2.1 Genome assembly and coverage depth analysis

We downloaded the short reads of all three Blumea species from the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) available under project numbers PRJNA438407 and PRJNA522689. The whole genome shotgun data of B. oxyodonta and B. balsamifera were generated using BGISEQ-500 with short reads of 100 bp from a single end (Liu et al., 2019), whereas the genome of B. tenella was sequenced using Illumina True-Seq with short reads of 250 bp. Data accession numbers and quantity are presented in Table 1.

To assemble the plastome, we first used the BWA-MEM algorithm with default parameters (Li & Durbin, 2009) and mapped all reads for each species to Aster hersileoides C.K. Schneid., Tagetes erecta L., Ambrosia trifida L., Bidens torta Sherff, and Artemisia ordosica Krasch. This approach was used to avoid contamination of the nuclear and mitochondrial origin reads. The plastomes were de novo assembled by Velvet v.1.2.10 (Zerbino & Birney, 2008) integrated in Geneious R8.1 (Kearse et al., 2012) using extracted reads. All generated contigs of Velvet were combined in a specific order using the de novo assembly option of Geneious R8.1 (Kearse et al., 2012). This helped us to assemble the plastome of the Blumea species in a single contig from total contigs generated by Velvet. The single contig of the Blumea species was assembled from 5–10 long contigs without any gap. The LSC, IR, and SSC regions were defined based on the manual inspection of scaffolds. The integrity of the assembled plastome was validated, and coverage depth analysis was performed by mapping the original reads of each genome to their respective genome using BWA-MEM (Li & Durbin, 2009). We did not predict any gap in the assembled plastome, which further confirmed the high quality of the assembled plastome. However, through mapping of short reads we avoid some nucleotide mismatches in the genomes.

Plastomes were annotated using GeSeq (Tillich et al., 2017), whereas transfer RNAs (tRNAs) were confirmed based on ARAGORN v.1.2.38 (Laslett & Canback, 2004) and tRNAscan-SE v.2.0.5 (Chan & Lowe, 2019). The annotations of protein-coding genes were further confirmed by comparison with other homologue genes in the public database. The five-column tab-delimited file was generated from annotated genomes using GB2Sequin (Lehwark & Greiner, 2019) and was submitted to NCBI GenBank under specific accession numbers (Table 1). The circular map of the Blumea plastome was drawn using Chloroplot to show genome organization and gene content (Zheng et al., 2020).

2.2 Reannotations of plastomes and comparative genomics among the species of the subfamily Asteroideae

For comparative genomics among species of the subfamily Asteroideae with the species of the genus Blumea, we retrieved 26 other species of the subfamily Asteroideae from the NCBI (Table 2). These species were selected to cover all the main tribes of Asteroideae, including Anthemideae, Astereae, Gnaphalieae, the Heliantheae alliance, and Senecioneae, whereas the species of Blumea cover the tribe Inuleae. For a good comparison, we first reannotated all the 26 species using an approach similar to that used for the annotation of Blumea species as previous studies showed certain errors exist in the annotation of the plastome available in a public database (Amiryousefi et al., 2018a). After that, we compared genome features including the size of the complete plastome, LSC, SSC, and IR along with the gene and intron content. The arrangement of genes in the genomes was determined using Mauve (Darling et al., 2004), whereas contraction and expansion of IRs were visualized using IRscope (Amiryousefi et al., 2018b).

2.3 Comparison of codon usage, amino acid frequency, microsatellites, and oligonucleotide repeats among the species of Blumea

The amino acid frequency and relative synonymous codon usage (RSCU) were determined among the Blumea species using Geneious R8.1. Microsatellite repeats were determined in Blumea plastomes using MISA-web (Beier et al., 2017) with the following parameters: ≥10 for mononucleotide, ≥5 for dinucleotide, ≥4 for trinucleotide, and ≥3 for tetra-, penta-, and hexanucleotide repeats. REPuter (Kurtz et al., 2001) was used to determine oligonucleotide repeats with a minimum size of ≥30 bp and at least 90% similarity. The maximum repeat count was set to 500.

2.4 Identification of polymorphic loci in the genus Blumea

Multiple alignment was formed among plastomes of all the Blumea species, after removal of IRa, using MAFFT (multiple alignments using fast Fourier transform) (Katoh & Standley, 2013). First, we manually checked for small inversions and removed them from the alignment to avoid false results. The intergenic spacer regions, intronic regions, and protein-coding regions were extracted from the alignment in Geneious R8.1 (Kearse et al., 2012) and visualized in DnaSP v.6 to determine the nucleotide diversity of each region (Rozas et al., 2017). The rates of transition and transversion substitutions were also determined from the pairwise alignment of MAFFT using B. balsamifera as a reference genome for B. oxyodonta and B. tenella (Katoh & Standley, 2013).

2.5 Phylogeny in the subfamily Asteroideae

We retrieved 95 species of the subfamily Asteroideae to include species of all the seven main tribes from the NCBI (Table S1) and used them in the inference of phylogeny along with the three species of Blumea. Sonchus acaulis Dum. Cours. was also retrieved from the NCBI and used as an out-group from the subfamily Cichorioideae of the family Asteraceae. The plastome sequence of Chrysanthemoides incana (Burm.f.) Norl. was extracted from Sequence Read Archive of NCBI under accession number SRR9119075 by mapping to Ambrosia trifida following previous approach (Henriquez et al., 2014) to include it as representative of tribe Calenduleae. The complete plastome of all species, after removal of IRb, was aligned using MAFFT. The phylogeny was inferred based on the alignment of 98,453 sites, after removal of indels, using IQ-TREE2 and related programs (Hoang et al., 2018; Kalyaanamoorthy et al., 2017; Nguyen et al., 2015). The IQ-TREE2 analyses were based on searching for the best maximum likelihood (ML) tree under the edge-unlinked partition model (Lopez et al., 2002) and a nonpartitioned dataset with general heterogeneous evolution on a single topology (GHOST) (Crotty et al., 2020). Branch supports values were estimated using the ultrafast bootstrap approximation approach with 10,000 bootstrap replicates (Hoang et al., 2018). The tree structure was drawn and improved by using Interactive Tree Of Life version 4 (Letunic & Bork, 2019).

3.1 Correction of annotations

The initial annotations of plastomes of the subfamily Asteroideae showed variations in both gene and intron content. The plastomes of all species showed similar features after the reannotation of all 26 species of the subfamily Asteroideae. We found various types of errors in annotations related to extra annotations of some genes, and missing annotations for tRNA, protein-coding genes, introns, and pseudogenes. The most common errors were linked to the genes which contained introns (Table S2).

3.2 Plastome organization and features of the genus Blumea and the subfamily Asteroideae

The plastomes were obtained with high average coverage depth ranging from 98× to 130× (Table 1). The three genomes of Blumea showed high similarities in genome size. The size of complete plastomes ranged from 150,829 bp (B. tenella) to 151,176 bp (B. balsamifera), LSC from 82,478 bp (B. tenella) to 82,746 bp (B. balsamifera), SSC from 18,438 bp (B. oxyodonta) to 18,466 bp (B. balsamifera), and IR from 24,907 bp (B. oxyodonta) to 24,982 bp (B. balsamifera) (Table 2). The plastome of every species contained 113 unique genes, including 29 tRNA, 4 ribosomal RNA (rRNA), and 80 protein-coding genes. We found 18 duplicated genes in the IR, including 7 tRNA, 4 rRNA, and 7 protein-coding genes (including rps12, a trans-spliced gene). The guanine-cytosine (GC) content showed high similarities among the three species throughout the plastome structure and genetic features (Table 2). The ycf1 gene also left a truncated copy at the junction of IR/SSC along with a functional copy. The genetic organization of the plastomes in Blumea species was shown as a circular map using Chloroplot (Figure 1).

The species of Asteroideae included in the analysis showed similar genetic features and the same number of unique genes. However, duplication of trnF-GAA was detected in Xanthium sibiricum. The pseudogene of rps19 was not observed in the species of Blumea, whereas other species of the subfamily Asteroideae also carry a pseudogene along with a functional copy of rps19 at the junction of IRa/LSC. Mauve-based analysis showed similarity in gene arrangement and gene content. The rearrangement of genes was not observed in the 29 analyzed species of Asteroideae (Figure 2). The size of complete plastomes ranged from 149,752 bp (Ismelia carinata (Schousb.) Sch. Bip.) to 153,318 bp (Symphyotrichum subulatum), LSC from 82,290 bp (I. carinata (Schousb.) Sch. Bip.) to 85,238 bp (S. subulatum), SSC from 17,768 bp (Dendrosenecio cheranganiensis (Cotton & Blakelock) E.B. Knox) to 18,488 bp (Anaphalis sinica Hance), and IR from 24,522 bp (Leucanthemum virgatum (Desr.) Clos) to 25,413 bp (Marshallia obovata) (Table 2). In addition, the average GC content of the complete plastome was found to be 37%–37.7%, of LSC 34.8%–35.8%, of SSC 30.4%–35.5%, and of IRs 42.7%–43.2%.

Comparative analysis of LSC/IR and SSC/IR junctions showed high similarities in the Blumea species and the species of the subfamily Asteroideae (Figure 3). At the LSC/IRb junction (JLB), rps19 exists entirely in the LSC region in the Blumea species and X. sibiricum, whereas in all other species rps19 starts from the IRb and enters LSC leaving a pseudo-copy of the rps19 gene at JLA (IRa/LSC). The rpl2 gene exists completely in the IR regions away from JLB and JLA. At the SSC/IRa junction (JSA), ndhF exists entirely in the SSC region, whereas ycf1 started in IR and ended in SSC, as shown at the JSBA (IRb/SSC) and JSA junctions. Hence, a pseudogene of ycf1 remains at JSA. At the junction of JLA, the trnH gene was found in all species (Figure 3). These data revealed a high resemblance of IR expansion and contraction among species of the subfamily Asteroideae.

3.3 Relative synonymous codon usage and amino acid frequency

RSCU and amino acid frequency revealed high similarities among species of Blumea. RSCU analysis showed high encoding efficacy of the codon that contained A/T at 3′ with an RSCU ≥1 compared with codons ending with C/G at 3′ with an RSCU <1 (Table S3). We found leucine to be the most frequent amino acid, whereas cysteine was the rarest (Table S4).

3.4 Analysis of substitutions and indels

We analyzed the substitution types of the complete plastome and the extent of substitutions and indels in three central plastome regions. We observed a great extent of transversion substitutions relative to transition substitutions and found a transition-to-transversion substitution ratio of 0.84–0.87 (Table 3). Most of the substitutions were found in the LSC, followed by SSC and IR (Table 3). Similar distributions were observed for indels. Most indels existed in LSC, followed by SSC and IR (Table 4).

3.5 Analysis of microsatellites and oligonucleotide repeats

We found 55–60 microsatellites among three Blumea species. Most of the microsatellites were made up of A/T motifs. Most of the repeats were located in LSC instead of IR and SSC regions (Figure 4a). The mononucleotide microsatellites were most abundant, followed by tetranucleotides (Figure 4b, Table S5). The analysis of oligonucleotide repeats by REPuter detected 75 oligonucleotide repeats in all three species. The highest number of repeats was found in B. oxyodonta (30), followed by B. tenella (26) and B. balsamifera (19). Most of the repeats were present in LSC, followed by IR, regions. Moreover, some repeats were also shared between the LSC, SSC, and IR regions (Figure 4c). Forward repeats showed an abundance relative to other types of repeats (Figure 4d). The intergenic spacer regions contained more repeats than the intronic and coding regions (Figure 4e). Most of the repeats were between 30 bp and 34 bp in size (Figure 4f). Details about oligonucleotide repeats are provided in Table S6.

3.6 Identification of polymorphic loci

We recorded the highest average polymorphism for intergenic spacer regions (0.0121) as compared with intronic regions (0.0096) or protein-coding sequences (0.0047). The polymorphism of all regions is shown in Figure 5. We ignored loci <200 bp and selected 10 polymorphic regions with nucleotide diversity >0.02, of which 6 belonged to intergenic spacer regions, 1 to intronic, 1 to protein-coding, and 1 to both intergenic spacer and coding regions. The 730 bp region of ycf1 was selected instead of the complete gene using the oligonucleotide repeat as a proxy. The chosen part showed a nucleotide diversity of 0.0252 and contained 28 substitutions events with zero missing data. A similar approach was used for ndhF-rpl32, selecting an 841 bp region, which had a nucleotide diversity of 0.0206 and contained 26 substitutions. The selected regions may act as suitable and cost-effective markers (Table 5).

3.7 Phylogenetic inference of the species of the genus Blumea with 95 other species

The species of the genus Blumea lay on the same node and share a node with a high bootstrapping support of 100 with Pluchea indica. Our result was based on sequences of the complete plastome and showed the placement of Blumea in the tribe Inuleae. Moreover, the phylogenetic relationship was also described between the seven tribes of the subfamily Asteroideae (Figure 6). Our phylogenetic inference showed that the Heliantheae alliance is the most recently diverged tribe of the subfamily Asteroideae, which forms a common node with the tribe Inuleae. The tribe Astereae was closely related to Anthemideae, whereas Gnaphalieae forms the first branching node of these two tribes. The tribe Calenduleae roots Gnaphalieae, which is rooted finally by Senecioneae. Hence, the species of the tribe Senecioneae lie in the first branching node of the subfamily Asteroideae in our phylogeny.

4.1 Plastome comparison of Blumea and Asteroideae

The plastome of the three Blumea species and the other 26 species of the subfamily Asteroideae showed high similarities in genome structure, gene organization, and genetic content without any inversion linked to the rearrangement of genes. Previous studies of angiosperms have also shown conserved plastomes in various plant lineages such as Solanaceae, Malvaceae, and Araceae in which the same gene content and gene order were previously reported (Abdullah, Henriquez, Mehmood, et al., 2020; Abdullah, Henriquez, Mehmood, Shahzadi, et al., 2020; Abdullah, Mehmood, et al., 2020; Amiryousefi et al., 2018a; Mehmood, Abdullah, Shahzadi, et al., 2020). The slow rate of evolution and typically conservative nature of the plastome is linked to various molecular mechanisms such as the organization of plastid genes in operons, uniparental inheritance (maternal or paternal), the presence of an active repair mechanism, and rarity of plastid fusion (Wicke et al., 2011). Nevertheless, a rearranged plastome and loss of certain genes are reported in various plant lineages such as the ferns, Pinaceae, and Cyperaceae (Daniell et al., 2016; Lee et al., 2020; Poczai & Hyvönen, 2011; Wicke et al., 2011). Hence, the plastomes of Asteroideae avoid the rearrangements by a certain mechanism. The GC content of the plastomes was also similar to previous Asteraceae genomes and of other plant lineages, and high GC content was observed in IRs, which might be due to the presence of rRNAs, as they have a GC content of up to 55% (Amiryousefi et al., 2018a; Jung et al., 2021; Kim et al., 2019; Lin et al., 2019; Poczai & Hyvönen, 2017).

We found various types of errors in the annotations of 26 species of Asteroideae. This shows that the reported genomes have certain errors in annotations as stated previously in a detailed study of the family Solanaceae (Amiryousefi et al., 2018a) and the comparative genomics of the two species of Malvaceae (Abdullah, Waseem, et al., 2020). The gene content was found to be the same in all the species of Asteroideae after correction of annotations, which is also in agreement with gene features in the previously reported plastome of other subfamilies such as Cichorioideae, Pertyoideae, and Carduoideae (Jung et al., 2021; Kim et al., 2019; Lin et al., 2019). Hence, these data showed the highly conservative plastomes of Asteraceae. If we considered the previously reported annotations accurately without repeating the annotations, large variations were evident in the analyzed species. Hence, based on these observations, together with a previous report on the Solanaceae family (Amiryousefi et al., 2018a), we suggest a correction of annotations before comparative genomics to ensure accurate data regarding the plastome structure, genetic content, intron content, and for various other analyses related to the study of plastome evolution.

The expansion and contraction of IRs showed much similarity among the species of the subfamily Asteroideae. This result agrees with previous studies of other angiosperms such as Malvaceae (Abdullah, Mehmood, et al., 2020) and Solanaceae (Amiryousefi et al., 2018a). The pseudogene of ycf1 originated at the junction of IR. The origination of the pseudogene due to IR expansion and contraction is expected and observed in other angiosperms (Abdullah, Henriquez, Mehmood, Carlsen, et al., 2020; Iram et al., 2019; Mehmood, Abdullah, Ubaid, et al., 2020). Previous studies have shown a resemblance at the junctions of the plastome in closely related species (Liu et al., 2018). A similar phenomenon can be seen in the closely related species of Blumea and the subfamily Asteroideae. Detailed studies of the family Araceae do not support this suggestion, however, instead showing a high level of variation at junctions of the plastome even in closely related species (Abdullah, Henriquez, Mehmood, Carlsen, et al., 2020; Abdullah, Henriquez, Mehmood, Shahzadi, et al., 2020). The expansion and contraction of IRs also led to duplication of single-copy genes (genes that travel from SSC or LSC to IRs become duplicated) or conversion of otherwise duplicated genes to single-copy genes (genes that move from IRs to LSC or SSC become single copy) (Zhu et al., 2016). This traveling of the gene also affects the rate of mutations; mostly, the genes that travel from LSC or SSC to IRs showed a low rate of evolution or vice versa (Abdullah, Henriquez, Mehmood, Carlsen, et al., 2020; Zhu et al., 2016). The high similarities in junctions also indicate the presence of the same genes in all the species, and the total number of genes does not vary due to IR expansion and contraction.

4.2 Repeat analysis and utilization of oligonucleotide repeats as proxy to identify polymorphic loci

Microsatellites are very important for the study of population genetics. Besides hexanucleotide, we detected mononucleotide, dinucleotide, trinucleotide, tetranucleotide, and pentanucleotide microsatellites in which mononucleotide repeats were abundant, followed by tetranucleotide repeats. A similar pattern of repeats was observed in other Asteraceae (Sablok et al., 2019). Most of the nucleotides were made up of A/T motifs instead of C/G motifs. This might be due to the A/T-rich plastome structure, as observed in other angiosperms (Iram et al., 2019; Mehmood, Abdullah, Shahzadi, et al., 2020). The identified microsatellites in the current study may be helpful in population genetic studies of Blumea.

Oligonucleotide repeats exist widely in the plastome (Abdullah, Mehmood, et al., 2021; Ahmed et al., 2012, 2013). These repeats play a role in generating mutations and have been suggested as a proxy to identify mutational hotspots (Abdullah, Mehmood, et al., 2020, 2021; Ahmed et al., 2012). Abdullah, Mehmood, et al. (2020) recently reported the co-occurrence of up to 90% of repeats with substitutions, whereas 36%–91% co-occurrence was recorded at the genus level. In the current study, we identified 10 highly polymorphic loci. Among these, five loci belong to the regions where repeats are present, including rps16-trnQ and ycf1, which showed the highest incidence of polymorphisms. Here, our findings support the use of repeats as a proxy, and this approach may also be helpful for the identification of suitable polymorphic loci for phylogenetic inference of other taxonomically complex genera. This approach is promising since the plastome of a single species can be used to identify polymorphic regions. Repeated coding regions and IR regions need to be avoided, however, due to the purifying selection pressure of protein-coding genes (Henriquez et al., 2020b) and the fact that copy-dependent repair mechanisms (Zhu et al., 2016) lead to low rates of mutation.

4.3 Suitable polymorphic loci for resolving phylogenetic discrepancies of Blumea

Regions of the plastome showed different polymorphisms, and certain regions are more predisposed to mutations (Abdullah, Mehmood, et al., 2020; Henriquez et al., 2020b; Mehmood, Abdullah, Ubaid, et al., 2020; Shahzadi et al., 2020). All regions are therefore not equally important for phylogenetic inference and barcoding of plant species (Daniell et al., 2016; Li et al., 2014). Pornpongrungrueng et al. (2007) used trnH-psbA and trnL-F of plastome along with ITS1 and ITS2 from the nuclear genome to resolve the phylogeny of Blumea. The regions of plastome showed very low polymorphism. The ITS region showed speedy evolution and its sequences generated a lot of missing data when aligned due to a high rate of insertion and deletion events. Hence, the phylogenetic tree drawn for the ITS region and concatenated sequences of plastome and ITS regions cannot resolve interspecific relationships and showed low bootstrapping support for various nodes. Hence, the authors suggested the use of highly polymorphic markers with broad sampling to resolve the phylogeny. Later, Pornpongrungrueng et al. (2009) again attempted to resolve the phylogeny of Blumea. They also validated the 5S-NTS region from the nuclear genome along with trnH-psbH, trnL-F, and ITS. The 5S-NTS showed speedy evolution and performed worse than the ITS and chloroplast regions. According to the authors, several distinct paralogous sequences within an individual may be the main reason for the low phylogenetic signal of 5S-NTS. The authors also could not resolve the phylogeny of several species, such as Blumea lacera (Burm.f.) DC., B. oxyodonta, Blumea megacephala (Randeria) C.T. Chang & C.H. Yu ex Y. Ling, Blumea mollis (D. Don) Merr., and Blumea saxatilis Zoll. ex Zoll. & Moritzi. Moreover, at the species level, several relationships were observed with low bootstrap / without bootstrap support. Hence, to resolve the phylogeny, the identification of suitable polymorphic loci was suggested by the authors. The complete plastome was suggested for barcoding and phylogenetic inference (Li et al., 2014), but the high cost hinders its use. Therefore, identifying species-specific suitable polymorphic loci can provide a quality resource for the phylogenetic inference and barcoding of plant species (Ahmed et al., 2013; Li et al., 2014). Here, we identified 10 polymorphic loci that are different and more polymorphic than previously employed loci (trnH-psbA and trnL-F) (Pornpongrungrueng et al., 2007; Zhang et al., 2019) of the plastome. Our identified loci also included intergenic spacer regions along with coding and intronic regions. Our approach agrees with recent studies in which intergenic spacer regions were also suggested at the low taxonomic level for phylogenetic inference in Bignonieae of the family Bignoniaceae and the genus Artemisia of Asteraceae (Shahzadi et al., 2020; Thode et al., 2020). Our identified polymorphic loci showed low polymorphism compared with ITS regions. Still, the generation of no/low missing data of our identified polymorphic loci relative to the ITS makes these loci appropriate for phylogenetic inference and barcoding of Blumea species. Moreover, the ITS regions show low amplification success during polymerase chain reaction and possible contamination with fungus sequences, rendering the downstream processes costly and time-consuming (Li et al., 2014). The species-specific markers of the plastome show high amplification success and are not contaminated with fungus sequences, being found to be authentic, robust, and cost-effective in recent studies (Abdullah, Henriquez, Croat, et al., 2021; Abdullah, Henriquez, Mehmood, et al., 2021; Ahmed, 2014; Ahmed et al., 2013; Li et al., 2014; Nguyen et al., 2018). Hence, our identified polymorphic loci may also be authentic, robust, and cost-effective for the barcoding and phylogenetic inference of the genus Blumea.

4.4 Conflicting signals in the phylogeny of Asteroideae

Phylogenetic analysis of 98 species of the subfamily Asteroideae based on the complete plastome shows that the genus Blumea lies in the tribe Inuleae and is closely related to the genus Pluchea. The same relationship was previously observed based on the ndhF gene of plastid (Anderberg et al., 2005). The tribal-level phylogenetic relationship of our study is similar to the previous studies of the subfamily Asteroideae reported based on plastome sequences (Fu et al., 2016; Panero & Crozier, 2016). However, similar to previous studies, the phylogenetic analysis in our recent study was based on the complete plastome, which also conflicts with the phylogenetic inference of the family performed based on the nuclear genome (Mandel et al., 2019). Mandel et al. (2019) stated a sister relationship between “Senecioneae and Anthemideae” and “Astereae and Gnaphalieae.” However, our study and previous reports (Fu et al., 2016; Panero & Crozier, 2016; Watson et al., 2020) based on plastome data show the sister relationship between “Astereae and Anthemideae,” while Gnaphalieae roots these two tribes which is rooted by Calenduleae, whereas Senecioneae presents at the base of Asteroideae. A recent phylogeny based on nuclear data showed a sister relationship between Astereae and Gnaphalieae rooted as Calenduleae, Anthemideae, and then Senecioneae (Watson et al., 2020). These results of nuclear phylogeny are also similar to the previous report (Huang et al., 2016) in which authors showed the same relationships based on transcriptomic data. However, they have not included the species of Anthemideae. Watson et al. (2020) demonstrated evidence for four reticulate events: three intratribal (within Astereae, Anthemideae, and Senecioneae) and one intertribal (between Anthemideae and Gnaphalieae). They suggested that ancient reticulating events within the five tribes may be possible, which further confounds any conclusion about the existing evolutionary history of these five tribes. Asteroideae diverged recently (~37 mya) (Mandel et al., 2019) during the Mid-Eocene Climatic Optimum, when the temperature of the globe decreased continuously, or somewhat earlier (~45 mya) (Panero & Crozier, 2016). Hence, the accelerated rate of diversification may be responsible for the conflicting signals due to the loss of some of the earliest lineages of the Asteroideae (Watson et al., 2020). Vargas et al. (2017) also observed conflicting signals for the data set of nuclear, plastome, and mitochondrial genomes in Diplostephium and in aligned genera of Astereae. They provided evidence for reticulate evolution in events of rapid diversification in the analyzed species of Astereae and suggested that the phylogeny based on plastome and mitochondria sequences contradict with nuclear due to uniparental inheritance of these genomes. In the current study, the conflicting signal among the aforementioned tribes may also be due to reticulate evolution in events of rapid diversification. Moreover, the uniparental inheritance of the plastome may also confound phylogenetic inference, which might require further investigation.

In conclusion, our study provides insight into plastome structure evolution of the genus Blumea and the subfamily Asteroideae. The identified polymorphic loci were linked to the location of oligonucleotide repeats and confirm the role of repeats as a proxy for the identification of polymorphic loci. The 10 identified loci may facilitate barcoding and phylogenetic inference of the genus Blumea. However, some practical validation may be required of the identified loci. Our study shows the conflicting signals between plastome and nuclear phylogeny at tribal levels, which also requires further investigation.