Sow Thistle Chloroplast Genomes: Insights into the Plastome Evolution and Relationship of Two Weedy Species, Sonchus asper and Sonchus oleraceus (Asteraceae)

genes G C A T T A C G G C A T Article Sow Thistle Chloroplast Genomes: Insights into the Plastome Evolution and Relationship of Two Weedy Species, Sonchus asper and Sonchus oleraceus (Asteraceae) Myong-Suk Cho 1, Jin Hyeong Kim 1, Chang-Seok Kim 2, JoséA. Mejías 3and Seung-Chul Kim 1,* 1Department of Biological Sciences, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Korea; [email protected] (M.-S.C.); [email protected] (J.H.K.) 2Highland Agriculture Research Institute, National Institute of Agricultural Sciences, Rural Development Administration (RDA), Gangwon-do 25342, Korea; [email protected] 3Department of Plant Biology and Ecology, Universidad de Sevilla, 41004 Seville, Spain; [email protected] *Correspondence: [email protected]; Tel.: +82-31-299-4499 Received: 20 September 2019; Accepted: 1 November 2019; Published: 1 November 2019   Abstract: Prickly sow thistle, Sonchus asper (L.) Hill, and common sow thistle, Sonchus oleraceus L., are noxious weeds. Probably originating from the Mediterranean region, they have become widespread species. They share similar morphology and are closely related. However, they differ in their chromosome numbers and the precise relationship between them remains uncertain. Understanding their chloroplast genome structure and evolution is an important initial step toward determining their phylogenetic relationships and analyzing accelerating plant invasion processes on a global scale. We assembled four accessions of chloroplast genomes (two S. asper and two S. oleraceus) by the next generation sequencing approach and conducted comparative genomic analyses. All the chloroplast genomes were highly conserved. Their sizes ranged from 151,808 to 151,849 bp, containing 130 genes including 87 coding genes, 6 rRNA genes, and 37 tRNA genes. Phylogenetic analysis based on the whole chloroplast genome sequences showed that S. asper shares a recent common ancestor with S. oleraceus and suggested its likely involvement in a possible amphidiploid origin of S. oleraceus. In total, 79 simple sequence repeats and highly variable regions were identified as the potential chloroplast markers to determine genetic variation and colonization patterns of Sonchus species. Keywords: Sonchus asper (L.) Hill; Sonchus oleraceus L.; chloroplast genome evolution; comparative genomic analyses; hybridization; invasive species 1. Introduction Prickly (or spiny) sow thistle, Sonchus asper (L.) Hill, and common (or annual) sow thistle, S. oleraceus L., are two well-known worldwide noxious weeds. The seeds can germinate throughout the year over a broad range of temperatures [ 1 , 2 ]. These species are considered to be particularly troublesome weeds across the grain growing regions because they have allelopathic potential to function as interference competition even in weed communities [ 3 ] and are prolific seed producers (up to 25,000 seeds per single plant in a fallow); the seeds possess pappi, which helps wind-mediated dispersal [ 4 ]. Moreover, the evolution of herbicide resistance found that populations of these species threatens the efficiency of weed control. For example, several populations of S. oleraceus have been reported as resistant to acetolactate synthase (ALS) inhibiting herbicides including chlorsulfuron (Group B) and glyphosate herbicides (Group M) in Australia [ 5 , 6 ]. In addition to being troublesome Genes 2019,10, 881; doi:10.3390/genes10110881 www.mdpi.com/journal/genes Genes 2019,10, 881 2 of 15 weeds worldwide, the tender leaves of common sow thistles are consumed as salads and potherbs and the extracts of both sow thistles have been used as medicinal herbs in Brazil, New Zealand, and China [7,8]. Prickly and common sow thistles belong to the subgenus Sonchus of the genus Sonchus L. (Asteraceae); the genus is a member of the subtribe Hyoseridinae Less. (formerly known as Sonchinae sensu K. Bremer) [ 9 , 10 ]. The current, redefined genus Sonchus in its wider circumscription is comprised of ca. 95 species, which are widely distributed globally. They mainly grow in native ranges from most of the Old World (Eurasia and Africa) and mid-Atlantic islands, avoiding the coldest and driest regions, and extend to Australia/New Zealand and the Southeast Pacific islands of the Juan Fern á ndez and Desventuradas [ 9 , 10 ]. Almost half of the species diversity is found in the oceanic insular ranges, which are examples of spectacular adaptive radiation (Macaronesia in the Atlantic and the Juan Fern á ndez and Desventuradas in the Pacific) [ 11 – 13 ]. Some of the most common species, both prickly and common sow thistles, have been introduced to America, being particularly widespread in northern temperate regions. The genus seems to have originated in the western Mediterranean region, including areas of western Morocco with a Mediterranean-type climate. The geographical origins of weedy sow thistles have not been formulated with any certainty, but they are also believed to have originated in the Mediterranean region, becoming one of the cosmopolitan species on planet Earth [ 14 ]. Both species are widespread and prevalent in Europe, Asia, Africa, and have also been introduced to America and Australia [ 1 , 11 , 15 ]. They are pioneer species, invade mainly open and disturbed areas, and occur in a wide variety of environments like cultivated fields, gardens, roadsides, pastures, and waste places. Although some diagnostic features are relevant for species identification, S. asper and S. oleraceus can sometimes be confused morphologically. Both species are erect herbs, annual or biennial weeds, and have yellow florets and hollow and smooth stems. They can be either low-growing rosettes or upright in their growth form and overwinter as achenes, but fall-germinated plants may overwinter as rosettes in milder climates. Despite their similar morphology and phenology, S. asper and S. oleraceus can be distinguished primarily based on the characteristics of their leaves and fruits (Figure 1). The leaves of S. asper are deep, shiny, and green, rarely divided, thick, and have very prickly leaf margins, while those of S. oleraceus are flatter, pinnatifid, and weakly spinous. In addition, the leaf base auricles of S. asper are often recurved or curled and rounded, while those of S. oleraceus are deltate to lanceolate, almost straight, and acute. However, the characteristic of leaf base and degree of prickly dentate leaf margin can be variable in both species, often confusing species identification based on these traits only. The most consistent reproductive diagnostic features are in their fruits: the cypselae of S. oleraceus are slightly compressed, with rounded margins, (2)3(4) ribs per side, two of them furrowed lengthwise, and transversely rugulose or tuberculate across and between ribs, while those of S. asper are strongly compressed, more or less winged, 3 ribs per side, and smooth across and between the ribs [ 4 ] (Figure 1). One important karyotype characteristic is their different ploidy levels. S. asper has been represented uniformly by diploid plants (somatic chromosome number 2n=18) in Europe, USA, and Canada, while S. oleraceus has typically been reported with a chromosome number of 2n=32 in Eurasia, Africa, and North America [ 11 , 16 – 20 ]. However, descending aneuploid (2n=16; from Eurasia) and tetraploid (2n=36; from North Dakota, USA) chromosome counts also have been rarely reported [ 16 ], although the taxonomic identity of the plant material studied remains unclear. Stebbins et al. [ 21 ] proposed an amphidiploid origin for S. oleraceus (n=16, 2 n=32), which has combined S. asper (n=9, 2n=18) and S. tenerrimus L. (n=7, 2n=14). Certainly, the morphological characteristics of S. oleraceus reflect a remarkable middle ground between the two putative parental species. It is also plausible that the hybrid origin of S. oleraceus increased ecological amplitude and evolutionary success via (re-)combining two different genomes. Previous molecular phylogenetic studies [ 13 , 22 – 24 ] provided evidence that S. asper and S. oleraceus are closely related to each other, but failed reciprocal monophyly due to poor resolution. It is of great interest to determine the origin of amphidiploid S. oleraceus based on extensive sampling strategy and rigorous molecular and cytological tools. Genes 2019,10, 881 3 of 15 Figure 1. Pictures of Sonchus asper (prickly sow thistle) and Sonchus oleraceus (common sow thistle). Sonchus asper ( A ). plant; ( C ). achene; ( E , G ). rounded leaf base auricle and prickly leaf margin. Sonchus oleraceus ( B ). plant; ( D ). achene; ( F , H ). pointed leaf base auricle and lobed leaf margin. Photo credits: (C,D) (Jose Mejías); (A,B,E–H) (Jin Hyeong Kim and Myong-Suk Cho). Chloroplasts in plant cells serve as metabolic centers and encode many key proteins that are involved in photosynthesis and other metabolic processes, primarily participating in photosynthesis, transcription, and translation [ 25 ]. Chloroplast (cp) sequence polymorphisms have been extensively used as useful genetic markers at wide ranges of taxonomic levels in plants. They have provided valuable insights into phylogenetic relationships and the origin and evolution of the crop species [ 26 ] or introduced/invasive species [ 27 – 30 ] that are facilitated by maternal inheritance of cp genomes. However, earlier phylogenetic analyses utilizing the partial cpDNA sequences from several regions often resulted in limited sequence variation owing to highly conservative genome evolution, particularly for closely related and recently radiated groups of species [ 31 ]. The advent of high-throughput sequencing technologies of next-generation sequencing (NGS) has helped to reveal considerable Genes 2019,10, 881 4 of 15 genome-wide variations in terms of sequences and structures of entire chloroplast genomes. The benefits of genome-wide data have increased phylogenetic resolution and significantly enhanced our understanding of plant evolution and diversity in the field of chloroplast genetics and genomics [ 25 ]. The chloroplast phylogeny based on several coding and noncoding cpDNA regions in previous studies has not provided enough resolution to elucidate the phylogenetic relationship among weedy Sonchus species as well as the origin of S. oleraceus. In both of the cpDNA and nuclear DNA phylogenies, S. oleraceus was more closely related to S. asper than to S. tenerrimus, even though the precise relationship between them was not clear [ 13 , 22 – 24 ]. Taking cpDNA phylogeny and maternal inheritance into consideration, it could be hypothesized that S. asper contributed as the maternal parent in the origin of amphidiploid S. oleraceus. To test this hypothesis and to better understand the evolutionary relationships of two weedy sow thistle species, we characterized four accessions of complete chloroplast genomes (two from S. asper and two from S. oleraceus) and conducted the comparative analyses of their whole chloroplast genomes. All but one accession of S. oleraceus was collected from the probable origin of diversity in the Mediterranean region. Considering their global distribution, one accession of S. oleraceus was collected from Dok-do Island in Korea and one more previously published accession of S. oleraceus from Australia (GenBank accession number MG878405) was also included in the comparative analyses. 2. Materials and Methods 2.1. Material Preparation, DNA Extraction, Genome Sequencing, and Annotation The silica-gel dried leaves of four prickly and common sow thistles which were sampled from natural habitats in Spain and Korea were used as sources of DNA. Two accessions of S. asper were sampled from Seville (VIL-1) and Huelva (MAR-1) in Spain, while S. oleraceus was sampled from Huelva (MAR-1) in Spain and Dok-do Island in the East Sea, Korea. Total genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s protocol. An Illumina paired-end (PE) genomic library was constructed and sequenced using the Illumina HiSeq platform (Illumina, Inc., San Diego, Ca, USA) at Macrogen Corporation (Seoul, Korea). The sequence reads of chloroplast genomes were assembled by the de novo genomic assembler, Velvet 1.2.10 [ 32 ] at coverages ranging from 789x to 1354x. Annotation was performed using the Dual Organellar GenoMe Annotator [ 33 ], ARAGORN v1.2.36 [ 34 ], and RNAmmer 1.2 Server [ 35 ]. Using Geneious v8.1.6 (Biomatters Ltd., Auckland, New Zealand), the draft annotation was inspected and corrected manually, performing blast search by comparison with homologous genes in Lactuca sativa (DQ383816), S. oleraceus (MG878405), S. canariensis (NC042381), S. acaulis (NC042382), and S. webbii (NC042383) from the GenBank database at the National Center for Biotechnology Information (NCBI) as references. The complete plastome sequences were registered in GenBank under the accession numbers MH908962 (S. oleraceus from Korea, Collection # Son0-uDS2), MK371006 (S. oleraceus from Spain, Population # MAR-1), MK371015 (S. asper from Spain, Population # VIL-1), and MK371016 (S. asper from Spain, Population # MAR-1). In addition, the raw HiSeq reads were deposited in the Short Read Archive (SRA) at NCBI under Bioproject ID PRJNA0577793 for MH908962 and PRJNA578572 for MK371006, MK371015, and MK371016. OGDRAW [ 36 ] was used to draw circular chloroplast genome maps (Figure 2). Genes 2019,10, 881 5 of 15 Figure 2. Merged gene map of four weedy Sonchus chloroplast genomes of two accessions each of S. asper and S. oleraceus that were sequenced in this study. The genes inside and outside of the circle are transcribed in the clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. The thick lines indicate the extent of the inverted repeats (IR A and IR B ) that separate the genomes into small single copy (SSC) and large single copy (LSC) regions. Large inversion and smaller inversion nested within the large inversion that is unique in Asteraceae are indicated with black lines outside the gene map. 2.2. Repeat Sequence Analysis Two types of repeat sequences were identified in the five chloroplast genomes of S. asper and S. oleraceus including previously reported S. oleraceus from Australia (GenBank accession number MG878405). REPuter [ 37 ] was used to detect the various types of repetitive sequences of the five Sonchus chloroplast genomes. Search parameters were set to: maximum computed repeats =50, minimum repeat size =8 bp, and hamming distance =0. Simple sequence repeats (SSRs) were identified using MISA web (http://pgrc.ipk-gatersleben.de/misa/) with search parameters of 1–15 (unit size-minimum repeats, i.e., mono-nucleotide motifs with 15 minimum numbers of repetition), 2-5, 3-3, 4-3, 5-3, and 6-3 with 0 interruption (maximum difference for two SSRs). 2.3. Identification of Highly Divergent Regions The five weedy Sonchus chloroplast genomes were compared to the reference genome of S. webbii at the entire chloroplast genomic level using DnaSP [ 38 ] and mVISTA [ 39 ]. S. webbii is a herbaceous perennial unlike other woody Sonchus species (S. acaulis and S. canariensis) in the Canary Islands and is Genes 2019,10, 881 6 of 15 sister to the clade containing five accessions of weedy Sonchus species. Overall sequence divergence was estimated for the five weedy Sonchus chloroplast genomes that were aligned and compared to the reference genome using the LAGAN alignment mode [ 40 ] in mVISTA. Nucleotide diversity (Pi) was calculated using the sliding window analysis (window length =1000 bp and step size =200 bp excluding sites with alignment gaps) to detect the most divergent regions (i.e., mutation hotspots) among the five weedy Sonchus genomes in DnaSP. The borders of large single copy (LSC), small single copy (SSC), and inverted repeats (IRs) were compared with the results of DnaSP and mVISTA. 2.4. Phylogenetic Analysis To investigate the taxonomic position and phylogenetic relationship of the newly sequenced accessions of S. asper and S. oleraceus, 26 complete chloroplast sequences representing major lineages of the family Asteraceae were obtained from GenBank. In total, full sequences of 30 chloroplast genomes were aligned using MAFFT v.7 [ 41 ]. A maximum likelihood tree was produced based on the relationships of whole chloroplast genomes by IQ-TREE [ 42 ] with 1000 replicate bootstrap (BS) analyses. The best fit evolutionary model was chosen as TVM +F+I+G4, which was scored according to the Bayesian information criterion (BIC) scores and weights by using ModelFinder [ 43 ] implemented in IQ-TREE. 3. Results and Discussion 3.1. Comparative Genomic Analysis of Five Weedy Sonchus Chloroplast Genomes in Content, Order, and Organization Despite morphological and cytological differences between two weedy Sonchus species (S. asper and S. oleraceus), gene content and arrangement were found to be identical in five chloroplast genomes, displaying 99.98% pairwise similarity in sequences (Table 1). The total length of five cp genomes ranged from 151,808 (S. oleraceus from Spain and Australia) to 151,849 (the two samples of S. asper from Spain and S. oleraceus from Dok-do, Korea) base pairs (bp) and consisted of four typical regions: LSC, SSC, and a pair of inverted repeat regions (IR A and IR B ). One large inversion with a size of 22.8 kb and a second smaller inversion of 3.3 kb, nested within the large inversion, were found in all five chloroplast genomes (Figure 2). These inversions are unique to Asteraceae and present in most of the family, but are absent in the species of the basal subfamily Barnadesioideae [ 44 , 45 ]. The overall guanine-cytosine (GC) content of each chloroplast genome was 37.6%, with LSC, SSC, and IR regions having 35.8%, 31.4–31.5%, and 43.1% GC contents, respectively. Each of the five cp genomes contained 130 genes, including 80 protein-coding genes (plus seven duplicated in IR), three rRNA genes (all duplicated in IR), and 30 tRNA genes (plus seven duplicated in IR). Eighteen genes contained introns, including seven tRNA genes. Three genes of clpP, rps12, and ycf3 exhibited two introns. The trnK tRNA gene harbored the largest intron, which contained the matK gene in between. In total, 17 genes were duplicated in the IR regions, including seven tRNAs, three rRNAs, and seven protein genes. The trans-splicing gene rps12, consisting of three exons, was located in the LSC region for exon 1, but exon 2 and exon 3 of the gene were imbedded in the IR regions. Part of ycf1 and rps19, for which we did not annotate in the four weedy Sonchus cp genomes that were sequenced in this study, were duplicated in the IR A region, forming pseudogenes. A pseudo ycf1 gene in IR was extended to the SSC region and overlapped with the near ndhF gene (Figures 2and 3; Tables 1and 2). The genomic features of five weedy Sonchus genomes were nearly identical to congeneric species in the woody Sonchus alliance in the Macaronesian Islands (Atlantic Ocean), S. canariensis (NC042381), S. acaulis (NC042382), and S. webbii (NC042383) in gene content and overall GC rate [ 46 ]. The cp genomes of other Asteraceae species were also highly conservative in gene order and content with minor variations in the gene prediction of several genes (e.g., ycf68 protein coding gene, 4.5 rRNA genes and pseudogenes), even though they represent morphologically and genetically diverse tribes of Asteraceae belonging to Anthemideae, Cardueae, Cichoroieae, Eupatorieae, Heliantheae, Millerieae, and Senecioneae [47–49]. Genes 2019,10, 881 7 of 15 Table 1. Summary of the complete chloroplast genome characteristics of five accessions of two weedy species of Sonchus:S. asper and S. oleraceus. Characteristics S. asper S. oleraceus GenBank Accession Number/Locality ID MK371015 Spain/VIL-1 MK371016 Spain/MAR-1 MG878405 Australia MK371006 Spain/MAR-1 MH908962 Korea/Dokdo Total length (bp) 151,849 151,849 151,808 151,808 151,849 LSC size (bp) 84,156 84,156 84,157 84,113 84,156 SSC size (bp) 24,738 24,738 24,707 24,739 24,738 IR size (bp) 18,217 18,217 18,237 18,217 18,217 Number of genes 130 (17 duplicated in IR) 130 (17 duplicated in IR) 130 (17 duplicated in IR) 130 (17 duplicated in IR) 130 (17 duplicated in IR) Number of protein coding genes 80 (+7 in IR) 80 (+7 in IR) 80 (+7 in IR) 80 (+7 in IR) 80 (+7 in IR) Number of tRNA genes 30 (+7 in IR) 30 (+7 in IR) 30 (+7 in IR) 30 (+7 in IR) 30 (+7 in IR) Number of rRNA genes 3 (+3 in IR) 3 (+3 in IR) 3 (+3 in IR) 3 (+3 in IR) 3 (+3 in IR) GC content of whole genome (frequency/%) 57,133/37.6% 57,134/37.6% 57,130/37.6% 57,130/37.6% 57,134/37.6% cpDNA: chloroplast DNA; LSC: large single copy region; SSC: small single copy region; IR: inverted repeat; GC: guanine-cytosine. Table 2. Genes present in the complete chloroplast genomes of five accessions of two weedy species of Sonchus:S. asper and S. oleraceus. Category Gene Name Photosystem I psaA, psaB, psaC, psaI, psaJ, ycf3b,ycf4 Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ Cytochrome b6/f complex petA, petBa,petD, petG, petL, petN Cytochrome C synthesis ccsA ATP synthase atpA, atpB, atpE, atpFa,atpH, atpI RuBisCO rbcL NADH oxidoreductase NdhAa,ndhBa,c,ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK Large subunit ribosomal proteins rpl2a,c,rpl14, rpl16a,rpl20, rpl22, rpl23c,rpl32, rpl33, rpl36 Small subunit ribosomal proteins rps2, rps3, rps4, rps7c,rps8, rps11, rps12 b,c,d,rps14, rps15, rps16 a, rps18, rps19 RNA polymerase rpoA, rpoB, rpoC1 a,rpoC2 Translation initiation factor infA Others accD, cemA, clpPb,matK Unknown function genes (conserved reading frames) ycf1, ycf 2c,ycf15 c Ribosomal RNAs rrn5c,rrn16 c,rrn23 c Transfer RNAs trnA-UGC a,c,trnC-GCA, trnC-ACA a,trnD-GUC, trnE-UUC, trnE-UUC a,c,trnF-GAA, trnG-GCC, trnG-UCC a,trnH-GUG, trnK-UUU a,trnL-CAAc,trnL-UAA a,trnL-UAG, trnM-CAU, trnM-CAU, trnM-CAU c,trnN-GUU c,trnP-UGG, trnQ-UUG, trnR-ACG c,trnR-UCU, trnS-CGA a,trnS-UGA, trnS-GGA, trnT-GGU, trnT-UGU, trnV-GAC c,trnW-CCA, trnY-GUA aGene containing a single intron. bGene containing two introns. cTwo gene copies in IRs. dTrans-splicing gene Genes 2019,10, 881 8 of 15 Figure 3. Comparison of the border positions of the large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions among five weedy Sonchus chloroplast genomes representing S. asper and S. oleraceus. Gene names are indicated in the boxes and their lengths in the corresponding regions are displayed above the boxes. Ψindicates a pseudogene. 3.2. SSRs and Large Repeat Sequences Repeat sequences are considered to play an important role in genome recombination and rearrangement [ 50 , 51 ]. Particularly, SSRs, which represent a unique type of tandemly repeated genomic DNA sequence, have high polymorphisms due to large variations in motifs and number of repetitions. Because of the high level of polymorphisms and genome-wide distribution, they have been considered as powerful tools to measure genetic diversity and address population genetic issues at the level of interand intra-specific variations, such as gene flow, parentage, and population structure [ 52 ]. We found that all the five cp genomes contained the same numbers and distribution patterns of repeated sequences in each cp genome. There were 79 SSRs detected by MISA [ 53 ] based on search parameters set for 1–15 (mono-nucleotide motifs with 15 minimum numbers of repetition), 2–5, 3–3, 4–3, 5–3, and 6–3. The majority of the SSRs were tri-nucleotide motifs (66 SSRs, 84%). Remarkably, there were low proportions of other SSR types, i.e., three mono-nucleotide SSRs (4%), four di-nucleotide SSRs (5%), and six tetra-nucleotide SSRs (7%) (Figure 4A). The abundance of tri-nucleotide SSRs was consistent with previous findings with similar parameter settings [ 54 ]; however, the frequency of mono-nucleotide SSRs was significantly lower because of the more stringent search parameter used in this study (i.e., minimum repeat of 15) than in previous studies (minimum repeat of 8 or 10) [ 47 , 54 ]. The most abundant repeat motif was “AAT/ATT” followed by “AAG/CTT” in all five genomes (Figure 4B, Table S1). Interestingly, SSRs were distributed most abundantly in the coding regions (57%), followed by intergenic regions (38%), however much lower numbers were distributed in the non-coding introns (5%) in each cp genome. The coding regions with the highest number of SSRs were ycf genes as shown in Cynara cardunculus (globe artichoke) and other Asteraceae species [ 47 ]. Gene ycf2 contained 10 SSRs (five duplicated in each IR) and ycf 1, three in SSC, emphasizing that these highly variable regions can be of specific interest to develop future cpSSR markers for phylogenetic studies of Sonchus species. Considering the quadripartite regional occupancy of SSRs, the IR and SSC regions were remarkably lower in overall SSR frequency compared with the LSC region: 19% from the SSC region and 10% from each of the two IR regions versus 61% from the LSC region (Table S2). Genes 2019,10, 881 9 of 15 Figure 4. Simple sequence repeat (SSR) number per distribution and repeat type of five accessions of weedy S. asper and S. oleraceus chloroplast genomes. ( A ) Variation in the numbers of SSRs detected in five chloroplast genomes of weedy S. asper and S. oleraceus. ( B ) Number of SSR motifs in different repeat motifs of each weedy Sonchus chloroplast genome. In the case of the large repeats, we found 49 pairs in each cp genome using the parameters of maximum computed repeats =50, minimum repeat size =8 bp, and hamming distance =0 by REPuter [ 37 ]. They contained 21 forward, 6 reverse, 1 complement, and 21 palindromic matches of repeats (Figure 5A). Similar results were reported in previous studies with the majority of repeats in forward (21) and palindromic (13) in other Asteraceae species, Taraxacum plastomes (dandelions) [ 48 ]. Most of these large repeats were present in the intergenic spacers, but a large proportion was found within the ycf gene as in Ambrosia trifida (giant ragweed) [ 54 ]. Lengths of 20–22 repeats were the most frequent (45%) followed by lengths of 23–26 repeats (37%), with repeats of 27–30 (6%) and >42 (12%) being quite rare (Figure 5B). Figure 5. Repeat numbers per repeat type and repeat length of five chloroplast genomes of two Sonchus species. ( A ) Variation in the distribution of forward, reverse, complement, and palindromic repeats in each chloroplast genomes of five weedy Sonchus genomes. ( B ) Number of different repeat lengths of each weedy Sonchus chloroplast genomes. 3.3. Sequence Divergence and Hotspots Nucleotide diversity among five weedy Sonchus cp genomes was estimated using DnaSP [ 38 ] with a sliding window analysis (window length =1000 bp and step size =200 bp excluding sites with alignment gaps). The divergence level was compared to the reference genome of S. webbii (Figure 6). Overall nucleotide diversity value (Pi) among Sonchus chloroplast genomes including the two closely related species, S. asper and S. oleraceus, was 0.00117 and ranged from 0 to 0.00807. The SSC region showed the highest nucleotide diversity (0.001629) among the regions of LSC, SSC, and IRs, while the