The complete chloroplast genome sequences of six Hylotelephium species: Comparative genomic analysis and phylogenetic relationships

Abstract

To evaluate the phylogenetic relationships between Hylotelephium and Orostachys, and to provide important information for further studies, we analyzed the complete chloroplast genomes of six Hylotelephium species and compared the sequences to those of published chloroplast genomes of congeneric species and species of the closely related genus, Orostachys. The total chloroplast genome length of nineteen species, including the six Hylotelephium species analyzed in this study and the thirteen Hylotelephium and Orostachys species analyzed in previous studies, ranged from 150,369 bp (O. minuta) to 151,739 bp (H. spectabile). Their overall GC contents were almost identical (37.7–37.8%). The chloroplast genomes of the nineteen species contained 113 unique genes comprising 79 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), and four ribosomal RNA genes (rRNAs). Among the annotated genes, fourteen genes contained one intron, and two genes contained two introns. The chloroplast genomes of the nineteen Hylotelephium and Orostachys species had identical structures. Additionally, the large single copy (LSC), inverted repeat (IR), and small single copy (SSC) junction regions were conserved in the Hylotelephium and Orostachys species. The nucleotide diversity between the Hylotelephium chloroplast genomes was extremely low in all regions, and only one region showed a high Pi value (>0.03). In all nineteen chloroplast genomes, six regions had a high Pi value (>0.03). The phylogenetic analysis showed that the genus delimitation could not be clearly observed even in this study because Hylotelephium formed a paraphyly with subsect. Orostachys of the genus Orostachys. Additionally, the data supported the taxonomic position of Sedum taqeutii, which was treated as a synonym for H. viridescens in previous studies, as an independent taxon.

Introduction

The family Crassulaceae DC. consists of approximately 1500 species in 35 genera that are mainly distributed throughout the Northern Hemisphere [1–3]. Among these, the genus Hylotelephum H. Ohba includes approximately 28 taxa that are distributed in Asia, Europe, and North America [3].

The taxa belonging to this genus are perennial herbaceous plants that are usually succulent. The morphological characteristics of this genus are as follows: the roots are fibrous or tuberous and often carrot shaped. The rhizomes are short and fleshy or woody. The stems are erect or decumbent and green or red. The young branches are not covered with scales. The stem leaves are alternate, opposite or 3-5-verticillate, and lanceolate, ovate, orbicular, or oblong; the blade is flat and glabrous, and the margin is serrate. The inflorescence is terminal and sometimes also subterminal and paniculate, cyme, often corymbiform, and sometimes umbel-like. The flowers are bisexual (rarely unisexual), subsessile or pedicellate, and pentamerous. The sepals are usually shorter than the petals. The petals are subconnate at the base and are purple, red, pink, white, occasionally yellowish, or greenish. The seeds are elliptical and have narrow wings [4, 5].

This genus was initially described as Sedum L. by Linne [6], and then Miller [7] and Hill [8] proposed the classification of Hylotelephium within the genera Anacampseros L. and genus Telephium L, respectively. Gray [9] recognized this genus as a section of Sedum, and many taxonomists [2, 10–12] agreed with Gray’s opinions, but Clausen [13] disagreed with this and classified it as a subgenus of Sedum. In addition, Ohba [14] recognized it as an independent genus because Hylotelephium differs from the other species of Sedum by having stipitate or attenuated ovaries, flat broad leaves, compound corymbose inflorescences, and nonyellow petals. However, Chung & Kim [15] proposed the classification of this genus as a section within Sedum.

Since then, various molecular phylogenetic studies [16–23] have been performed and revealed a large phylogenetic distance between the two genera; Hylotelephium formed a clade with Sinocrassula A. Berger, Orostachys, and Meterostachys Nakai (Telephium clade), and Sedum was in the Acre clade. However, in most studies, including studies using ITS of nuclear DNA [16, 18] and some marker regions of the chloroplast genome [19], as well as whole chloroplast genome sequences [23], the taxonomic position of Hylotelephium as an independent genus was not supported because it formed a paraphyletic clade with Orostachys species. Therefore, studies are needed to evaluate an accurate taxonomic position by identifying the exact phylogenetic relationship between two genera.

Furthermore, it is known as a very difficult group to classify [15, 24] because members of Hylotelephium have very similar external morphological characteristics between species, and each taxon has wide variation in external morphological characteristics. Because of this, the identities of many taxa are unclear, and scientific names have been repeatedly mixed and misused.

With the development of next-generation sequencing (NGS) technology that has reduced the time and cost required for sequencing, many studies have performed whole chloroplast genome sequencing. These studies have provided much information about plant systematics and evolution. The rapidly evolving loci identified by these studies are very important for resolving unclear phylogenetic relationships because they have higher resolution than traditional molecular markers [25, 26]. Therefore, many studies have focused on finding genetic regions among specific families or genera to provide useful information about molecular markers for further studies [25–32].

We obtained the whole chloroplast genome sequences of six Hylotelephium species (H. pallescens (Freyn) H. Ohba, H. spectabile (Boreau) H. Ohba, H. ussuriense (Kom.) H. Ohba, H. viridenscens (Nakai) H. Ohba, H. viviparum (Maxim.) H. Ohba and S. taquetii Praeger, and compared the sequence to those of thirteen published congeneric and closely related genera (Orostachys) chloroplast genomes, i.e., those from H. erythrostictum (Miq.) H. Ohba, H. ewersii (Ledebour) H. Ohba, H. verticillatum (L.) H. Ohba, O. chongsunensis Y.N. Lee, O. iwarenge (Makino) H. Hara, O. iwarenge f. magnus Y.N. Lee, O. japonica (Maxim.) A. Berger, O. japonica f. polycephala (Makino) H. Ohba, O. latielliptica Y.N. Lee, O. malacophylla (Pall.) Fisch, O. margaritifolia Y.N. Lee, O. minuta (Kom.) Berger. and O. ramosa Y.N. Lee. Our main goal was (1) to evaluate the phylogenetic relationships between Hylotelephium and Orostachys at the whole chloroplast genome level and (2) to identify the taxonomic position of S. taquetii, which was treated as a synonym of H. viridescens by Ohba. Furthermore, we also aimed (3) to provide important information about the most suitable chloroplast molecular markers for further studies to solve unclear phylogenetic relationships of Hylotelephium.

Materials and methods

Taxon sampling, DNA extraction, and sequencing

Since the six Hylotelephium taxa examined in this study were not endangered or protected species, plant materials were collected without permission. The plant materials for this study were collected from the native habitats of each taxon, and the voucher specimens were deposited in the Sangji University Herbarium (SJUH) (S1 Table). Total DNA was extracted from approximately 100 mg of fresh leaves using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA), and LabChip GXII (PerkinElmer, Inc., MA, USA) was used to quantify the DNA concentration and quality. Genomic libraries were prepared using a TruSeq DNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) and paired-end sequencing was performed on a MiSeq platform at LabGenomics, (Seongnam, Korea). The DNA of the Hylotelephium taxa was sequenced to produce 3,750,814–4,573,271 raw reads with lengths of 301 bp (S1 Table).

Assembly and annotation

Low-quality sequences (Phred score < 20) were trimmed using CLC Genomics Workbench (version 6.04; CLC Inc., Arhus, Denmark). Then, de novo assembly was implemented using the Geneious assembler with a medium sensitivity option via Geneious Prime v.2022.1.1 (Biomatters Ltd., Auckland, New Zealand). A total of 124,975–421,454 reads were aligned (S1 Table) and selected from chloroplast contigs using Geneious Prime v.2022.1.1. The draft genome contigs were merged into a single contig by joining the overlapping terminal sequences of each contig. The protein-coding genes (PCGs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) in the chloroplast genome were predicted and annotated using Geneious Prime v.2022.1.1 and manually edited by comparison with the published chloroplast genome sequences of Hylotelephium. The tRNAs were confirmed using tRNAscan-SE [33]. A circular chloroplast genome map was drawn using the OGDRAW program [34].

Comparative analyses in Hylotelephium and allied genera

The newly complete chloroplast genome sequences of six Hylotelephium taxa were used along with the following chloroplast genome sequences from GenBank of NCBI for comparative analysis: three published Hylotelephium, H. ewersii (MN794014), H. erythrostictum (MZ519882), and H. verticillatum (MT558730); and ten Orostachys, O. choungsunensis (ON979333), O. iwarenge (ON979332), O. iwarenge f. magnus (MW851201), O. japonica (MW579549), O. japonica f. polycephala (ON979327), O. latielliptica (ON979328), O. malacophylla (ON979331), O. margaritifolia (ON979329), O. minuta (OK094425) and O. ramose (ON979330).

Relative synonymous codon usage (RSCU) and amino acid frequency in the protein coding gene region were determined by DnaSP 6 [35]. The genome structures of the nine species were compared using the MAUVE program [36]. Additionally, the program mVISTA was used to compare similarities between the nine species using the shuffle-LAGAN mode [37]. The annotated H. verticillatum chloroplast genome was used as a reference. The large single copy/inverted repeat (LSC/IR) and inverted repeat/small single copy (IR/SSC) boundaries of these species were also compared and analyzed.

Nucleotide diversity and repeat analysis

To assess the nucleotide diversity (Pi) between the nineteen chloroplast genomes, including nine Hylotelephium and ten Orostachys genomes, the complete chloroplast genome sequences were aligned using the MAFFT [38] aligner tool and manually adjusted with BioEdit [39]. We then performed sliding window analysis to calculate the nucleotide variability (Pi) values using DnaSP 6 [35] with a window length of 600 bp and a step size of 200 bp [40].

The REPuter program [41] was used to identify repeats: forward, reverse, palindrome, and complement sequences. The following settings for repeat identification were used: (1) Hamming distance equal to 3; (2) minimal repeat size set to 30 bp; and (3) maximum computed repeats set to 90 bp. The simple sequence repeats (SSRs) were identified by MISA software (http://pgrc.ipk-gatersleben.de/misa/) with the parameters set as follows: 10 for mono-, 5 for di-, 4 for tri-, and 3 for tetra-, penta-, and hexanucleotides [42].

Phylogenetic analysis

The whole chloroplast genome sequences from 41 Crassulaceae species were compiled into a single file of size 165,758 bp and aligned using MAFFT [37]. Thirty-nine Telephium clade [19] species were selected as the ingroups, and two species from subfam. Kalachoideae (Cotyledon tomentosa Harv. and Kalanchoe delagoensis Eckl. & Zeyh.) were chosen as the outgroups (S2 Table). Maximum likelihood (ML) analyses were performed using raxmlGUI v.2.0.6 with 1000 bootstrap replicates and the GTR+I+Γ model [43]. Bayesian inference (ngen = 1,000,000, samplefreq = 200, burninfrac = 0.25) was carried out using MrBayes v3.0b3 [44], and the best substitution model (GTR+I+Γ) was determined by the Akaike information criterion (AIC) in jModeltest version 2.1.10 [45].

Results

Chloroplast genome features

The chloroplast genomes of six Hylotelephium species have been submitted to GenBank of the National Center for Biotechnology Information (NCBI) (Table 1 and S1 Table). The total length of the chloroplast genomes of the nineteen species, i.e., the six Hylotelephium species analyzed in this study and the thirteen species analyzed in previous studies, ranged from 150,369 bp (O. minuta) to 151,739 bp (H. spectabile). Among the Hylotelephium species, H. viviparum was the smallest (150,430 bp) (Table 1 and Fig 1). All nineteen chloroplast genomes exhibited a typical quadripartite structure, including a large single copy (LSC) region, a small single copy (SSC) region and a pair of inverted repeat (IR) regions. The length of the LSC region ranged between 81,991–83,252 bp, and the GC content of the LSC regions was similar in nineteen species, ranging from 35.7–35.9%. The length of the SSC region was distributed between 16,702 and 17,808 bp, with a GC content of 31.5–31.8%. The length range of the IR region of nineteen species was 25,285–25984 bp, which contained 42.8–43.0% GC content. The chloroplast genomes of the nineteen species contained 113 unique genes comprising 79 PCGs, 30 tRNAs, and four rRNAs (Table 1 and S3 Table). In addition, nineteen genes, including eight protein-coding genes (rpl23, ycf2, ndhB, rps7, 3’-end rps12, and part of rps19 and ycf1), seven tRNAs (trnI-CAU, trnL-CAA, trnV-GAC, trnI-GAU, trnA-UGC, trnR-ACG and trnN-GUU) and four rRNAs (16S rRNA, 23S rRNA, 4.5S rRNA and 5S rRNA), were duplicated in IR regions. The rps12 gene had trans-splicing, and its 3’-end was duplicated in the IR region, while its 5’-end was present in the LSC region (Fig 1). Among the annotated genes, fourteen genes (atpF, ndhA, ndhB, petB, petD, rpl2, rps12, rpl16, rpoC1, trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) contained one intron, and two genes (clpP and ycf3) contained two introns (S3 Table).

[Figure omitted. See PDF.]

Genes inside the circle are transcribed clockwise, and genes outside are transcribed counterclockwise. The dark gray inner circle corresponds to the GC content, and the light gray circle corresponds to the AT content.

[Figure omitted. See PDF.]

A total of 23,968–26,063 codons were identified in nine Hylotelephium species (S4 Table). Among them, AUU (4.1–4.2%, isoleucine), AAA (4.1%, lysine), and GAA (3.9%, glutamic acid) were the most frequently used codons, while CGC (0.4–0.5, arginine) and UGC (0.3%, cysteine) had the lowest usage rates. There were 29 codons with relative synonymous codon usage (RSCU) values greater than 1, 2 of which were equal to 1, and 30 were less than 1. In addition, the codons containing A or T at the 3’-end mostly had RSCU > 1 (26 out of 29 codons), and most of the codons containing G or C at the 3’-end were less than 1 or equal to 1 (28 out of 30 codons).

The results of the chloroplast genome structure comparison between the nineteen Hylotelephium and Orostachys species using MAUVE [35] showed that all chloroplast genomes were the same (S1 Fig). The pairwise cp genomic alignments between all 19 Hylotelephium and Orostachys species showed very high similarity in all sequences (Fig 2). The LSC and SSC regions were more variable than the IR regions, and noncoding regions were more susceptible to mutations than coding regions. In addition, clpP, ndhA and ycf1 were the most different from each other among the coding regions, and in the noncoding regions, rps16-trnQ(UUG), atpH-atpI, trnE(UUC)-trnT(GGU), psbZ-trnG(UCC), ycf4-cemA and ycf2-trnL(CAA) of were quite different from each other.

[Figure omitted. See PDF.]

The vertical scale indicates the percent identity, ranging from 50% to 100%. Coding genes, RNAs, and noncoding regions are marked in purple, sky blue, and red, respectively.

The border regions and adjacent genes of chloroplast genomes were compared to analyze the expansion and contraction variation in junction regions, which are common phenomena in the evolutionary history of terrestrial plants [46]. A comparison of the LSC/IR and IR/SSC boundaries in the nineteen species is shown in Fig 3. The rps19, ndhF and ycf1 genes of all nineteen species spanned the LSC and IRb, IRb and SSC, and SSC and IRa regions, respectively. At the junction of IRa/LSC (JLA), trnH-GUG and part of the rps19 genes were in the LCS and IRa regions, respectively. The junctions of LSC/IRb (JLB), IRb/SSC (JSB) and JLA were highly conserved with no contraction or expansion, whereas a small length change was identified in the SSC/IRa (JSA) junction. The length of ycf1 in the IRa region was the same, but ycf1 in the SSC region varied from 4065 to 4074 bp.

[Figure omitted. See PDF.]

Repeats and SSR analyses

The four types of repeat structures, including forward, reverse, complement and palindromic repeats, were identified using REPuter software [41] in the nine Hylotelephium chloroplast genomes. Overall, 18 (H. ewersii, H. verticillatum and H. viviparum) to 22 (H. pallescens) repeat sequences were identified in each chloroplast genome, of which 7 to 8 were forward repeats and 10 to 12 were palindromic repeats. Additionally, reverse and complement repeats were present only in H. ewersii (1 repeat) and H. pallescens (2 repeats), respectively (Fig 4A). The length of repeats ranged from 30 to 48 bp, and a repeat with a length of 41 bp was the most abundant, followed by those with lengths of 31, 37 and 30 bp (Fig 4B).

[Figure omitted. See PDF.]

(A) Types and number of repeats in the nine chloroplast genomes, (B) frequency by length of repeats in the nine chloroplast genomes, (C) frequency by type of SSRs in the nine chloroplast genomes.

A total of 41 (H. erythrostictum, H. pallescens, H. viridescens and S. taquetii) to 57 (H. spectabile) SSRs (also called microsatellites) were examined for the Hylotelephium species and ranged in size from 10 to 16 bp. All nine chloroplast genomes had no hexanucleotide repeats, and four species (H. erythrostictum, H. spectabile, H. ussuriense, and H. verticillatum) had five types of SSRs, i.e., mono-, di-, tri-, tetra- and pentanucleotides. Mononucleotide repeats ranged from 27 (H. erythrostictum) to 42 (H. spectabile) and were the most abundant in the nine Hylotelephium chloroplast genomes, with A/T repeats being the only represented repeats (Fig 4C).

Sequence divergence and mutational hotspots

The nucleotide diversity (Pi) between the nine Hylotelephium species and all nineteen species is shown in Fig 5. The Pi values in the IR regions were much lower than those in the LSC and SSC regions. The average Pi values were estimated to be 0.003 and 0.007. Among the Hylotelephium species, the Pi values were extremely low in all regions, and only one region (rpl32-ccsA) showed relatively high Pi value (>0.03). In all nineteen species, six regions had a high Pi value (>0.03). Among these, four (rps16-trnQ, trnC-petN, ycf4-cemA, and cemA) and two (rpl32-ccsA and ycf1) were in the LSC and SSC regions, respectively. Moreover, corebarcode regions, rbcL and matK, and molecular markers used in previous studies, rps16, and trnL-trnF [17–20], of nine Hylotelephium species and all nineteen Hylotelephium and Orostachys species pad very low Pi values of 0.0074 and 0.0176 or less, respectively (S5 Table).

[Figure omitted. See PDF.]

(A) Pi values of nine Hylotelephium species and (B) Pi values of all nineteen species.

Phylogenetic analyses

The ML tree constructed with whole chloroplast genome sequences was well supported at the genus level, except for those of Hylotelephium and Orostachys. The ML tree was divided into two subclades. In the first clade, the genera Rhodiola L. and Phedimus Raf. were clustered into a well-supported, monophyletic clade with high bootstrap (BP) and Bayesian posterior probability (PP) values. The second clade consisted of Hylotelephium, Orostachys, Meterostachys, Sinocrassula A. Berger and Umbilicus DC. Umbilicus was the earliest-diverging lineage, and Sinocrassula was sister to Hylotelephium, Orostachys and Meterostachys. Hylotelephium was paraphyletic and formed a clade with subsect. Orostachys of Orostachys (Fig 6).

[Figure omitted. See PDF.]

Bootstrap (BP) values greater than 50% are below the clades, and Bayesian posterior probabilities (PP) are shown above the clades.

Discussion

Comparison of the chloroplast genomes

In this study, we collected six species of Hylotelephium and obtained their complete chloroplast genome sequences. The chloroplast genome of terrestrial plants is highly conserved in its nucleotide sequence and its gene content and order [47–52]. However, the gene orders of the chloroplast genome are sometimes rearranged in independent plant groups [26, 49, 50, 53, 54], and its structural rearrangements provide important systematic data. We found that the genome structure, gene content and gene order of nine Hylotelephium species, including the six species analyzed in this study and three published species, were identical, and the sequence identity was also very similar between species in most of the chloroplast regions (Fig 2 and S1 Fig). Therefore, we believe that the chloroplast genomes of this group are very conserved. In the Hylotelephium species, the genome size differed by less than 500 bp except for H. viviparum (Table 1). The chloroplast genome of H. viviparum was approximately 1000 bp shorter than that of other Hylotelephium species (Table 1), which was confirmed because most of the sequences of the intergenic spacer between rps16-trnQ(UUG) were deleted. It is considered a relatively large event in the highly conserved Hylotelephium chloroplast genome, and it is speculated that it can be used as a specific marker region that distinguishes H. viviparum from other species.

In many previous studies analyzing codon usage bias, it was confirmed that isoleucine and cysteine are the most common and the least common codons, respectively [55–58], and most codons showed higher A/T preference in the third codon [55, 59–62], probably because of the A or T abundance in the IR region [63]. As a result of this study, it was confirmed that the chloroplast genomes of Hylotelephium species had the same characteristics as those of general higher land plants.

The contraction and expansion of IR regions during evolution is a relatively common occurrence and has been employed as an evolutionary locus for phylogenetic studies [64–66]. In the chloroplast genome of Hylotelephium and Orostachys, however, there is little change in the IR regions (Figs 2 and 5B), so it will not be suitable for phylogenetic studies.

The number of repeats in the nine Hylotelephium species ranged from 18 (H. ewersii, H. verticillatum and H. viviparum) to 22 (H. pallescens), and the number of repeats according to type and length showed slight differences between species (Fig 5A and 5B). The presence and abundance of repetitive sequences in the chloroplast or nuclear genome are likely to involve many phylogenetic signals [67–69], and may, therefore, provide additional evolutionary information. In addition, the SSRs identified in this study may provide various markers for population genetic studies of Hylotelephium species because SSRs are considered to play an important role in population genetics [58, 70].

Selection of useful molecular marker regions for phylogeny

Hylotelephium is known to be a very difficult group to classify due to the high external morphological variation of each taxon and the very similar morphological characteristics between species [15, 24]. According to these findings, many morphological and molecular phylogenetic studies have been conducted, but the delimitation of this genus and phylogenetic relationships between its species remain insufficiently resolved [16–23, 71].

Previous studies have mostly used the ITS region of nuclear DNA and matK, rps16, psbA-trnH and trnL-trnF of the regions in the chloroplast genome for phylogenetic analyses [16–20]. Additionally, the CBOL Plant Working Group has recommended matK and rbcL genes as core plant barcodes [72]. The Pi values of these regions were calculated in this study, and all showed a very low Pi of 0.0074 (matK) or less in Hylotelephium chloroplast genomes and 0.0176 (matK) or less in Hylotelephium and Orostachys species. Therefore, the low phylogenetic resolution of the previous studies was due to the selection of molecular marker regions with very low Pi values. Additionally, it may be challenging to obtain high resolution even in the core barcode regions (matK and rbcL).

The results of this study showed that only one region (rpl32-ccsA) had a high Pi value (>0.03) in Hylotelephium species, and three regions (rps16-trnQ, accD and ycf1) had relatively high Pi values (>0.015) (Fig 5A). Therefore, it is considered the most suitable region to evaluate the phylogenetic relationships between Hylotelephium species. Furthermore, six regions (rps16-trnQ, trnC-petN, ycf4-cemA, cemA, rpl32-ccsA and ycf1) were the most suitable chloroplast regions for resolving the unclear phylogenetic relationships between Hylotelephium and Orostachys due to their high Pi values (Fig 5B).

Assessment of phylogenetic relationships

Ohba [14] recognized two sections, Hylotelephium H. Ohba and Populisedum (A. Berger) H. Ohba, in the genus Hylotelephium based mainly on the insertion point of the flowering stem. He then divided the genus into three sections by treating Sieboldia, which was classified as a series of sect. Hylotelephium, as its own section [73]. Hylotelephium formed a polytomy or was polyphyletic with subsect. Orostachys of the genus Orostachys in many previous phylogenetic studies based on nrITS [16, 18] and some chloroplast markers [19]. Unfortunately, the genus delimitation could not be clearly observed even in this study because Hylotelephium formed a paraphyly with subsect. Orostachys of the genus Orostachys. We do not believe these results can be interpreted as a conclusion that the two genera should be combined into one genus because the morphological characteristics such as radical leaves and inflorescences, between the two genera greatly differ. Further studies are needed that include various species and nuclear DNA to clarify the delimitation of the genus. Additionally, the classification system below the genus level of Hylotelephium will also need to be reconsidered in future studies because the taxa belonging to sect. Hylotelephium were polyphyletic, and H. ewersii, which is considered in sect. Populisedum [18], did not form an independent clade (Fig 6).

Moreover, Sedum taquetii, a Korean endemic species that is distributed only on Jeju-do Island, was first described by Praeger [74] because of its larger flowers (especially carpels and petals reaching approximately 10 mm and 9 mm, respectively) compared to the taxa belonging to the same section. Since then, this species has been treated as a synonym for H. viridescens without taxonomic studies [13, 75], and Ohba [14] also accepted these opinions when first describing the genus Hylotelephium and treated it as a synonym. However, Chung and Kim [15] argued that S. taquetii should be recognized as an independent species because it is distinguished from H. viridescens in that the anther is purple. The results of this study showed that H. viridescens had the closest relationship with H. pallescens, and S. taquetii was the sister to the two species mentioned above. Therefore, we strongly agree with Chung and Kim [15] that S. taquetii should be treated as an independent taxon.

Conclusion

In this study, we assembled the chloroplast genomes of six Hylotelephium species, which had a total length ranging from 150,430 to 151,717 bp. The chloroplast genomes of Hylotelephium had identical structures and were highly conserved. The Hylotelephium species are not easy to classify because the morphological variations of each taxon are very high and there are remarkably similar morphological characteristics between species. Therefore, the four regions (rpl32-ccsA, rps16-trnQ, accD and ycf1) and six regions (rps16-trnQ, trnC-petN, ycf4-cemA, cemA, rpl32-ccsA and ycf1) presented in this study will presumably be useful for resolving the many unclear phylogenetic relationships between Hylotelephium species and between Hylotelephium and Orostachys, respectively. The results of the phylogenetic analysis of this study do not resolve the unclear relationships of the Hylotelephium species, so additional studies are needed. Furthermore, the results supported the taxonomic position of S. taquetii, which was treated as a synonym of H. viridescens in previous studies, as an independent taxon.

Supporting information

S1 Fig. Comparison of nine Hylotelephium chloroplast genome structures using the MAUVE program.

https://doi.org/10.1371/journal.pone.0292056.s001

(JPG)

S1 Table. Information for sample collection sites, voucher specimens, sequencing results and genome assembly.

https://doi.org/10.1371/journal.pone.0292056.s002

(XLSX)

S2 Table. The list and GenBank accession numbers used for phylogenetic analyses in this study.

https://doi.org/10.1371/journal.pone.0292056.s003

(XLSX)

S3 Table. List of genes in the chloroplast genomes of nine Hylotelephium species.

https://doi.org/10.1371/journal.pone.0292056.s004

(DOCX)

S4 Table. Codon usage bias and relative synonymous codon usage (RSCU).

https://doi.org/10.1371/journal.pone.0292056.s005

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S5 Table. Pi values of four genetic regions, including corebarcode regions or molecular markers used in previous studies.

https://doi.org/10.1371/journal.pone.0292056.s006

(XLSX)

Citation: An S-M, Kim B-Y, Kang H, Lee H-R, Lee Y-B, Park Y-J, et al. (2023) The complete chloroplast genome sequences of six Hylotelephium species: Comparative genomic analysis and phylogenetic relationships. PLoS ONE 18(10): e0292056. https://doi.org/10.1371/journal.pone.0292056

About the Authors:

Sung-Mo An

Contributed equally to this work with: Sung-Mo An, Bo-Yun Kim

Roles: Data curation, Investigation, Validation, Writing – original draft

Affiliation: Department of Biological Science, Sangji University, Wonju, South Korea

Bo-Yun Kim

Contributed equally to this work with: Sung-Mo An, Bo-Yun Kim

Roles: Data curation, Formal analysis, Software, Visualization, Writing – original draft

Affiliation: Plant Resources Division, National Institute of Biological Resources, Incheon, South Korea

Halam Kang

Roles: Formal analysis, Investigation, Software, Visualization

Affiliation: Department of Biological Science, Sangji University, Wonju, South Korea

Ha-Rim Lee

Roles: Formal analysis, Investigation, Software, Visualization

Affiliation: Department of Biological Science, Sangji University, Wonju, South Korea

Yoo-Bin Lee

Roles: Formal analysis, Investigation, Visualization

Affiliation: Department of Biological Science, Sangji University, Wonju, South Korea

Yoo-Jung Park

Roles: Conceptualization, Formal analysis, Investigation, Visualization

Affiliation: Department of Biological Science, Sangji University, Wonju, South Korea

Kyeong-Sik Cheon

Roles: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing – original draft, Writing – review & editing

E-mail: [email protected] (KSC); [email protected] (KAK)

Affiliation: Department of Biological Science, Sangji University, Wonju, South Korea

ORICD: https://orcid.org/0000-0002-2182-7379

Kyung-Ah Kim

Roles: Conceptualization, Formal analysis, Investigation, Software, Visualization, Writing – original draft, Writing – review & editing

E-mail: [email protected] (KSC); [email protected] (KAK)

Affiliation: Environmental Research Institute, Kangwon National University, Chuncheon, South Korea

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