whereas genes associated with ATP synthase (ATP), photosystems I (PSA), and photosystems II (PSB) had lower Pi median value (Figure 4D).

Phylogenetic analyses
The length of the alignment matrix for the trimmed plastome dataset was 161,260 bp, and the length of the ITS sequence matrix was 593 bp.In our phylogenetic analyses (Figure 5, Supplementary  Figure 7), although several conflicts existed between the plastome phylogenetic tree and the ITS phylogenetic tree, such as Sanicula sp., SBN2022073001 solely formed a clade in the plastome tree (Figure 5A), whereas it was sister to S. astrantiifolia in the ITS tree (Figure 5B), as well as Sanicula sp.SBN2023041201 was resolved as sister to S. orthacantha + S. lamelligera in the plastome phylogenetic tree (Figure 5A), whereas it formed a clade with S. elongata, S. tienmuensis, and S. tienmuensis var.pauciflora in the ITS phylogenetic trees (Figure 5B); both strongly supported that all Sanicula species involved in the current study were well clustered together.The phylogenetic trees also showed that three varieties of our main focus, S. giraldii var.ovicalycina, S. tienmuensis var.pauciflora, and S. orthacantha var.stolonifera, were clearly distant from S. giraldii, S. tienmuensis, and S. orthacantha.In addition, the accessions of Sanicula sp.SBN2022073001 and Sanicula sp.SBN2023041201 formed their own clades in both trees (Figure 5, Supplementary Figure 7).For plastome trees, the phylogenetic topologies of ML and BI analyses were highly identical (Figure 5A).The 21 Sanicula members scattered in two clades: clade I included three species (S. rubriflora, S. flavovirens, S. chinensis) (PP = 1.00,BS = 100), and the remainders were placed in clade II (PP = 1.00,BS = 100) (Figure 5A).In clade II, eighteen Sanicula taxa were divided into two subclades (PP = 1.00,BS = 100).Among them, S. giraldii was located in subclade A and was sister to S. rugulosa + S. hacquetioides, whereas S. giraldii var.ovicalycina nested in subclade B and formed a clade with S. pengshuiensis (PP = 1.00,BS = 100).Sanicula tienmuensis failed to gather with S. tienmuensis var.pauciflora but formed a separate clade (PP = 1.00,BS = 100).Instead, S. tienmuensis var.pauciflora was sister to S. oviformis X. T. Liu & Z. Y. Liu + S. caerulescens Franch.(PP = 0.97, BS = 67).Sanicula orthacantha was more closely related to the S. lamelligera (PP = 1.00,BS = 91), whereas S. orthacantha var.stolonifera clustered with S. orthacantha var.brevispina H. Boissieu and both formed a separate clade with strong support (PP = 1.00,BS = 100) and was far from S. orthacantha.As for the two new species, Sanicula sp.SBN2022073001 formed an individually monophyletic clade (PP = 1.00,BS = 100) and Sanicula sp.SBN2023041201 made a sister to S. orthacantha + S. lamelligera (PP = 0.99, BS = 58) (Figure 5A).

Morphological characteristics
Fruits (mericarps) of eight Sanicula taxa were mapped to the two phylogenetic trees (Figure 5).The detailed information of fruit anatomical and micromorphological characteristics of the eight Sanicula species were shown in Table 1.The other morphological features were also presented in Supplementary Table 8.
The key morphological features of S. giraldii were the fruit densely covered with developed yellow or purplish red, long and hard uncinate bristles as well as ovate and small calyx teeth, the tip mucronate (Figures 1A, 5, Table 1); the inflorescence was 2-4trichotomously branched.All branches elongate, and the leaf vein surface was smooth (Figure 1A, Supplementary Table 8).Sanicula giraldii var.ovicalycina had the unique characteristics of the fruits rarely covered with purplish red short bristles, proximal end with tubercles, obscure, distal end with uncinate bristles or straight, calyx teeth broadly ovate and large (Figures 1B, 5, Table 1), inflorescence dichotomously cymose-branched, leaf veins distinctly concave in adaxial surface, distinctly prominent in abaxial surface, gridded (Figure 1B, Supplementary Table 8).
The distinctive features of S. orthacantha were as follows: the fruit was narrowly ovoid, densely covered with short, straight and sharp spines, and sometimes the base formed a thin layer, fruit ribs and furrows spinulose, narrowly lanceolate calyx teeth (Figures 1E,  5, Table 1), the inflorescence was 2-3-branched; umbels 3-8, sometimes 1 shortened branch between forks or on lateral The morphological characteristics of S. orthacantha var.brevispina were based on the study of Li and Song (2022)."/" represented the information was missing.Song et al. 10.3389/fpls.2024.1351023Frontiers in Plant Science frontiersin.orgbranches, its rootstock short, tuberlike, woody, bearing a fascicle of thinly fibrous roots (Figure 1E, Supplementary Table 8).Sanicula orthacantha var.stolonifera had the ovoid fruit with proximal end with degenerated to disappeared the prickles, nearly smooth, whereas distal end with prickles and formed a thin layer, ovate calyx teeth, tip sharp (Figures 1F, 5, Table 1), slender, elongate and lignified nodes stoloniferous rhizomes (Figure 1F, Supplementary Table 8).

Phylogenetic inference and taxonomic implication
In the present study, we performed phylogenetic analyses using complete plastomes and ITS sequences.Unfortunately, the plastome-based and ITS-based phylogenetic trees yielded incongruent topologies.The phenomenon of conflict was also frequently observed in other genera of Apiaceae (Ren et al., 2020;Cai et al., 2022;Ren et al., 2022;Wen et al., 2021;Gui et al., 2023;Qin et al., 2023;Song et al., 2023;Tian et al., 2023;Song et al., 2024).This conflict was likely attributed to the biparental inheritance, higher mutation rate, and the insufficient sequence length of ITS data, whereas plastid DNA was maternal inheritance and has lower mutation rate (Wolfe et al., 1987;Koch et al., 2001).Moreover, the hybridization/introgression and incomplete lineage sorting (ILS) may be responsible for the inconsistent relationships between plastome-based and ITS-based phylogenies (Wen et al., 2021).Further study is needed to identify the cause of the nuclearplastome conflict in Sanicula.Although the conflicts existed between the plastome tree and ITS tree, both strongly suggested that S. giraldii var.ovicalycina, S. tienmuensis var.pauciflora, and S. orthacantha var.stolonifera were extremely distant from S. giraldii, S. tienmuensis, and S. orthacantha, respectively, which implied that these three varieties should be regarded as three independent species.Moreover, the morphological characteristics of three varieties also supported the above phylogenetic results.Furthermore, we also clarified the species relationships with ambiguous systematic position, such as S. pengshuiensis and S. lamelligera, and suggested that S. pengshuiensis should be regarded as an independent species rather than a synonymy of S. lamelligera.
Both phylogenetic analyses (Figure 5) showed that S. giraldii var.ovicalycina was distant from S. giraldii, which implied that this variety should not be regarded as a variety but rather as an independent species.Multiple morphological characteristics also further supported the above phylogenetic results (Figure 1, Table 1, Supplementary Table 8).Previously, Shan (1943) described a species (S. subgiraldii) of the genus based on the nomenclatural type specimen of S. giraldii var.ovicalycina.Subsequently, Pimenov (2017) reduced S. subgiraldii to the synonym S. giraldii var.ovicalycina in his checklist of Chinese Umbelliferae.By examination of herbarium specimens and observations on living plants in field, we found that the S. subgiraldii and S. giraldii var.ovicalycina was identical in morphology, such as the smaller primary polyphylla, the longer flowering branches and the basally obsoletely setulous-crenate leaf segments, the fruit was broadly ovate, vittae obscure.Therefore, according to the International Code for Nomenclature for plants, we reinstated the independent specific status of S. subgiraldii and suggested that S. subgiraldii should be as a legitimately accepted name and treated S. giraldii var.ovicalycina as a synonym of S. subgiraldii.Li and Song (2022) found that S. orthacantha var.stolonifera was identical with S. orthacantha var.brevispina in morphology, especially in the erose-spinulose ribs and spinulose or smooth furrows of the fruits and the number of staminate flowers per umbellule, and thus merged S. orthacantha var.stolonifera into S. orthacantha var.brevispina.Our plastome evidence and morphological data also strongly supported this treatment (Figure 5, Table 1, Supplementary Table 8).Moreover, our phylogenetic tree showed that S. orthacantha var.brevispina clustered together with S. orthacantha var.stolonifera and formed a separate clade, which was distant from S. orthacantha.These findings indicated that S. orthacantha var.brevispina should also be considered as an independent species, rather than a variety of S. orthacantha, which was further verified by morphological evidence (Figure 1, Table 1, Supplementary Table 8).Therefore, treating S. orthacantha var.brevispina as an independent species was reasonable and convincing, and a new independent species of the genus was presented.
Phylogenetic analyses based on plastome data and ITS sequences showed that S. tienmuensis var.pauciflora was clearly distant from S. tienmuensis (Figure 5), implying that the variety should not be regarded as a variety, but rather as an independent species.Moreover, the morphological characteristics also further supported the above phylogenetic results.For example, the key morphological features that distinguished S. tienmuensis and S. tienmuensis var.pauciflora were the calyx teeth and fruit.S. tienmuensis had broadly ovate calyx teeth; subglobose fruit, covered with short and obtuse prickles, slight formed scales and tubercles; endosperm slightly concave on commissural side, whereas long-lanceolate calyx teeth; long ovate fruit, densely covered with sharp prickles; flat endosperm on commissural side were existed in S. tienmuensis var.pauciflora (Figures 1D, E, Table 1, Supplementary Table 8).
The another new species (Sanicula sp.SBN2023041201) was resolved as sister to S. orthacantha + S. lamelligera in the plastome phylogenetic tree (Figure 5A), whereas it formed a clade with S. elongata, S. tienmuensis, and S. tienmuensis var.pauciflora in the ITS phylogenetic trees (Figure 5B).It noticed that Sanicula sp.SBN2023041201 can be discriminated from these five Sanicula species by its clearly different morphological characteristics, such as inflorescence dichotomously cymose-branched, extremely shortened peduncle, staminate flowers 9-10 per umbellule, usually 9, calyx teeth narrowly ovate, fruit long ellipsoid, proximal end with scalariform prickles, not acute, distal end with acute prickles (Figure 3, Table 1, Supplementary Table 8).Hence, there is no doubt that Sanicula sp.SBN2023041201 was also a new member of Sanicula and we described it here as a new species, Sanicula langaoensis B.N.Song, T. Ren & X.J.He, sp.nov.Sanicula hanyuanensis B.N.Song, C.K.Liu & X.J.He, sp.nov.(Figure 2).
Etymology: The species is named after Hanyuan County, Sichuan Province, China, where it is the type locality.
Phenology: The flowering and fruiting period is from June to September.
Distribution and habitat: This species is distributed in Hanyuan County, Sichuan Province, China, and grows under the mixed forest or roadsides at an altitude of 2000-2200 m.
Etymology: The species is named after Langao country, Shanxi Province, China, where it is the type locality.
Phenology: The flowering and fruiting period is from March to June.
Distribution and habitat: This species is distributed in Langao country, Shanxi Province, China, and grows in stream banks in mixed forests at an altitude of 1,400 m-1,660 m.
The reviewer HW declared a shared affiliation with the author B-NS to the handling editor at the time of review.

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FIGURE 4 Comparative analysis of the nucleotide diversity (Pi) values among the 21 Sanicula plastomes.(A) Protein-coding genes.(B) Non-coding and intron regions.(C) The nucleotide diversity (Pi) in chloroplast regions (IR/SSC/LSC).(D) The nucleotide diversity (Pi) of different functional groups.

TABLE 1
Fruit morphological and anatomical characteristics of nine Sanicula species.