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. 2024 Aug 7;25:769. doi: 10.1186/s12864-024-10663-x

Comparative analysis of complete chloroplast genomes of Synotis species (Asteraceae, Senecioneae) for identification and phylogenetic analysis

Xiaofeng Liu 1,2, Junjia Luo 2, Hui Chen 2, Tingyu Li 2, Tianmeng Qu 2, Ming Tang 3,✉, Zhixi Fu 1,2,4,✉
PMCID: PMC11308156  PMID: 39112930

Abstract

Background

The Synotis (C. B. Clarke) C. Jeffrey & Y. L. Chen is an ecologically important genus of the tribe Senecioneae, family Asteraceae. Because most species of the genus bear similar morphology, traditional morphological identification methods are very difficult to discriminate them. Therefore, it is essential to develop a reliable and effective identification method for Synotis species. In this study, the complete chloroplast (cp.) genomes of four Synotis species, S. cavaleriei (H.Lév.) C. Jeffrey & Y.L. Chen, S. duclouxii (Dunn) C. Jeffrey & Y.L. Chen, S. nagensium (C.B. Clarke) C. Jeffrey & Y.L. Chen and S. erythropappa (Bureau & Franch.) C. Jeffrey & Y. L. Chen had been sequenced using next-generation sequencing technology and reported here.

Results

These four cp. genomes exhibited a typical quadripartite structure and contained the large single-copy regions (LSC, 83,288 to 83,399 bp), the small single-copy regions (SSC, 18,262 to 18,287 bp), and the inverted repeat regions (IR, 24,837 to 24,842 bp). Each of the four cp. genomes encoded 134 genes, including 87 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes (ycf1 and rps19). The highly variable regions (trnC-GCA-petN, ccsA-psaC, trnE-UUC-rpoB, ycf1, ccsA and petN) may be used as potential molecular barcodes. The complete cp. genomes sequence of Synotis could be used as the potentially effective super-barcode to accurately identify Synotis species. Phylogenetic analysis demonstrated that the four Synotis species were clustered into a monophyletic group, and they were closed to the Senecio, Crassocephalum and Dendrosenecio in tribe Senecioneae.

Conclusions

This study will be useful for further species identification, evolution, genetic diversity and phylogenetic studies within this genus Synotis and the tribe Senecioneae.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-024-10663-x.

Keywords: Synotis, Chloroplast genome, Super-barcode, Comparative analysis, Phylogenetic analysis

Background

The genus Synotis (C.B. Clarke) C. Jeffrey & Y.L. Chen belongs to tribe Senecioneae of the family Asteraceae. It typically comprises subshrubs and perennial herbs. The leaves of Synotis are simple, either petiolate or sessile, with broadly ovate-cordate to narrowly oblong-lanceolate plates. Its capitula are heterogamous and radiate, always arranged in terminal or axillary [1]. It consists of approximately 60 species, mainly distributed in Bhutan, China, India, Myanmar, Nepal, Thailand, and Vietnam [1–13]. China, the diversity center of Synotis, harbors approximately 50 species, and approximately 30 species are endemic [6–9, 11–13]. The genus was separated from Senecio L. mainly because of its conspicuously caudate anthers [14]. Tang identified five series within Synotis, including ser. Synotis, ser. Erectae (C. B. Clarke) C. Jeffrey & Y. L. Chen, ser. Oliganthae (J. F. Jeffrey) C. Jeffrey & Y. L. Chen, ser. Fulvipapposae C. Jeffrey & Y. L. Chen, and ser. Hieraciifoliae M. Tang & Q. E. Yang [6]. Most species in this genus exhibit relatively similar morphological variations, making it challenging to distinguish them using traditional identification methods.

During our fieldwork in southwestern China, the materials of four species of Synotis, including S. cavaleriei (ser. Hieraciifoliae), S. duclouxii (ser. Hieraciifoliae), S. nagensium (ser. Erectae) and S. erythropappa (ser. Oliganthae) were collected. Among these species, S. nagensium is mainly distributed in China (Chongqing, Gansu, Guangdong, Guangxi, Guizhou, Hubei, Hunan, Sichuan, Xizang, Yunnan provinces), as well as in India, Nepal, Bhutan, Myanmar, Thailand and Vietnam [1, 11]. The other three species are endemic to China [1], of which S. cavaleriei is mainly distributed in Guizhou, Sichuan, and Yunnan provinces [1, 6, 8], S. duclouxii is found mainly in Sichuan and Yunnan provinces [1, 8], and S. erythropappa mainly occurs in Guizhou, Hubei, Sichuan, Xizang, and Yunnan provinces [1]. These four species often grow in mixed forests, woods, thickets and meadows [1]. They play a crucial role in ecological restoration and soil and water conservation efforts. The species of S. nagensium has disciform capitula and paniculoid thyrses, whereas S. erythropappa has broadly pyramidal compound thyrses, which two are easily distinguished from each other. However, the other two species bear the same habit, leaf and inflorescence shape, making them difficult to distinguish by traditional identification (Fig. 1).

Fig. 1.

Fig. 1

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Images of Synotis cavaleriei, S. duclouxii, S. nagensium, and S. erythropappa. (A, B,C, D) Inflorescence of plant growing in natural habitat; (E, F,G, H) morphology of leaf. ((A, E) S. cavaleriei, voucher LXF0170, SCNU, Butuo county, Liangshan prefecture, Sichuan province, China). ((B, F) S. duclouxii, voucher LXF0219, SCNU, Butuo county, Liangshan prefecture, Sichuan province, China). ((C, G) S. nagensium, voucher FZX5549, SCNU, Tongchuan District, Dazhou City, Sichuan Province, China). ((D, H) S. erythropappa, voucher FZX1038, SCNU, Puge county, Liangshan prefecture, Sichuan Province, China). All photographs by Ming Tang except B and F provides by Yi Yang

The chloroplast (cp.) is a vital organelle in green plants. It converts light energy to chemical energy through photosynthesis [15]. The chloroplast contains its own genomes [16]. In general, a typical cp. genome of the family Asteraceae contains a quadripartite architecture, with two inverted repeat regions (IRs) that separate a large single-copy region (LSC) and a small single-copy region (SSC) [17–19]. The cp. genomes generally contain 100–120 genes with lengths of 120–160 kb [15, 20, 21]. Compared with the partial cp. genes and nuclear genes, the complete cp. genomes exhibit unique characteristics, such as conserved genetics, slow evolution, maternal inheritance, and numerous mutation site information [18, 19, 22]. Consequently, the complete cp. genomes have become an efficient tool for phylogenetic studies within the family Asteraceae [23, 24] and serve as a super-barcode for distinguishing species [25–29]. The advent of high-throughput sequencing has facilitated the reporting of several cp. genomes within the Asteraceae family, including Artemisia L. [13, 18, 22, 30], Aster L. [31–36], Cavea W. W. Smith and J. Small [24], Conyza L. [26, 37], Chrysanthemum L. [38], Crepidiastrum Nakai [39], Dolomiaea DC. [40], Erigeron L. [41], Gynura Cass. [42], Heteroplexis C.C.Chang [43], Ligularia Cass. [25], Nouelia Franch. [44], Saussurea DC. [17, 45], Sinosenecio B. Nordenstam [19], Xanthium L. [46]. These studies have contributed to enhancing our understanding of the cp. genome characteristics and evolutionary relationships within the Asteraceae family.

With the rapid development of molecular technology, external transcribed spacers (ETS) [47–50], internal transcribed spacers (ITS) [47–51] and cp. fragments, such as matK-trnK-rpS16, rpS16-trnQ-psbK have been employed to determine the molecular phylogeny of Synotis [6]. However, the complete cp. genomes of genus Synotis have not yet been reported. Compared to nuclear markers and partial cp. genes, the complete cp. genomes possess highly conserved DNA sequences and many genetic information. Therefore, analyzing the complete cp. genomes may be an effective approach to solve the problem of the evolutionary relationships and taxonomic identification of species [18, 19, 22]. Meanwhile, with advances in next-generation sequencing technology, it has recently become quicker, cheaper and easier to achieve the complete plastomes than before [52–54]. Therefore, it is crucial to obtain the complete cp. genomes of Synotis for reconstructing phylogenetic relationships and species identification.

In this study, the complete cp. genomes of Synotis cavaleriei, S. duclouxii, S. nagensium, and S. erythropappa were sequenced using Illumina technology, and their gene features were characterized. The aims of this study were to: (1) assemble, annotate, and conduct a comparative analysis of the complete cp. genomes of four species of Synotis, (2) identify similarities and differences in the structural characteristics of the cp. genomes between Synotis and its related species, (3) reconstruct phylogenetic relationships among Synotis species and its related species, (4) utilize the complete cp. genomes as a super-barcode for the identification of Synotis species. These results will provide valuable insights into the DNA barcoding and phylogenetics of genus Synotis.

Results

Cp genome features of the four Synotis species

The gene map of the circular complete cp. genomes of four Synotis species is shown in Fig. 2. The GenBank numbers, GC content, gene numbers, and lengths of cp. genomes are given in Table 1. The cp. genome lengths of S. cavaleriei, S. duclouxii, S. nagensium, and S. erythropappa were 151,341 bp, 151,305 bp, 151,298 bp, and 151,228 bp, respectively (Table 1). These cp. genomes had a typical quadripartite circular structure, containing a LSC region (83,288 to 83,399 bp), an SSC region (18,262 to 18,287 bp) and two IR regions (24,837 to 24,842 bp) (Table 1; Fig. 2). The overall GC content was 37.4%, with the IR regions displaying significantly higher GC content (43.0%) compared to the LSC region (35.6%) and SSC region (30.6–30.7%).

Fig. 2.

Fig. 2

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Gene maps of the cp. genomes of Synotis species. Genes inside of the circle are transcribed clockwise and those on the outside are transcribed counter-clockwise

Table 1.

The basic cp. Genomes information of four Synotis species

Characteristics S. cavaleriei S. duclouxii S. nagensium S. erythropappa
GenBank accession OM912601 OM912602 OM912603 OQ985056
Genome size (bp) 151,341 151,305 151,298 151,228
LSC size (bp) 83,399 83,334 83,343 83,288
SSC size (bp) 18,268 18,287 18,277 18,262
IRa/IRb size (bp) 24,837 24,842 24,839 24,839
Number of genes 134 134 134 134
Protein-coding genes 87 87 87 87
Pseudogene 2 2 2 2
tRNA genes 37 37 37 37
rRNA genes 8 8 8 8
Overall GC content (%) 37.4 37.4 37.4 37.4
GC content in LSC (%) 35.6 35.6 35.6 35.6
GC content in IRa/IRb (%) 43.0 43.0 43.0 43.0
GC content in SSC (%) 30.6 30.6 30.7 30.6
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Each of the four cp genomes contained 134 genes, including 87 protein-coding, 37 tRNA, 8 rRNA genes, and 2 pseudogenes (ycf1 and rps19, Table 1). The cp genes of the four Synotis species can be divided into four types: named photosynthesis-related genes, self-replication-related genes, other genes and genes of unknown function. Among these genes, 7 protein-coding genes (ndhB, rpl2, rpl23, rps12, rps7, ycf15, and ycf2), 7 tRNAs (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC), and all 4 rRNA genes (rrn16, rrn23, rrn4.5 and rrn5) were duplicated in the IR regions (Table 1). In total, there were 19 intron-containing genes (Table 1). 16 genes (ndhA, ndhB, petB, petD, atpF, rbcL, rpl16, rpl2, rps16, rpoC1, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA and trnV-UAC) contained only 1 intron and 3 genes (clpP, rps12 and ycf3) contained 2 introns (Table 1). The rps12 gene was trans-splicing, with the 5’ end located in the LSC region and 3’ end located in the IR regions. The majority of protein coding genes had the standard ATG start codon, but some genes had alternative start codons, such as ACG in psbL and ndhD. Four cp genome sequences of the species of Synotis were submitted to NCBI (accession number: OM912601, OM912602, OM912603, OQ985056).

SSRs analysis

The number and types of SSRs were generally similar among the cp. genomes of four Synotis species (Fig. 3). The four sequenced plastomes contained 195 SSRs, including 111 Mononucleotide repeats (57%), 16 Dinucleotide repeats (8%), 24 Trinucleotide repeats (12%), 43 Tetranucleotide repeats (22%), and 1 Pentanucleotide repeats (1%) (Fig. 3A). The most common type of SSRs were Mononucleotide repeats composed of A/T bases (Fig. 3B). Specifically, there were 52 SSRs in S. cavaleriei, including 31 Mononucleotides, 4 Dinucleotides, 6 Trinucleotides, and 11 Tetranucleotides, 48 SSRs in S. duclouxii, including 27 Mononucleotides, 4 Dinucleotides, 6 Trinucleotides, and 11 Tetranucleotides, 45 SSRs in S. nagensium, including 24 Mononucleotides, 4 Dinucleotides, 6 Trinucleotides, 11 Tetranucleotides, and 1 Pentanucleotide, 50 SSRs in S. erythropappa, including 29 Mononucleotides, 4 Dinucleotides, 6 Trinucleotides, and 11 Tetranucleotides (Fig. 3C).

Fig. 3.

Fig. 3

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Analysis of SSRs in the four newly sequenced Synotis cp. genomes. (A) Frequencies of identified SSR types in all four plastomes, (B) Number of different SSR motifs, (C) Number of SSR repeat types

Structure comparison among the cp genomes of Synotis and its related species

The boundaries of the IR/SC were comprehensively compared (Fig. 4) in four species of Synotis and their related species. The genes located at these boundaries included rps19, rpl2, ndhF, ycf1, and trnH genes. The length of the IR regions ranged from 24,821 to 24,852 bp in Synotis and its related species, with some degree of expansion. The cp. genomes of these species were relatively conserved, except for one major divergences in Senecio vulgaris. In the cp. genomes of Ligularia jaluensis, Sinosenecio oldhamianus, S. cavaleriei, S. duclouxii, S. nagensium and S. erythropappa, the ycf1 gene crossed the SSC/IRa boundary with the larger part located in the SSC region, and the ndhF gene located at the IRb/SSC boundary. However, in S. vulgaris, the ycf1 gene crossed the IRb/SSC boundary, with 4,494 bp located in SSC region, and the ndhF gene was near the SSC/IRa boundary. All species contained a functional copy of the rps19 gene at the LSC/IRb junction and a pseudo copy (rps19Ψ) at the IRa/LSC junction. The length of the rps19 gene was conserved at 279 bp. The rpl2 gene was present completely in the IRb region and was located away from the junction of LSC/IRb, with a length ranging from 113 to 119 bp. The trnH gene was commonly located at the LSC region, with variable gap lengths observed in the four species of Synotis and its related species.

Fig. 4.

Fig. 4

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Comparison of borders in the LSC, SSC and IR regions of the seven Senecioneae cp. genomes. Genes are denoted by colored boxes. The gaps between the genes and the boundaries are indicated by the base lengths

With reference to Ligularia jaluensis, the structural differences among cp. genomes of the four Synotis species and its related species were compared by mVISTA (Fig. 5). The LSC and SSC regions were more significantly divergent than the two IR regions. Additionally, the non-coding regions showed greater variability than the coding regions.

Fig. 5.

Fig. 5

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Sequence alignment of seven Senecioneae cp. genomes using the mVISTA program with Ligularia jaluensis as a reference. X-axis: the coordinates in the cp. genome, Y-axis: percent identity within 50–100%. The transcriptional direction of genes indicated by grey arrows

Codon usage patterns in Synotis cp genomes

The RSCU values were calculated for the cp. genomes of Synotis cavaleriei, S. duclouxii, S. nagensium and S. erythropappa based on the protein-coding sequences (Table 2; Fig. 6). The total sequence size coding for protein genes was 77,616–78,381 bp in the cp. genomes of four species. These protein sequences encoded 22,424–22,679 codons. The cp. genomes of the four Synotis species contained 64 codons. Among these, 61 codons encoded 20 amino acids, and the other 3 were termination codons. Among these amino acids, Leucine was the most abundant amino acid in the cp. genomes of four Synotis species, whereas cysteine was the least (Table 2). The RSCU values of all codons are shown in Fig. 6. There were 32 codons with RSCU values less than 1 (RSCU < 1), which showed a lower usage frequency than expected. A total of 30 codons had RSCU values greater than 1 (RSCU > 1). Both methionine (Met) and tryptophan (Trp) exhibit no codon bias and have RSCU values of 1.

Table 2.

Codon usage and codon-anticodon recognition patterns of four Synotis species

Amino acid Symbol Codon Numbers and RSCU
S. cavaleriei S. duclouxii S.nagensium S. erythropappa
* Ter UAA 47/1.7625 47/1.7625 47/1.7625 47/1.7625
* Ter UAG 18/0.675 18/0.675 18/0.675 18/0.675
* Ter UGA 15/0.5625 15/0.5625 15/0.5625 15/0.5625
A Ala GCA 352/1.1043 362/1.1173 363/1.1178 362/1.1156
A Ala GCC 203/0.6369 207/0.6389 210/0.6467 208/0.641
A Ala GCG 144/0.4518 145/0.4475 145/0.4465 147/0.453
A Ala GCU 576/1.8071 582/1.7963 581/1.7891 581/1.7904
C Cys UGC 69/0.5542 70/0.5578 70/0.5578 69/0.552
C Cys UGU 180/1.4458 181/1.4422 181/1.4422 181/1.448
D Asp GAC 180/0.4114 182/0.4113 183/0.4131 184/0.4144
D Asp GAU 695/1.5886 703/1.5887 703/1.5869 704/1.5856
E Glu GAA 879/1.509 882/1.509 885/1.5128 883/1.512
E Glu GAG 286/0.491 287/0.491 285/0.4872 285/0.488
F Phe UUC 409/0.6472 414/0.6454 414/0.6444 414/0.6444
F Phe UUU 855/1.3528 869/1.3546 871/1.3556 871/1.3556
G Gly GGA 594/1.5389 602/1.5347 602/1.5377 601/1.5361
G Gly GGC 175/0.4534 180/0.4589 180/0.4598 181/0.4626
G Gly GGG 259/0.671 260/0.6628 257/0.6564 259/0.662
G Gly GGU 516/1.3368 527/1.3435 527/1.3461 524/1.3393
H His CAC 126/0.4961 129/0.5 129/0.499 128/0.4961
H His CAU 382/1.5039 387/1.5 388/1.501 388/1.5039
I Ile AUA 626/0.9812 628/0.9782 632/0.9829 633/0.9844
I Ile AUC 351/0.5502 351/0.5467 352/0.5474 351/0.5459
I Ile AUU 937/1.4687 947/1.4751 945/1.4697 945/1.4697
K Lys AAA 884/1.5021 889/1.5004 887/1.4996 889/1.5004
K Lys AAG 293/0.4979 296/0.4996 296/0.5004 296/0.4996
L Leu CUA 323/0.8095 324/0.8013 323/0.7995 321/0.7939
L Leu CUC 141/0.3534 140/0.3462 141/0.349 142/0.3512
L Leu CUG 145/0.3634 146/0.3611 147/0.3639 148/0.366
L Leu CUU 521/1.3058 525/1.2984 525/1.2995 524/1.296
L Leu UUA 761/1.9073 778/1.9242 775/1.9183 777/1.9217
L Leu UUG 503/1.2607 513/1.2688 513/1.2698 514/1.2712
M Met AUG 544/1 550/1 551/1 550/1
N Asn AAC 233/0.4283 234/0.4251 237/0.4301 236/0.4279
N Asn AAU 855/1.5717 867/1.5749 865/1.5699 867/1.5721
P Pro CCA 275/1.1752 278/1.1767 277/1.175 277/1.175
P Pro CCC 170/0.7265 170/0.7196 170/0.7211 169/0.7169
P Pro CCG 125/0.5342 128/0.5418 129/0.5472 130/0.5514
P Pro CCU 366/1.5641 369/1.5619 367/1.5567 367/1.5567
Q Gln CAA 625/1.5432 626/1.5381 626/1.5381 625/1.5375
Q Gln CAG 185/0.4568 188/0.4619 188/0.4619 188/0.4625
R Arg AGA 414/1.8455 417/1.8465 415/1.839 413/1.8315
R Arg AGG 139/0.6196 139/0.6155 139/0.616 139/0.6164
R Arg CGA 297/1.3239 300/1.3284 301/1.3338 301/1.3348
R Arg CGC 93/0.4146 93/0.4118 91/0.4032 93/0.4124
R Arg CGG 102/0.4547 101/0.4472 101/0.4476 101/0.4479
R Arg CGU 301/1.3418 305/1.3506 307/1.3604 306/1.357
S Ser AGC 98/0.3513 99/0.3511 98/0.3475 98/0.3477
S Ser AGU 358/1.2832 360/1.2766 359/1.273 358/1.2703
S Ser UCA 349/1.2509 354/1.2553 354/1.2553 355/1.2596
S Ser UCC 240/0.8602 243/0.8617 243/0.8617 243/0.8622
S Ser UCG 128/0.4588 130/0.461 132/0.4681 131/0.4648
S Ser UCU 501/1.7957 506/1.7943 506/1.7943 506/1.7954
T Thr ACA 348/1.2395 354/1.2443 357/1.2559 356/1.2513
T Thr ACC 215/0.7658 216/0.7592 213/0.7493 214/0.7522
T Thr ACG 110/0.3918 111/0.3902 109/0.3835 110/0.3866
T Thr ACU 450/1.6028 457/1.6063 458/1.6113 458/1.6098
V Val GUA 472/1.5375 476/1.533 473/1.5233 472/1.5189
V Val GUC 143/0.4658 144/0.4638 145/0.467 147/0.473
V Val GUG 164/0.5342 165/0.5314 166/0.5346 165/0.531
V Val GUU 449/1.4625 457/1.4718 458/1.475 459/1.4771
W Trp UGG 383/1 400/1 400/1 400/1
Y Tyr UAC 160/0.3778 160/0.3738 160/0.3747 161/0.3766
Y Tyr UAU 687/1.6222 696/1.6262 694/1.6253 694/1.6234
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Fig. 6.

Fig. 6

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Codon content of 20 amino acids and stop codons in the protein-coding genes of the cp. genomes of the four Synotis species. Each bar represents a species, from left to right: S. cavaleriei, S. duclouxii, S. nagensium, and S. erythropappa

Nucleotide diversity analysis

The cp. genomes contain numerous nucleotides. It can be used as the valuable DNA barcoding to resolve the phylogenetic relationships of closely related species or genera. In this study, highly variable regions were identified in Synotis and its related species using DnaSP (Fig. 7). Among the seven species, polymorphism information (Pi) values ranged from 0.02232 (petN gene) to 0.03 (ccsA-psaC region). The Pi analyses revealed that the IR regions displayed significantly lower variation compared to the LSC and SSC regions (Fig. 7). Six mutational hotspots had remarkably higher Pi values (> 0.022), including three genes (ycf1, ccsA and petN) and three intergenic regions (trnC-GCA-petN, ccsA-psaC and trnE-UUC-rpoB).

Fig. 7.

Fig. 7

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Nucleotide diversity (Pi) values among the seven Senecioneae species. X-axis: position of the midpoint of a window, Y-axis: Pi value, Pi: polymorphism information

Phylogenetic analysis

Based on the analysis of the complete cp. genome sequences, the Maximum likelihood (ML) phylogenetic tree of 42 Asteraceae species was constructed, with Anthriscus cerefolium (Apiaceae) and Kalopanax septemlobus (Araliaceae) used as outgroups (Fig. 8). In our sampling of Asteraceae, the species formed a monophyletic group and were grouped into 5 monophyletic clades, corresponding to the 5 subfamilies of Asteraceae (Asteroideae, Cichorioideae, Gymnarrhenoideae, Carduoideae and Pertyoideae). In the subfamily Asteroideae, 6 subgroups were separated, corresponding to the tribe Senecioneae, Astereae, Gnaphalieae, Anthemideae, Helenieae and Inuleae, respectively. The tribe Senecioneae contains two clades: subtribe Tussilagininae and Senecioninae. The subtribe Tussilagininae consists of 13 species in 8 genera, including Gynoxys Cass., Nordenstamia Lundin, Roldana La Llave, Telanthophora H.Rob. & Brettell, Arnoglossum Raf., Sinosenecio B.Nord., Ligularia Cass. and Petasites Mill. The subtribe Senecioninae consists of 11 species from 4 genera, including Senecio L., Crassocephalum Moench, Dendrosenecio (Hauman ex Hedberg) B.Nord. and Synotis. From the perspective of complete cp. genomes, the species of S. cavaleriei, S. duclouxii, S. nagensium and S. erythropappa were sister species, supported by a 100 bootstrap value. Among the four Synotis species, the species of S. cavaleriei clustered with S. duclouxii, and S. nagensium clustered with S. erythropappa. The genus of Synotis was phylogenetically close to Senecio, Crassocephalum and Dendrosenecio.

Fig. 8.

Fig. 8

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Phylogenetic tree was reconstructed using ML method based on complete cp. genomes of the Asteraceae. Numbers at nodes are bootstrap support values. The phylogenetic position of Synotis species is marked with black circles

Molecular age estimation

Divergence time estimates of Synotis species based on the complete cp. genomes were shown in Fig. 9. The molecular age estimation suggested that Synotis originated at 3.81 Mya. The divergence time between the S. cavaleriei and S. duclouxii was estimated at 0.87 Mya. The divergence time between the S. nagensium and S. erythropappa was estimated at 2.78 Mya.

Fig. 9.

Fig. 9

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Divergence time estimates of Synotis based on complete cp. genomes. The numbers above or below branches represent median divergence time estimates. Pal., Pli. and Ple. indicate Paleocene, Pliocene and Pleistocene, respectively. The Synotis species were marked in red

Discussion

Cp genomes structure analysis

In this study, the cp. genomes of four Synotis species were sequenced, assembled, and annotated (Fig. 2; Tables 1 and 3). This is the first report of complete cp. genomes from the genus Synotis. The complete cp. genomes also exhibited a highly conserved characteristic in terms of genomic numbers, orders, structures, GC contents and intron numbers. The length of these cp. genomes ranged from 151,228 (S. erythropappa) to 151,341 bp (S. cavaleriei). They are similar to other cp. genomes of tribe Senecioneae, such as Cremanthodium rhodocephalum [55], Ligularia veitchiana (151,253 bp) [25], Parasenecio palmatisectus (151,263 bp) [56], Sinosenecio albonervius (151,224 bp) [19], and Senecio roseiflorus (151,228 bp) [57]. Among three series within Synotis in this study, the length of the cp. genome of ser. Hieraciifoliae is the longest. The cp. genomes of four Synotis species encoded 134 genes, which is similar to the cp. genomes of Ligularia [25], Aster [33, 34], Gynura [42], and Sinosenecio [19]. Like most other species of Asteraceae, pseudogenes ycf1 and rps19 were also detected [19, 25, 30, 32, 35, 43, 56]. The ACG start codons of psbL and ndhD genes were also detected in angiosperms [38, 58–60]. The clpP, rps12, and ycf3 genes included two introns, while the other genes included one intron. All these features are consistent with the cp. genomes of most family Asteraceae [22, 25, 43, 56]. The total GC contents of the complete cp. genomes among the three series of the Synotis was 37.4% (Table 1), which is similar to previous study [19, 22, 25, 37, 55]. Among the LSC, SSC, and IR regions, the IR regions had the highest GC contents (43.0%), followed by the LSC (35.6%) and SSC regions (30.6 to 30.7%). The IR regions had the highest GC contents among the four regions, which is likely due to the presence of two copies of rRNAs (rrna4.5, rrna5, rrna23, and rrna16) within this region [22, 43].

Table 3.

The basic cp. Genomes information of four Synotis species

Category Gene group Gene name
Photosynthesis Subunits of photosystem I psaA, psaB, psaC, psaI, psaJ
Subunits of photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
Subunits of NADH dehydrogenase ndhA*, ndhB*(2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complex petA, petB*, petD*, petG, petL, petN
Subunits of ATP synthase atpA, atpB, atpE, atpF*, atpH, atpI
Large subunit of rubisco rbcL*
Self-replication Proteins of large ribosomal subunit rpl14, rpl16*, rpl2*(2), rpl20, rpl22, rpl23(2), rpl32, rpl33, rpl36
Proteins of small ribosomal subunit #rps19, rps11, rps12**(2), rps14, rps15, rps16*, rps18, rps19, rps2, rps3, rps4, rps7(2), rps8
Subunits of RNA polymerase rpoA, rpoB, rpoC1*, rpoC2
Ribosomal RNAs rrn16(2), rrn23(2), rrn4.5(2), rrn5(2)
Transfer RNAs trnA-UGC*(2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC*, trnH-GUG, trnI-CAU(2), trnI-GAU*(2), trnK-UUU*, trnL-CAA(2), trnL-UAA*, trnL-UAG, trnM-CAU, trnN-GUU(2), trnP-UGG, trnQ-UUG, trnR-ACG(2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(2), trnV-UAC*, trnW-CCA, trnY-GUA, trnfM-CAU
Other genes Maturase matK
Protease clpP**
Envelope membrane protein cemA
Acetyl-CoA carboxylase accD
c-type cytochrome synthesis gene ccsA
Translation initiation factor infA
Genes of unknown function Conserved hypothetical chloroplast ORF #ycf1, ycf1, ycf15(2), ycf2(2), ycf3**, ycf4
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Notes: *: Gene with one introns, **: Gene with two introns, #: Pseudogene, (2): Gene with two copies

Repeat sequences analysis

SSRs, also called microsatellites, are widely utilized as valuable molecular markers in species identification and phylogenetic studies due to their high substitution rates [61]. In this study, a total of 195 SSRs were identified in the complete cp. genomes of four Synotis species, 111 were found to be Mononucleotide repeats, constituting the majority (57%) of all SSRs (Fig. 3A). The most common SSRs are Mononucleotide repeats composed of A/T (Fig. 3B). It is worth noting that the number of SSRs varied across different species (Fig. 3C). These findings are consistent with previous reports within the Asteraceae family, such as Artemisia [18, 38]. In this study, we analyzed the number and composition of SSRs in the cp. genomes of four Synotis species. It will serve as valuable references for further research on molecular markers and population genetics of Synotis and the tribe Senecioneae.

Genomes comparison and nucleotide diversity

Comparative analysis of the complete cp. genomes provides a pivotal reference in understanding genome evolution and evolutionary relationships within the family Asteraceae [18, 19, 22, 23, 56]. Through the comparative analysis of the IR boundary regions among Synotis and its related species, some expansion or contractions were detected, with the IR regions ranging from 24,821 to 24,852 bp (Fig. 4). Similar large-scale IR expansion had also been reported in other Asteraceae, such as Saussurea [17], Aster [35], Xanthium [46]. Furthermore, the SSC and LSC regions showed higher sequence divergence than the IR regions, and the non-coding region was more divergence than the coding region (Fig. 5), the same result was also obtained in Senecioneae [19, 44, 57]. It should be noted that mutation hotspots can serve as specific DNA barcodes for species identification [62]. In this study, six highly variable regions (Pi > 0.022) were identified, including trnC-petN, ccsA-psaC, trnE-rpoB, ycf1, ccsA and petN. These variable regions could be utilized as potential molecular barcodes for species identification and phylogenetic research in genus Synotis and tribe Senecioneae.

Phylogenetic relationship, super barcode and molecular dating analysis

In recent years, several molecular phylogenetic analyses have been conducted to resolve the phylogenetic relationships of genus Synotis. Liu et al. (2006) studied the radiation and diversification within the Ligularia-Cremanthodium-Parasenecio complex in the Qinghai-Tibetan Plateau, they used Synotis lucorum as the outgroup and inferred a phylogenetic tree of the LCP complex using ITS, ndhC-F and trnL-F [63]. Pelser et al. (2007) constructed the phylogenetic relationships for the Senecioneae using ITS data. The results showed that the species of Synotis was nested within the Cissampelopssis-Crassocephalum clade [51]. However, these studies were based on small taxon sampling and small fragments of barcoding markers. Tang (2014) reconstructed the Phylogenetic tree of the Synotis using ITS and seven cpDNA sequence regions. In his analyses, the monophyly of Synotis is strongly supported and the species of Senecio kumaonensis is also included [6]. Although some studies of phylogenetic relationships have been reported using limited nuclear and cpDNA fragments in the above studies, the research progress of genus Synotis at the complete cp. genome-scale level is relatively slow. The result indicated that all species were grouped into five monophyletic clades (Fig. 8), corresponding to the five subfamilies of Asteraceae, which was consistent with the previous study [64]. In this study, the phylogenetic trees among the three series of Synotis were constructed using complete cp. genomes for the first time. In the ML tree, the Synotis was a branch of family Asteraceae, the four Synotis species are sister groups within tribe Senecioneae and subfamily Asteroideae, and the bootstrap support values for these were 100. Our study also confirms the monophyly of Synotis. In the tribe Senecioneae, two subgroups were separated, corresponding to the subtribe Tussilagininae and Senecioninae, respectively. The genus Synotis belongs to the Senecioninae subtribe, which is consistent with the backbone of Senecioneae in previous studies [47, 51, 64].

The complete cp. genomes have been widely served as super barcode for species identification and classification [25–29]. In this study, the Senecioneae contains two clades. Within subtribe Senecioninae, a sub-clade consisting of four Synotis species was identified with high bootstrap support values (bootstrap = 100) (Fig. 8). Four Synotis species were discriminated completely based on complete cp. genome sequences. This topology shows the separation of Synotis at intra-species level, which suggested that the complete cp. genomes of Synotis might be a super barcode for species identification.

Previous studies have estimated the divergence time of the Senecioneae tribe during the Eocene period using various molecular markers. For instance, Panero and Crozier (2016) used a few cp. genomes markers [65], Han et al. (2019) utilized complete cp. genomes [42], and Mandel et al. (2019) employed nuclear phylogenomics data [66]. Huang et al. (2016) estimated the divergence time of the tribe Senecioneae during the early Miocene based on multiple nuclear genes [67]. In this study, the divergence time of the tribes Senecioneae was early Oligocene period. This study provides further support and traces the divergence time of the Senecioneae tribe.

In this study, it is an efficient attempt to utilize the complete genomes as super-barcode for the identification of genus Synotis. Our research provides a foundation for developing a reliable and efficient method for species identification of Synotis. In the future, it is necessary to further investigate and verify this assumption. In addition, Synotis is a large genus with ca. 60 species [1], and this study only analyzes the cp. genome sequences of four species. Therefore, a more comprehensive analysis of cp. genomes and a broader taxon sampling are essential in order to fully develop the super-barcode approach. The development of such a method will greatly contribute to biodiversity assessments, conservation efforts, and various fields of plant research.

Conclusions

This study reported the complete cp. genomes from four Synotis species, ranging from 151,228 (S. erythropappa) to 151,341 bp (S. cavaleriei). The structure and composition, SSRs, codon usage of the complete cp. genomes were highly conserved. The complete cp. genomes of four Synotis species encoded 134 genes, including 87 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 Pseudogenes (ycf1 and rps19). Phylogenetic analysis showed that four species were clustered into a monophyletic group, and they were close to the genus of Senecio, Crassocephalum and Dendrosenecio in tribe Senecioneae. The ML tree showed that the complete cp. genomes could be used as a super-barcode for the identification of Synotis species.

Materials and methods

Plant materials and DNA extraction

Fresh leaves of Synotis cavaleriei and S. duclouxii were collected from Butuo County, Liangshan Prefecture, Sichuan Province, China. Fresh leaves of S. nagensium were collected from Tongchuan District, Dazhou City, Sichuan Province, China. Fresh leaves of S. erythropappa were collected from Puge County, Liangshan Prefecture, Sichuan Province, China. (Fig. 1). These specimens were identified by Dr. Zhixi Fu at Sichuan Normal University. The voucher specimen of S. cavaleriei (LXF0170), S. duclouxii (LXF0219), S. nagensium (FZX5549) and S. erythropappa (FZX1038) were deposited in the herbarium of Sichuan Normal University (SCNU) (Contact: Prof. Dr. Zhixi Fu, fuzx2017@sicnu.edu.cn). We achieved all required permits for the protected areas from the local governments and National Park Services. This research was carried out in compliance with the relevant laws of China. Total genomic DNA was extracted from fresh leaves using the cetyltrimethylammonium bromide (CTAB) method, following the manufacturer’s instructions [68]. The Illumina Paired-End (PE) DNA Library Kit (Illumina, San Diego, CA, USA) was used to construct the DNA libraries. The genomic library was sequenced using the Illumina NovaSeq 6000 platform with a 150 bp read length (NovoGene Inc., Beijing, China).

Genomes assembly and annotation

The complete cp. genome sequences of Synotis were assembled using SPAdes software (v3.10.1) with default parameters [69]. The assembly quality was assessed using Bandage software to identify circular maps [70]. The cp. sequences were then annotated using PGA based on reference cp. genome sequences of Ligularia jaluensis (MF539931.1) [71]. The annotation results were manually inspected and adjusted using Geneious [72]. Circular maps of the cp. genomes were drawn using Organellar Genome Draw (OGDraw) (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html (Accessed August 6, 2022)) [73]. Various plastome characteristics, such as the number of genes, gene length, GC contents and intron numbers were analyzed using Geneious [72]. The obtained cp. genome sequences were deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/) with the GenBank accession numbers OM912601 (S. cavaleriei), OM912602 (S. duclouxii), OM912603 (S. nagensium), and OQ985056 (S. erythropappa).

Simple sequence repeats (SSRs) analysis

SSRs were analyzed using the MISA software (http://pgrc.ipkgatersleben.de/misa/ (Accessed January 3, 2023)) [74]. The minimum number of repeats for Mono-, Di-, Tri-, Tetra-, Penta- and Hexanucleotide motifs were set to 8, 5, 3, 3, 3, and 3, respectively.

Comparative analysis of cp genomes

Three cp. genomes from tribe Senecioneae were downloaded from GenBank to conduct comparative cp. genomes analysis. The species included in the analysis were Ligularia jaluensis Kom., Senecio vulgaris L. and Sinosenecio oldhamianus (Maxim.) B.Nord. The boundary differences among these genomes were visualized using mVISTA in Shuffle-LAGAN mode, with the Ligularia jaluensis as the reference (http://genome.lbl.gov/vista/mvista/submit.shtml (Accessed August 15, 2022)) [75]. The contraction and expansion of the IR boundaries among the four regions (LSC/IRa/SSC/IRb) of the cp. genome sequences were observed using the online software IRSCOPE (https://irscope.shinyapps.io/irapp/ (Accessed January 3, 2023)) [76].

Codon usage analysis

The distribution of codon usage was studied using CodonW (http://downloads.fyxm.net/ CodonW-76666.html (Accessed February 4, 2023)) with the relative synonymous codon usage (RSCU) ratio [77]. A value of RSCU > 1 indicates that the codon is used more frequently, a value of 1 indicates no codon usage preference, and a value of < 1 indicates less frequent codon usage.

Genetic divergence analysis

To identify and analyze highly variable regions of the cp. genomes sequence, the sequences of seven Senecioneae cp. genomes were aligned using the MAFFT program [78]. The nucleotide diversity (Pi) was then analyzed through the sliding window using DnaSP v5 (http://www.ub.edu/dnasp/ (Accessed January 6, 2023)) [79]. The step size was set to 200 bp with an 800 bp window length.

Phylogenetic analysis

Phylogenetic analysis was inferred based on 44 complete cp. genomes, including the 4 newly sequences of Synotis in this study, 38 cp. genomes of family Asteraceae downloaded from the NCBI database (Table S1), and Anthriscus cerefolium (Apiaceae) and Kalopanax septemlobus (Araliaceae) as outgroup. To align the sequences, the MAFFT program was used with the auto strategy [78]. The initial alignment was further manually adjusted in BioEdit to ensure accuracy [80]. The maximum-likelihood (ML) phylogenetic tree was constructed in RaxML 8.2.10 tool at CIPRES Science Gateway web (https://www.phylo.org/ (Accessed January 7, 2023)) with 1000 bootstrap replicates [81, 82].

Estimation of divergence times

The divergence times of genus Synotis were estimated using BEAST2 by lognormal relaxed clock [83]. The GTP model was chosen as a substitution model. The GTR model was chosen to generate the tree. As a calibration point, the pairwise divergence time of Asteroideae was set at 37.7 Mya [66]. A total of 10,000,000 generations were run. Tracer v.1.7 was used to check the correctness [84]. Finally, the maximum clade credibility tree was calculated in TreeAnnotator v1.8.4.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (56KB, doc)

Acknowledgements

The authors would like to thank the editor and anonymous reviewers for the constructive criticism of the original manuscript. We also thank Dr. Yi Yang from Jiangxi Agricultural University provides the color images of Synotis duclouxii.

Author contributions

M.T. and Z.F. conceived and designed the research. X.L. and J.L. performed bioinformatic analyses. X.L., H.C. and J.L. carried out wet-lab experiments. T.L. and T.Q. contributed to data interpretation. X.L. and Z.F. wrote the manuscript. M.T. and Z.F. revised the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was financially supported by the National Natural Science Foundation of China (No. 32000158, No. 31500166, No. 31960043), the National Science & Technology Fundamental Resources Investigation Program of China (No. 2021XJKK0702), and the Foundation of Sustainable Development Research Center of Resources and Environment of Western Sichuan, Sichuan Normal University (No. 2020CXZYHJZX03). Jiangxi Provincial Key Laboratory of Conservation Biology (No. 2023SSY02081).

Data availability

All annotated chloroplast genomes have been deposited in GenBank as OM912601 (Synotis cavaleriei), OM912602 (S. duclouxii), OM912603 (S. nagensium), and OQ985056 (S. erythropappa).

Declarations

Ethics approval and consent to participate

The study was conducted the plant material that complies with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Ming Tang, Email: tangming@jxau.edu.cn.

Zhixi Fu, Email: fuzx2017@sicnu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (56KB, doc)

Data Availability Statement

All annotated chloroplast genomes have been deposited in GenBank as OM912601 (Synotis cavaleriei), OM912602 (S. duclouxii), OM912603 (S. nagensium), and OQ985056 (S. erythropappa).

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