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. 2023 Feb 27;29(3):409–420. doi: 10.1007/s12298-023-01292-x

Comparative analyses of five complete chloroplast genomes from the endemic genus Cremanthodium (Asteraceae) in Himalayan and adjacent areas

Weihong Zhong 1,2, Xiaolang Du 1,3,✉, Xiaoyun Wang 1,3, Lan Cao 1,3, Zejing Mu 1,3, Guoyue Zhong 1,3,4,✉
PMCID: PMC10073364  PMID: 37033762

Abstract

Cremanthodium Benth. is an endemic genus in the Himalayas and adjacent areas. Some plants of the genus are traditional medicinal plants in Tibetan medicine. In this study, the chloroplast genomes of five species (Cremanthodium arnicoides (DC. ex Royle) Good, Cremanthodium brunneopilosum S. W. Liu, Cremanthodium ellisii (Hook. f.) Kitam., Cremanthodium nervosum S. W. Liu, and Cremanthodium rhodocephalum Diels) were collected for sequencing. The sequencing results showed that the size of the chloroplast genome ranged from 150,985 to 151,284 bp and possessed a typical quadripartite structure containing one large single copy (LSC) region (83,326–83,369 bp), one small single copy (SSC) region (17,956–18,201 bp), and a pair of inverted repeats (IR) regions (24,830–24,855 bp) in C. arnicoides, C. brunneopilosum, C. ellisii, C. nervosum, and C. rhodocephalum. The chloroplast genomes encoded an equal number of genes, of which 88 were protein-coding genes, 37 were transfer ribonucleic acid genes, and eight were ribosomal ribonucleic acid genes, and were highly similar in overall size, genome structure, gene content, and order. In comparison with other species in the Asteraceae family, their chloroplast genomes share similarities but show some structural variations. There was no obvious expansion or contraction in the LSC, SSC or IR regions among the five species, indicating that the chloroplast gene structure of the genus was highly conserved. Collinearity analysis showed that there was no gene rearrangement. The results of the phylogenetic tree showed that the whole chloroplast genomes of the five species were closely related, and the plants of this genus were grouped into one large cluster with Ligularia Cass. and Farfugium Lindl.

Keywords: Cremanthodium Benth., Chloroplast genomes, Endemic genera, Sequencing

Introduction

The genus of Cremanthodium Benth. belongs to perennial herbaceous plants of Asteraceae, is distributed primarily in the Tibetan Plateau and southwest mountain area, and contains approximately 64 species in China. The Cremanthodium genus is an endemic genus of the Himalayas and adjacent areas, growing in alpine bushwood, grassy marshland, and screes (Flora Reipublicae Popularis Sinicae 1989). Some plants of this genus are traditional medicinal plants in Tibetan medicine.

At present, studies on this genus have mainly focused on chemicals and pharmacology. Chemical constituents of C. ellisii (Hook. f.) Kitam., C. potaninii C. Winkl., C. discoideum Maxim., C. rhodocephalum Diels, C. lineare Maxim., C. helianthus (Franch.) W. W. Smith, C. stenactinium Diels ex Limpr., and C. brunneopilosum S. W. Liu were studied. Sesquiterpenoids (Chen et al. 1996; Saito et al. 2012; Tori et al. 2012), phenylpropanoids (Zhu et al. 2001), lignin (Yang et al. 1995; Su et al. 1999, 2020; Wang et al. 2004) and steroids (Zhu et al. 2000) are the main constituents of the Cremanthodium genus, as well as fatty acids (Tu et al. 2006) and volatile oils. Recent studies have tapped into the potential of the Cremanthodium genus as antibacterial and antitumour plants (Li et al. 2007). Specifically, the volatile oils of C. discoideum have been well known for their ability to relieve coughing and repell mosquitoes (Wu et al. 2003), while the ether extract of C. humile exhibits significant activities for inducing HeLa cell apoptosis (Li et al. 2007).

Although the Cremanthodium genus has many species, their chloroplast genome has not been used for species identification. This study focused on characterizing the chloroplast genome sequences of C. arnicoides, C. brunneopilosum, C. ellisii, C. nervosum, and C. rhodocephalum (A-B-E-N-R) by the Illumina sequencing platform and discussing the structures and features of the five newly sequenced chloroplast genomes to provide evidence for species identification of the Cremanthodium genus.

Materials and methods

Plant material

Fresh leaves from five species of Cremanthodium were collected for DNA extraction (Fig. 1). The leaf material of C. nervosum (Voucher No. JXZY187) was collected in Yadong County, Tibet, China (27° 36′ 28.7″ N, 89° 02′ 28.65″ E, 3520 m). The leaf material of C. rhodocephalum (Voucher No. JXZY113) was collected from Jiangzi County, Tibet, China (28° 57′ 29.83″ N, 89° 30′ 4.15″ E, 4630 m), and the leaf material of C. ellisii (Voucher No. JXZY496) was collected from Nyalam, Tibet, China (28° 19′ 06.2″ N, 86° 02′ 19.5″ E, 4172 m). The leaf material of C. arnicoides (Voucher No. JXZY286) and C. brunneopilosum (Voucher No. JXZY491) were collected from Chefei Township, Bailang County, Tibet, China (29° 20′ 50.0″ N, 89° 38′ 30.3″ E, 3776 m).

Fig. 1.

Fig. 1

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Five species of Cremanthodium genus. a C. arnicoides, b C. brunneopilosum, c C. ellisii, d C. nervosum, e C. rhodocephalum

DNA extraction and sequencing

The quality of isolated genomic DNA was verified using two methods: (1) DNA degradation and contamination were monitored on 1% agarose gels; and (2) DNA concentration was measured in a Qubit® 3.0 Fluorometer with a Qubit® DNA Assay Kit (Invitrogen, USA).

A total amount of 0.2 μg DNA per sample was used as input material for the DNA library preparations. The sequencing library was generated using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina sequencing (NEB, USA) following the manufacturer’s recommendations, and index codes were added to each sample. Briefly, genomic DNA samples were fragmented by sonication to a size of 350 bp. Then, DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. After the PCR products were purified by an AMPure XP system (Beckman Coulter, Beverly, USA), the DNA concentration was measured by a Qubit®3.0 Fluorometer (Invitrogen, USA) and the libraries were analysed for size distribution by NGS3K/Calliper and quantified by real-time PCR (3 nM).

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using an Illumina PE Cluster Kit (Illumina, USA) according to the manufacturer’s instructions. After cluster generation, the DNA libraries were sequenced on an Illumina platform, and 150 bp paired-end reads were generated.

Genome sequencing, assembly, and annotation

Pair-end Illumina raw reads were cleaned, adaptors and barcodes were removed, and then quality filtering was performed using Trimoraic. Individual bases with Phred quality score < 20 were removed from both ends of reads, as well as more than three consecutive uncalled bases. Entire reads with a median quality score lower than 21 or less than 40 bp in length after trimming were discarded. After quality filtering, reads were mapped to the chloroplast genome of the closest species with a chloroplast genome available (NCBI download) using Bowtie2 v.2.2.6 (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.6/) to exclude reads of nuclear and mitochondrial origins. All putative chloroplast reads mapped to the reference sequence above were then used for de novo assembly to reconstruct the chloroplast genomes using Get Organelle (Jin et al. 2020). Automatic annotation of the chloroplast genomes was generated by CpGAVAS2 (Shi et al. 2019), and a circular representation of both sequences was drawn using the online tool OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw. html). The draft annotations given by CpGAVAS2 were then manually corrected using Artemis software and other plastid genomes for comparison.

Analysis of chloroplast genomic characteristics

Relative synonymous codon usage (RSCU) analysis was used to express the ratio of the actual codon usage value to the theoretical codon usage value of five species. The characteristics of scattered repeat sequences were analysed by Reputer software (Kurtz et al. 2001). Simple repeat sequences were identified by the MISA tool (http://pgrc.ipk-gatersleben.de/misa/) (parameters: 1-10 2-53-4 4-3-3 6-3).

Comparative genomic analysis

Based on the genome map, the known genes and genome structures were compared to reveal gene function, expression regulation mechanisms, species evolution and other aspects. IR expansion and contraction were analysed by online IR scope (http://irscrope.shinyapps.io/irapp/). The Mauve tool (http://darlinglab.org/mauve/mauve.html) was used to analyse multiple sequences to determine the local collinearity between genomes. The chloroplast genomes of 25 species of Asteraceae were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/), and the chloroplast genomes were constructed by RAxML software (Stamatakis 2014).

Results

Genomic characteristics of chloroplasts

After assembly, the lengths of the A-B-E-N-R chloroplast genomes were 151,192 bp, 151,158 bp, 151,159 bp, 150,985 bp, and 15,1284 bp, respectively. The chloroplast genomes of five Cremanthodium species (A-B-E-N-R) all had a typical quadripartite structure: a large single copy region (LSC), a small single copy region (SSC), and two inverted repeat regions (IRs) (Fig. 2). In the A-B-E-N-R chloroplast genomes, the lengths of the LSC region were 83,357 bp, 83,351 bp, 83,326 bp, 83,369 bp, and 83,423 bp; the lengths of the SSC regions were 18,125 bp, 18,107 bp, 18,173 bp, 17,956 bp, and 18,201 bp; and the lengths of the pair of inverted repeats (IRs) were 24,855 bp, 24,850 bp, 24,830 bp, 24,830 bp, and 24,830 bp, respectively (Fig. 2). The GC content in the A-B-E chloroplast genome was 37.45%, that in the N chloroplast genome was 37.48%, and that in the R chloroplast genome was 37.46%. The chloroplast genomes were highly conserved in structure, which was basically the same among species. All the chloroplast genome sequences have been uploaded to NCBI (GenBank: OM386855, OM386856, OM386857, OM386858, OM386859).

Fig. 2.

Fig. 2

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The assembly, size, and features of A-B-E-N-R chloroplast genomes (Cremanthodium). The genes outside the circle are transcribed in the counter clockwise direction, and the genes inside the circle are transcribed in the clockwise direction. Different colors in genes represent different functions. The dark gray area and light gray area of the inner circle represent the GC content and AT content of the genome, respectively

Through gene annotation, we found that the chloroplast genomes of five Cremanthodium species showed similar genome structures, containing 133 unique genes (88 protein coding genes, 37 tRNA genes, and 8 rRNA genes) (Table 1). There was no significant difference in gene sequence, gene type or quantity among the five chloroplast genomes. All five chloroplast genomes had 19 genes containing two copies distributed in the IR region, including eight protein editing genes (ndhB, rpl2, rpl23, rps12, rps7, ycf1, ycf15, and ycf2), seven tRNA genes (trnA-UGC, trnG-UCC, trnI-CAU, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) and four rRNA genes (rrn16, rrn23, rrn4.5, and rrn5). A total of 18 genes had introns, of which nine protein-coding genes (ndhA, ndhB, petB, petD, atpF, rpl16, rpl2, rps16, and rpoC1) and six tRNA genes contained one intron (trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC), and three protein-coding genes (rps12, clpP, and ycf3) contained two introns (Table 2). In all five chloroplast genomes, the ycf1 gene spanned the SSC and IRb junction.

Table 1.

Chloroplast genome characteristics of five species in Cremanthodium genus

Species Size (bp) No. of PCGs No. of tRNAs No. of rRNAs No. of genes GC content (%) LSC size (bp) SSC size (bp) IR size (bp)
C.arnicoides 151,192 88 37 8 133 37.45 83,357 18,125 24,855
C. brunneopilosum 151,158 88 37 8 133 37.45 83,351 18,107 24,850
C. ellisii 151,159 88 37 8 133 37.45 83,326 18,173 24,830
C. nervosum 150,985 88 37 8 133 37.48 83,369 17,956 24,830
C. rhodocephalum 151,284 88 37 8 133 37.46 83,423 18,201 24,830
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Table 2.

Encoded genes in chloroplast genome of five Cremanthodium species

Category Group Genes
Photosynthetic Subunits of photosystem I psaA, psaB, psaC, psaI, psaJ
Subunits of photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbG, psbH, psbI, psbJ, psbK, 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 Large subunit of ribosomal rpl14, rpl16†, rpl2 (× 2)†, rpl20, rpl22, rpl23 (× 2), rpl32, rpl33, rpl36
Samll subunit of ribosomal 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
Tanslational initiation factor infA
Other Protease clpP†
Maturase matK
Envelope membrane protein cemA
c-type cytochrome synthesis gene ccsA
Subunit of Acetyl-CoA- carboxylase accD
Hypothetical chloroplast reading frames ycf1 (× 2), ycf15 (× 2), ycf2 (× 2), ycf3†, ycf4
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(× 2) indicates that the gene has two copies. †Indicate genes containing introns

The codon-anticodon recognition pattern and codon usage

The codon preference analysis of the five chloroplast genomes showed that there was no significant difference in the overall size, base composition, or AT/GC content of the protein coding region. There were few differences in codon usage preferences among the chloroplast genomes of the five species (Table 3, Fig. 3).

Table 3.

The codon-anticodon recognition pattern and codon usage for five Cremanthodium species

Amino acid Codon C. arnicoides C. brunneopilosum C. ellisii C. nervosum C. rhodocephalum
No RSCU No RSCU No RSCU No RSCU No RSCU
Ter UAA 51 1.7385 51 1.7385 51 1.7385 51 1.7385 51 1.7385
UAG 21 0.7158 21 0.7158 21 0.7158 21 0.7158 21 0.7158
UGA 16 0.5454 16 0.5454 16 0.5454 16 0.5454 16 0.5454
Ala GCA 409 1.144 409 1.144 409 1.1448 409 1.144 412 1.1524
GCC 231 0.646 232 0.6488 232 0.6496 232 0.6488 231 0.646
GCG 160 0.4476 160 0.4476 160 0.448 160 0.4476 159 0.4448
GCU 630 1.7624 629 1.7596 628 1.758 629 1.7596 628 1.7568
Cys UGC 91 0.6066 90 0.598 91 0.6066 91 0.6046 91 0.6108
UGU 209 1.3934 211 1.402 209 1.3934 210 1.3954 207 1.3892
Asp GAC 213 0.4068 213 0.4064 213 0.4064 212 0.4046 213 0.405
GAU 834 1.5932 835 1.5936 835 1.5936 836 1.5954 839 1.595
Glu GAA 986 1.473 986 1.4738 985 1.4724 986 1.475 988 1.4768
GAG 352 0.262 352 0.5262 353 0.5276 351 0.525 350 0.5232
Phe UUC 517 0.6888 515 0.688 517 0.688 517 0.6898 518 0.6944
UUU 984 1.3112 982 1.312 986 1.312 982 1.3102 974 1.3056
Gly GGA 704 1.582 704 1.582 704 1.582 704 1.582 702 1.58
GGC 193 0.4336 194 0.436 194 0.436 194 0.436 196 0.4412
GGG 302 0.6788 301 0.6764 301 0.6764 301 0.6764 301 0.6776
GGU 581 1.3056 581 1.3056 581 1.3056 581 1.3056 578 1.3012
His CAC 155 0.5008 155 0.5024 155 0.5024 155 0.5024 154 0.5
CAU 464 1.4992 462 1.4976 462 1.4976 462 1.4976 462 1.5
Ile AUA 720 0.9711 723 0.9753 721 0.9726 722 0.9738 720 0.9717
AUC 428 0.5772 425 0.5733 425 0.5733 424 0.5718 425 0.5736
AUU 1076 1.4514 1076 1.4514 1078 1.4541 1078 1.4541 1078 1.4547
Lys AAA 1042 1.4738 1042 1.4738 1043 1.4742 1042 1.4728 1031 1.4708
AAG 372 0.5262 372 0.5262 372 0.5258 373 0.5272 371 0.5292
Leu CUA 387 0.8184 388 0.8196 387 0.819 388 0.8202 383 0.813
CUC 188 0.3978 187 0.3948 187 0.396 187 0.3954 187 0.3972
CUG 192 0.4062 192 0.4056 192 0.4062 192 0.4056 191 0.4056
CUU 616 1.3026 617 1.3038 617 1.3056 617 1.3038 612 1.2996
UUA 859 1.8168 861 1.8192 859 1.818 861 1.8198 856 1.8174
UUG 595 1.2582 595 1.257 593 1.2552 594 1.2552 597 1.2678
Met AUG 639 1.9938 639 1.9938 641 1.9938 639 1.9938 638 1.9968
GUG 2 0.0062 2 0.0062 2 0.0062 2 0.0062 1 0.0032
Asn AAC 285 0.4388 287 0.4416 287 0.4412 286 0.44 285 0.4378
AAU 1014 1.5612 1013 1.5584 1014 1.5588 1014 1.56 1017 1.5622
Pro CCA 328 1.206 329 1.2096 328 1.206 328 1.206 325 1.198
CCC 203 0.7464 203 0.7464 203 0.7464 203 0.7464 202 0.7448
CCG 146 0.5368 145 0.5332 146 0.5368 146 0.5368 149 0.5492
CCU 411 1.5112 411 1.5112 411 1.5112 411 1.5112 409 1.508
Gln CAA 712 1.5116 714 1.5144 713 1.5122 713 1.5122 714 1.5144
CAG 230 0.4884 229 0.4856 230 0.4878 230 0.4878 229 0.4856
Arg AGA 504 1.89 503 1.8888 504 1.8912 503 1.8888 501 1.881
AGG 177 0.6636 177 0.6648 177 0.6642 177 0.6648 177 0.6648
CGA 354 1.3278 353 1.3254 353 1.3248 352 1.3218 353 1.3254
CGC 108 0.405 108 0.4056 108 0.405 109 0.4092 108 0.4056
CGG 116 0.435 116 0.4356 116 0.435 116 0.4356 116 0.4356
CGU 341 1.2786 341 1.2804 341 1.2798 341 1.2804 343 1.2876
Ser AGC 117 0.3468 118 0.3504 118 0.3498 118 0.3498 120 0.3576
AGU 413 1.2252 412 1.2228 412 1.221 412 1.2222 410 1.2222
UCA 424 1.2576 424 1.2582 425 1.2594 425 1.2606 425 1.2666
UCC 308 0.9132 307 0.9108 308 0.9126 307 0.9108 303 0.903
UCG 164 0.4866 165 0.4896 165 0.489 165 0.4896 164 0.489
UCU 597 1.7706 596 1.7688 597 1.7688 596 1.7676 591 1.7616
Thr ACA 413 1.2524 413 1.2524 413 1.2524 413 1.2524 412 1.254
ACC 247 0.7492 247 0.7492 246 0.746 247 0.7492 243 0.7396
ACG 133 0.4032 133 0.4032 133 0.4032 133 0.4032 132 0.402
ACU 526 1.5952 526 1.5952 527 1.598 526 1.5952 527 1.6044
Val GUA 525 1.4968 525 1.4968 525 1.4988 525 1.4968 523 1.4976
GUC 175 0.4988 175 0.4988 175 0.4996 175 0.4988 175 0.5012
GUG 195 0.556 195 0.556 193 0.5512 195 0.556 194 0.5556
GUU 508 1.4484 508 1.4484 508 1.4504 508 1.4484 505 1.446
Trp UGG 455 1 455 1 454 1 454 1 451 1
Tyr UAC 181 0.3668 181 0.366 181 0.366 182 0.368 181 0.3676
UAU 806 1.6332 808 1.634 808 1.634 807 1.632 804 1.6324
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Fig. 3.

Fig. 3

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Codon RSCU clustering in chloroplast genome of five Cremanthodium species

The protein coding sequences of C. arnicoides and C. brunneopilosum were 79,005 bp, and 88 protein coding genes encoded 26,335 codons. The protein coding sequence of the C. ellisii was 79,017 bp, and 88 protein coding genes encoded 26,339 codons. The protein coding sequence of the C. nervosum was 79,008 bp, and 88 protein coding genes encoded 26,336 codons. The protein coding sequence of the C. rhodocephalum was 78,807 bp, and 88 protein coding genes encoded 26,269 codons. There were three stop codons (UAA, UAG and UGA) in the protein coding sequences of five chloroplast genomes (Table 3). UAA appeared 51 times, with more than 50% frequency; UAG appeared 21 times, and UGA appeared 16 times.

In the coding protein sequences of the A-B-E-N-R chloroplast genomes, the most frequent amino acid encoded by codons was leucine (Leu), which appeared 2837, 2840, 2835, 2839 and 2826 times, respectively, and the most frequents codon was AUU of isoleucine (Ile), which appeared 1076 times, 1076 times, 1078 times, 1078 times and 1078 times, respectively. Only tryptophan (Trp) has one codon, and other amino acids have 2–6 synonymous codons. RSCU > 1 indicates codon preference, RSCU < 1 indicates low usage rate, and RSCU = 1 indicates no codon preference.

Detection of chloroplast repeat sequences and SSRs

The scattered repeat sequences were analysed by Repeater software. In this study, we identified 38, 39, 36, 38, and 39 interspersed repeat sequences (LTRs) and 62, 63, 62, 60 and 61 simple repeated sequences (SSRs) in the A-B-E-N-R chloroplast genomes (Table 4), respectively. There were two main types of LTRs: forwards LTRs, accounting for 43.6–50.0% of all repeats, and palindromic LTRs, accounting for 50.0–56.4% of all repeats.

Table 4.

Distribution of chloroplast genomes LTR in chloroplast genome of five Cremanthodium species

Type C. arnicoides C. brunneopilosum C. ellisii C. nervosum C. rhodocephalum
Forward 19 18 17 18 17
Palindromic 19 21 19 20 22
Reverse 0 0 0 0 0
Complement 0 0 0 0 0
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All single nucleotide repeats were A/T homopolymers. Single nucleotide repeats accounted for 62.3–65.0% of the SSR, and 10–14 bp repeats accounted for 80.0–89.5% of the single nucleotide repeats. There were 6–7 dinucleotide repeats, accounting for 9.5–11.3% of the SSRs (Table 5). All dinucleotide repeats were AT/TA. The number of trinucleotides in all repeats was 5. The types of trinucleotides were ATG, ATT, TTA, and TTC. The number of tetranucleotides in all repeats was 11. The types of trinucleotides were ATAA, AAAT, ACTA, TATT, TTTC, AATT, ATAG, AATA, AAAT, and AATC. The only hexanucleotide was ACTCCT, and it was detected in the chloroplast genome of C. rhodocephalum.

Table 5.

Classification of cp SSR in five Cremanthodium species

type C. arnicoides C. brunneopilosum C. ellisii C. nervosum C. rhodocephalum
Mono- 40 41 39 38 38
Di- 6 6 7 6 6
Tri- 5 5 5 5 7
Tetra- 11 11 11 11 11
Penta- 0 0 0 0 0
Hexa- 0 0 0 0 1
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IR expansion and contraction

There was no obvious expansion or contraction in the LSC, SSC or IR regions among the five species, indicating that the chloroplast gene structure of the genus was highly conserved (Fig. 4).

Fig. 4.

Fig. 4

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Chloroplast boundary characteristics of five Cremanthodium species

Collinearity analysis

The chloroplast genomes of five species of the Cremanthodium genus were compared. The results showed that the chloroplast genomes of the five species were collinear, and there was no gene rearrangement (Fig. 5). There still are some differences in their chloroplast genomes which the code gene near the site of 110,000. It can be used as a mutation hotspot, and this area was in the middle between ndhF and rpl32.

Fig. 5.

Fig. 5

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Chloroplast boundary characteristics of five Cremanthodium species. Colinear analysis of chloroplast genomes of five Cremanthodium species. Color bands represent genes, and different colors represent different blocks. Blocks with the same color between different genes represent homologous regions. Two rows of small blocks below the color band represent genes. The top one is on the positive chain, and the below one is on the complementary chain. Of which, the white block represents the coding genes, a thin line inside the white blocks denotes intron. Green and red blocks represent tRNA and rRNA, respectively

Phylogenetic tree

In addition to the five species of Cremanthodium (A-B-E-N-R) in this study, 25 published chloroplast genomes of the Compositae family were selected to construct phylogenetic trees using the maximum likelihood (ML) method to explore phylogenetic relationships. From the ML tree, C. arnicoides and C. ellisii formed a sister group, and C. brunneopilosum and C. nervosum formed a sister group. Five species of Cremanthodium (A-B-E-N-R) formed a monophyletic group (Fig. 6). C. rhodocephalum was the first differentiated species among the five species of Cremanthodium (A-B-E-N-R). Based on the ML tree, there was a closer genetic relationship between the five species of Cremanthodium (A-B-E-N-R), and their next closest genetic relationships were with Ligularia stenocephala, L. fischeri, L. jaluensis, L. intermedia, L. mongolica, L. veitchiana, Farfugium japonicum, and Petasites japonicus.

Fig. 6.

Fig. 6

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Phylogenetic tree for 30 species in Asteraceae family using maximum likelihood (ML), based on alignments of complete chloroplast genomes

Discussion

Senecioneae Cass. belongs to subfamily Asteroideae (Asteraceae), which contains about 3500 species and 152 genera, and is widely distributed around the world (Nordenstam 2007; Nordenstam et al. 2009). However, due to the possible rapid diversification in the early evolution of Asteraceae, the specific system location of Senecioneae Cass. has not been determined (Kim et al.2005; Panero and Funk 2008). Different genus of Senecioneae Cass. formed a large complex (Ligularia-Cremanthodium-Parasenecio complex; L-C-P complex) except for Tussilago L. and Petasites Mill.. Ligularia Cass., Cremanthodium Benth., Parasenecio W. W. Sm. and J. Small, and Sinosenecio B. Nord. which are defined according to morphological characters instead of monophyletic groups, and the boundaries between genera need to be revised (Liu et al. 2006). The position and genetic relationship of Ligularia Cass., Cremanthodium Benth., and Parasenecio W. W. Sm. in the system are not clear, which needs more experimental verification at the molecular level. In this study, the whole chloroplast genomes of five Cremanthodium species were sequenced and annotated for the first time, which enriched the chloroplast genome data of L-C-P complex and provided a basis for their intergeneric boundaries. According to the expeimental results, the chloroplast genomes length of five Cremanthodium species are similar to that of other species of Asteraceae. Due to the narrow distribution of Cremanthodium plants, the chloroplast genome showed obvious conservation. The assembled chloroplast genome sequences of C. arnicoides, C. brunneopilosum, C. ellisii, C. nervosum, and C. rhodocephalum, with lengths of 150,985–151,284 bp (Fig. 2), were similar to the most sequenced chloroplast genomes: Chrysanthemum L. (151,010–151,098 bp) (Tyagi et al. 2020a, b), Senecio L. (150,000–151,000 bp) (Gichira et al. 2019), Ligularia Cass. (151,118–151,253 bp) (Chen et al. 2018; Lee et al. 2016), Farfugium Lindl. (151,222 bp) (Gu et al. 2016), Taraxacum F. H. Wigg. (151,307 and 151,451 bp) (Kim et al. 2016), Saussurea involucrate (152,490 bp) (Wang et al. 2020), Aster flaccidus (151,329 bp) (Tyagi et al. 2020a, b), and Carpesium abrotanoides L. (151,394 bp) (He et al. 2022). The chloroplast genomes encoded equal number of 133 unique genes, of which 88 were protein-coding genes, 37 were transfer ribonucleic acid genes, and eight were ribosomal ribonucleic acid genes (Table 1), and were highly similar in overall size, genome structure, gene content, and order. The GC content is also similar, indicating that there is little variation among the species in the genus.

Codon usage analysis showed that there were 31 codons with RSCU > 1 in the five species, of which 16 ended in U, 13 in A, and 2 in G, which indicated that more codons ended with U or A (Table 3, Fig. 3). In Cremanthodium plants, the identification of microsatellite loci in the intergenic spacer region and introns can show a potential polymorphism since coding regions were conserved across other genomes. There is a predominance of mononucleotides, followed by tetranucleotides. The number of SSRs identified for these five Cremanthodium chloroplast genomes ranged from 60 to 63.

Comparing the junction of four parts of chloroplast genome of five Cremanthodium species, it was found that the composition and distribution of genes at the boundary were highly similar (Fig. 4). The gene rps19 is located at the junction of the LSC region and the IRb region, and the sequence length distribution in the two regions is stable. The gene length of rps19 entering the IRb region is 60 bp, and the length retained in the LSC region is 219 bp. The gene ycf1 is located at the junction of IRb region and SSC region and between SSC region and IRa region, while ycf1 gene is highly conserved at the boundary.

The collinearity of chloroplast genes (Fig. 5) showed that the sequences of protein coding genes, tRNA and rRNA genes were similar, and the gene structure was conservative. There is an obvious difference in 112 kb between Cremanthodium rhodocephalum and other species, and it is speculated that there is a relatively distant relationship between this and other species, which is also consistent with the relationship on ML tree.

Based on the chloroplast genome sequences of five Cremanthodium species, 23 species of Asteraceae, and 2 species of Campanulaceae were compared and analyzed, and the taxonomic position and evolutionary relationship of the plants sequenced in this study were evaluated. According to ML tree (Fig. 6), as part of the L-C-P complex, five Cremanthodium species are grouped into one group, which C. arnicoides is resolved as sister to C. ellisii, C. brunneopilosum is resolved as sister to C. nervosum, and C. rhodocephalum is resolved as a separate group. This is different from the results of classical morphological classification, and the taxonomic position of C. nervosum is determined by chloroplast genome data. According to the characteristics of leaf veins, C. nervosum, C. arnicoides and C. ellisii were divided into a sister group (pinnate vein group), but the whole chloroplast genome sequence showed that C. nervosum did not belong to this sister group. Five Cremanthodium species and eight Ligularia species constituted a morphologically distinct with high support rate, the existing chloroplast genomes data support the location of genera, and show that the evolutionary relationship between the two genera is relatively close, which may be derived from the same ancestor (Liu et al. 2006).

Conclusion

The complete chloroplast genome sequences of five Cremanthodium Benth. and their phylogenetic relationship were reported to provide evidence for species identification of the Cremanthodium genus. The structure and composition of the chloroplast genomes are highly similar and their overall sequence, gene content and gene order were conserved. Phylogenetic analyses using other Compositae species and other species supported the taxonomic status of the Cremanthodium within the tribe. This study provides invaluable data for species identification, allowing for future studies on phylogenetic evolution, as well as for further biological discoveries.

Acknowledgements

We thank Prof. Xiang Liu and Prof. Huarong Zhou for their assistance during leaves collection.

Author contributions

WZ performed the experiments, data processing and manuscript draft preparation, XD contributed to analyzing the data, LC and ZM performed sample collection, XW and GZ designed the project and approved the final manuscript version.

Funding

This work was supported by National Key Research and Development Program of China (No. 2019YFC1712300) and Jiangxi University of Chinese Medicine Science and Technology Innovation Team Development Program (CXTD22002).

Data Availability

The data that support the findings of this study have been deposited in the NCBI database (GenBank accession: OM386855, OM386856, OM386857, OM386858, OM386859) (http://www.ncbi.nlm.nih.gov/).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

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Contributor Information

Xiaolang Du, Email: 20131059@jxutcm.edu.cn.

Guoyue Zhong, Email: zgy1037@163.com.

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Associated Data

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

Data Availability Statement

The data that support the findings of this study have been deposited in the NCBI database (GenBank accession: OM386855, OM386856, OM386857, OM386858, OM386859) (http://www.ncbi.nlm.nih.gov/).

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