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Article

Comparative Chloroplast Genomes and Phylogenetic Relationships of True Mangrove Species Brownlowia tersa and Brownlowia argentata (Malvaceae)

by
Panthita Ruang-areerate
1,
Duangjai Sangsrakru
1,
Thippawan Yoocha
1,
Wasitthee Kongkachana
1,
Sonicha U-Thoomporn
1,
Onnitcha Prathip Na Thalang
2,
Pranom Chumriang
2,
Poonsri Wanthongchai
2,
Sithichoke Tangphatsornruang
1 and
Wirulda Pootakham
1,*
1
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand
2
Department of Marine and Coastal Resources, 120 The Government Complex, Chaengwatthana Rd., Thung Song Hong, Bangkok 10210, Thailand
*
Author to whom correspondence should be addressed.
Curr. Issues Mol. Biol. 2025, 47(2), 74; https://doi.org/10.3390/cimb47020074
Submission received: 19 December 2024 / Revised: 21 January 2025 / Accepted: 21 January 2025 / Published: 23 January 2025
(This article belongs to the Special Issue Functional Genomics and Comparative Genomics Analysis in Plants, 2nd Edition)
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Abstract

:
Brownlowia tersa and Brownlowia argentata are two true mangroves in the genus Brownlowia in Malvaceae, and they are a near-threatened and a data-deficient species, respectively. However, the genomic resources of Brownlowia have not been reported for studying their phylogeny and evolution. Here, we report the chloroplast genomes of B. tersa and B. argentata based on stLFR data that were 159,478 and 159,510 base pairs in length, respectively. Both chloroplast genomes contain 110 unique genes and one infA pseudogene. Sixty-eight RNA-editing sites were detected in 26 genes in B. argentata. A comparative analysis with related species showed similar genome sizes, genome structures, and gene contents as well as high sequence divergence in non-coding regions. Abundant SSRs and dispersed repeats were identified. Five hotspots, psbI-trnS, trnR-atpA, petD-rpoA, rpl16-rps3, and trnN-ndhF, were detected among four species in Brownlowioideae. One hotspot, rps14-psaB, was observed in the two Brownlowia species. Additionally, phylogenetic analysis supported that the Brownlowia species has a close relationship with Pentace triptera. Moreover, rpoC2 was a candidate gene for adaptive evolution in the Brownlowia species compared to P. triptera. Thus, these chloroplast genomes present valuable genomic resources for further evolutionary and phylogenetic studies of mangroves and plant species in Malvaceae.

1. Introduction

Malvaceae, commonly known as the mallows, is the largest family in Malvales, comprising a diverse group of flowering plants with at least 4225 species in nine subfamilies, namely Bombacoideae, Brownlowioideae, Byttnerioideae, Dombeyoideae, Grewioideae, Helicteroideae, Malvoideae, Sterculioideae, and Tilioideae [1,2]. In the subfamily Brownlowioideae, the genus Brownlowia contains approximately 30 species which are widely distributed in Southeast Asia [3,4,5]. Notably, two Brownlowia species, Brownlowia tersa (Dungun air or the Durian of the sea) and Brownlowia argentata (Dungun or Durian Laut), are classified as non-viviparous mangrove species, which grow in inland mangrove forests [3,4,5]. They are known to occur in Borneo and Peninsular Malaysia. B. tersa is distributed in Asia including Bangladesh, Brunei, Cambodia, India, Indonesia, Malaysia, Myanmar, Philippines, Singapore, and Thailand [5,6]. B. argentata is distributed in Brunei, Indonesia, Malaysia, Myanmar, Papua New Guinea, Philippines, Solomon Islands, and Thailand, and is extinct in Singapore [7]. The two Brownlowia species are clearly different in morphological characteristics. For example, B. tersa is a shrub that grows to 2/5 meters (m); leaves are narrow and lanceolate are up to 5 centimeters (cm) wide. In contrast, B. argentata is a tree up to 10 m; leaves are broad and ovate to 10 cm wide [3,4]. B. tersa is commonly used in traditional medicines for the treatment of several symptoms such as boils, diarrhea, dysentery, and wounds because it contains several bioactive compounds that serve for anti-inflammatory, antibacterial, antinociceptive, and antioxidant activities [5,8,9]. It is also used as fuel and materials for house construction in local areas. Based on the IUCN Red List, B. tersa and B. argentata are listed as a near-threatened (NT) and a data-deficient (DD) species, respectively [6,7,10]. B. tersa populations have been decreasing due to habitat loss from shrimp and fish farm construction and coastal development [6,11]; therefore, this species might be at a higher risk of extinction in the future.
Chloroplasts play essential roles in photosynthesis, carbon fixation, and various metabolic pathways in plants. They contain their own genomes with a typically quadripartite structure including a large single copy (LSC), a pair of inverted repeats (IR), and a small single copy (SSC). The size of chloroplast genomes has varied among plant species. In Malvaceae, chloroplast genome sizes range from 143 to 169 kilobases (kb) [12,13,14,15,16,17,18,19,20]. For the subfamily Brownlowioideae, only two species, Pentace triptera [16] and Diplodiscus trichospermus [18], have had their chloroplast genomes reported. The presence of variations in chloroplast genomes could serve as molecular markers for species identification, phylogenetic analysis, and adaptative evolution [21,22].
Advances in next-generation sequencing technologies have facilitated the exploration of chloroplast genomes in plant species [14,16,18,23,24,25,26]. Recently, single-tube long-fragment read (stLFR) is an efficient technology that enables sequencing from long DNA molecules using second-generation sequencing technology such as BGISEQ-500 and MGISEQ-2000 [27]. The stLFT data is a cost-effective replacement for long reads and could increase accuracy for de novo assembly to be near complete and smaller misassembled regions [27]. This technology has been used to complete a chloroplast genome of Calamus tetradactylus [26].
Recently, no study has focused on the genome sequence of Brownlowia species, which is important for studying evolution and species identification. To generate genetic resources for Brownlowia species and to reveal evolutionary relationships of Brownlowia and related species, we report the complete chloroplast genome and genetic variation of B. tersa and B. argentata based on stLFR data. Comparative chloroplast genomes between the two Brownlowia species and related non-mangrove and mangrove species were conducted. A phylogenetic tree among forty-one plant species within Malvaceae and three outgroups was performed. Moreover, genes under purifying selection or positive selection in the two Brownlowia species were identified to investigate whether genes related to adapt in environments of inland coastal areas. Therefore, these Brownlowia chloroplast genomes provide valuable insights into their genomic features, evolutionary phylogenetic relationships, and gene selective pressures in the subfamily Brownlowioideae in Malvaceae.

2. Materials and Methods

2.1. Plant Materials, DNA Extraction, and Sequencing

Young fresh leaves from a mature Brownlowia tersa (L.) Kosterm (N 8.280251, E 98.737898) and a mature Brownlowia argentata Kurz (N 9.233155, E 99.238231) were collected in 2023 from the mangrove forest in Ao Luek Noi District in Krabi Province and Phunphin District in Surat Thani Province, Thailand, respectively, under the supervision of Thailand’s Department of Marine and Coastal Resources. They were flash-frozen and maintained in liquid nitrogen until use. Genomic DNA in each sample was extracted from the young leaves using CTAB [28]. Purified DNA was used to generate stLFR sequencing library that was constructed following the protocol of the MGIEasy stLFR Library Prep Kit (MGI, Shenzhen, China), and sequenced on MGISEQ-2000RS (MGI, Shenzhen, China) in house.

2.2. Chloroplast Genome Assembly and Annotation

The stLFR reads of B. tersa and B. argentata were used for assembling the Brownlowia chloroplast genomes using GetOrganelle v.1.4.1 [29] based on a reference genome-based strategy with Diplodiscus trichospermus from the GenBank sequence database under accession number OP572286 following a pipeline in Figure S1. These Brownlowia chloroplast genomes were then annotated using GeSeq online tool v.2.03 [30]. The start–stop loci and intron–exon junctions of coding genes were manually examined by comparing them with the chloroplast genomes of D. trichospermus and other mangrove species. Apart from the coding genes, transfer RNAs (tRNAs) were identified using ARAGORN v1.2.38 [31] and tRNAscan-SE v2.0.3 [32] in the GeSeq software v.2.03. Circular chloroplast genomes were visualized using OGDRAW v1.3.1 [33]. Finally, the annotated chloroplast sequences of B. tersa and B. argentata were deposited in GenBank with accession numbers PP419968 and PP419969, respectively.

2.3. RNA Sequencing and RNA Editing Site Identification

For RNA sequencing, RNA from leaf tissues of the two Brownlowia species in the same sample collection was extracted following the protocol in Pootakham et al. (2021) [34]. Poly(A) mRNA was subsequently purified using the Dynabeads mRNA purification Kit (ThermoFisher Scientific, Waltham, Massachusetts, USA). The integrity of RNA was evaluated using the Fragment Analyzer System (Agilent, Santa Clara, CA, USA). Libraries were constructed in each sample with 200 ng of poly(A) mRNA using the MGIEasy RNA Library Prep Kit V3.0 (MGI Tech, Shenzhen, China) and were sequenced using the MGISEQ-2000RS Sequencing Flow Cell v3.0 (MGI Tech, Shenzhen, China). The short-read RNA sequences of B. tersa and B. argentata were obtained and deposited in sequence read archive (SRA) with SRR31035998 and SRR31066870, respectively.
To identify candidate RNA editing sites in all protein-coding genes of both Brownlowia species, the RNA sequences of each species were mapped to each chloroplast genome using Burrows–Wheeler aligner (BWA) v.0.7.17-r1188 with default parameters [35]. A sequence alignment map (SAM) file was converted to a binary alignment map (BAM) file using samtools v1.9 [36]. The index of BAM files was also created using samtools v1.9. To examine RNA-editing sites of the genes, the alignment results of BAM files were visualized using integrative genomics viewer (IGV) v2.17 [37]. RNA-editing sites where cytidine (C) to uridine (U) conversion events were present were identified with read coverage by at least 30 reads and editing events by at least 20%.

2.4. Chloroplast Genome Comparative Analysis

Chloroplast genome comparative analysis in eight plant species in Malvaceae including two Brownlowia species, two reported plant species in Brownlowioideae (Pentace triptera: BGT7001 and D. trichospermus: OP572286), two mangrove species in Sterculioideae (Heritiera fomes: OM022247 and Heritiera littoralis: NC_043923), and two mangrove associates in Malvoideae (Talipariti tiliaceum: MN533969 and Thespesia populnea: NC_048518) was performed using mVistra with the Shuffle-LAGAN mode [38]. The chloroplast sequence of P. triptera is available in DRYAD (https://datadryad.org/stash/share/LkbLwUlzW_GJ5rBDMHYZz69S19HzkNWY6fPOySH9tBQ, accessed on 2 May 2024) [16], whereas D. trichospermus is available in the GenBank database. The chloroplast genome of B. tersa was used as a reference for comparison. In addition, the junctions and borders of the IR regions among the eight plant species were visualized using the CPJSdraw software v1.0.0 [39].

2.5. Nucleotide Diversity Analysis

Two datasets were used to estimate nucleotide diversity (Pi). The first dataset was four species, B. tersa, B. argentata, P. triptera, and D. trichospermus. The second dataset was only two Brownlowia species. Both protein-coding genes and interspace regions of the two datasets were aligned in each region using MUSCLE. The nucleotide diversity values (Pi) in each aligned sequence region were calculated using DnaSP version 6.12.03 [40].

2.6. Analysis of Simple Sequence Repeats and Dispersed Repeats

Simple sequence repeats (SSRs) were identified among the four chloroplast genomes in Brownlowioideae, including B. tersa, B. argentata, P. triptera, and D. trichospermus, using MISA v2.1 [41]. The identified SSRs included mononucleotide repeats ≥ 10 bases, dinucleotides ≥ 10 bases (five repeats), trinucleotides ≥ 12 bases (four repeats), tetranucleotides ≥ 12 bases (three repeats), pentanucleotide ≥ 15 bases (three repeats), and hexanucleotides ≥ 18 bases (three repeats). Moreover, dispersed repeat sequences, including complement, forward, palindrome, and reverse, were identified using REPuter online tool (https://bibiserv.cebitec.uni-bielefeld.de/reputer/, accessed on 6 August 2024) [42].

2.7. Phylogenetic Analysis

To confirm the position of the Brownlowia species, a phylogenetic analysis of 44 plant species was performed using a maximum likelihood (ML) method based on 68 shared protein-coding genes (Table S1). The 44 plant species were four species in Brownlowioideae (B. argentata, B. tersa, P. triptera, and D. trichospermus), thirty-seven plant species in other subfamilies in Mavalceae, and three outgroup species (Hopea hainanensis: MN533970, Vatica mangachapoi: NC_041485, and Vativa odorata: MN533976). The 68 conserved genes include accD, atpA, atpB, atpE, atpF, atpH, atpI, ccsA, cemA, clpP, matK, ndhA, ndhB, ndhC, ndhD, ndhE, ndhG, ndhH, ndhI, ndhJ, petA, petB, petD, petG, petL, petN, psaB, psaC, psaI, psaJ, psbA, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, rbcL, rpl2, rpl14, rpl16, rpl20, rpl23, rpl32, rpl33, rpl36, rpoA, rpoB, rpoC1, rpoC2, rps2, rps3, rps4, rps7, rps8, rps11, rps12, rps14, rps15, rps18, ycf3, and ycf4. Each gene sequence was aligned using MUSCLE with default in MEGA X [43], and the aligned sequences were concatenated in each species. The GTR + I + G model as the best-fit model was identified using the best DNA/protein model tool in MEGA X. A maximum likelihood (ML) analysis was used to construct a phylogenetic tree using RAxML version 8.2.10 [44] with a GTRGAMMAI (GTR + I + G) model. Node support was estimated by performing 1000 bootstrap replicates. Finally, the phylogenetic tree was visualized using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 23 August 2024). In addition, all chloroplast genomes were blasted against with an infA gene of T. tiliaceum (LC605014) to check this gene in their chloroplast genomes. Then, the chloroplast infA gene loss and gain in plant species in Malvaceae were plotted onto clades of the phylogenetic tree.

2.8. Gene Selective Pressure Analysis

The sixty-eight shared chloroplast protein-coding genes were used to investigate selection pressures for the two Brownlowia species. Compared species pairs contained between each Brownlowia species and two terrestrial plant species in Brownlowioideae (P. triptera and D. trichopermus). Each protein-coding gene that was translated to amino acid sequences was aligned using MEGA X [43]. The ratios of the rate of non-synonymous substitutions (Ka) to the rate of synonymous substitutions (Ks) or Ka/Ks ratios of each gene in each species pair were calculated using KaKs-calculator v2.0 [45]. R with the heatmap function [46] was used to visualize the Ka/Ks ratios of genes in each species pair. Genes with Ka/Ks ratios greater than 1 were determined to be under position selection, whereas genes with Ka/Ks ratios less than 1 were determined to be under purifying selection. Notably, genes with Ka/Ks ratios ≥ 40 or not available (NA) were replaced and set to zero because they had extremely low Ks values or no substitution, respectively.
Furthermore, genes with Ka/Ks ratios > 1 were tested statistically with the Chi-square test to identify potential positive selection sites across lineages using the codeml program from PAML v4.9 [47] based on the branch–site model [48]. The Brownlowia species and others were set as a foreground and background branches, respectively.

3. Results

3.1. Characteristics of B. tersa and B. argentata Chloroplast Genomes

Two chloroplast genomes of Brownlowia were assembled based on stLFR reads. A total of 13.3 and 14.2 Gb stLFR raw reads were used for the chloroplast genome assemblies of B. tersa and B. argentata, respectively. The chloroplast genome sizes of B. tersa (159,478 bp) and B. argentata (159,510 bp) were similar, and GC contents were all 37.05% (Figure 1 and Table 1). They exhibited a conserved quadripartite structure, consisting of a large single copy (LSC: 88,394–88,435 bp, 34.89% GC content), a small single copy (SSC: 19,984–19,985 bp, 31.35–31.36% GC content), and two inverted repeats (IRs: 25,545–25,550, 43.01% GC content) (Table 1). Each chloroplast genome contained 130 genes, with 84 protein-coding genes, 8 ribosomal RNAs (rRNAs), 37 transfer RNAs (tRNAs), and 1 infA pseudogene. Among those genes, three genes (clpP, rps12 and ycf3) had two introns, and nine genes (atpF, ndhA, ndhB, petB, petD, rpoC1, rpl2, rpl16, and rps16) had one intron (Table 2). Notably, the rps12 gene was trans-spliced because of the location of the first exon at the LSC and the other two exons at the IRs (Figure 1). In total, 16 genes were duplicated in the IRs, including 5 protein-coding genes (ndhB, rpl2, rpl23, rps7, and ycf2), 4 rRNA genes (rrn4.5, rrn5, rrn16, and rrn23), and 7 tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC). One infA pseudogene was identified in the two Brownlowia species due to partial deletion (1–22 bp).

3.2. Comparative Chloroplast Genomes

Comparing chloroplast genomes between two Brownlowia species and six plant species in Malvacaea including two non-mangrove chloroplast genomes in Brownlowioideae (P. triptera and D. trichospermus) and four mangrove chloroplast genomes in Malvaceae (H. littoralis, H. fomes, T. populnea, and T. tiliaceum), chloroplast genomes, GC contents, and gene contents were relatively similar (Table 1 and Table S2). The size of the chloroplast genomes ranged from 158,570 (D. trichospermus) to 168,778 (H. littoralis) bp in length, and all had ~37% GC contents. They contained 128−132 genes, including 83−86 protein-coding genes, 8 rRNAs, 36−37 tRNAs, and 1 infA pseudogene.
The differences among the eight chloroplast genomes were also evaluated using mVISTA, with the annotated B. tersa chloroplast genome as a reference (Figure 2). The chloroplast genomes of the two Brownlowia species were highly conserved with non-mangrove species in Brownlowioideae (P. triptera and D. trichospermus) when compared with other mangroves species in Malvaceae. IR sequences in all chloroplast genomes had a low divergence level. High levels of divergence between the Brownlowia species and other species were mainly concentrated in non-coding regions of LSC and SSC regions such as trnH-psbA, petN-psbM, ycf3-trnS, trnT-trnF, trnT-psbD, ndhF-rpl32, and rpl32-trnL.
In addition, the comparison of the junctions of the LSC, SSC, IRa, and IRb among eight chloroplast genomes in Malvaceae are presented in Figure 3. The SSC orientation of B. tersa was designed as a reference. By aligning the genomes to the reference sequence, the SSC sequence in most species has a forward-read orientation, whereas P. triptera possesses a reverse orientation. Rpl2 and trnH genes were found at the IRa/LSC boundary in all chloroplast genomes. The rpl2 gene is located in the IRa region and the trnH gene is located in the LSC region, 0–2 bp from the IRa/LSC boundary. The other boundaries of the Heritiera species were different from those of the others due to their long IR regions. At the junction of LSC/IRb, rps19 was found in the Brownlowia species and other four species, P. triptera, D. trichospermus, T. tiliaceum, and T. populnea. It was located in the IRb region with 3 bp (T. tiliaceum and T. populnea), 6 bp (B. tersa, B. argentata and P. triptera) and 8 bp (D. trichospermus) interval after the LSC/IRb boundary. The ycf1 gene crossed the SSC and IR boundary with 33 bp (B. tersa and B. argentata), 36 bp (P. triptera), 78 bp (T. populnea), 109 bp (D. trichospermus) and 960 bp (T. tiliaceum) of the 3′ end of this gene in the IR region. The ndhF gene was located in the SSC region with a 39 bp (B. tersa and B. argentata), 51 bp (D. trichospermus), 56 bp (P. triptera), 175 bp (T. populnea), and 330 bp (T. tiliaceum) interval to the boundary of the IR and SSC. The rpl2 and trnN genes were located in IR regions near the LSC/IRs junction and the SSC/IRs junction in all species in Brownlowioideae and T. populnea, respectively.

3.3. RNA Editing Sites in Chloroplast Genes of Brownlowia

In the B. argentata chloroplast genome, there are 68 C-to-U sites in 26 protein-coding genes with 9, 56, and 3 sites at the first, second, and third codon positions, respectively (Table 3). The RNA-editing sites resulted in sixty-five nonsynonymous and three synonymous editing sites. A majority of editing events were observed in codon changes in the second position, including CCU to CUU and CCA to CUA (Pro to Leu), UCU to UUU (Ser to Phe), UCA to UUA and UCG to UUG (Ser to Leu), and ACG to AUG (Thr to Met). Approximately half of the amino acid changes from the RNA-editing events were Ser to Leu. In addition, the start codon of the chloroplast ndhD gene had the RNA editing event, an ACG codon (84%) to an ATG codon (16%). Of these editing events, 86% of the first-position edits consisted of His (CAC and CAU) to Tyr (UAC and UAU) codon changes. All the third-position editing sites were nonsynonymous UUC to UUU (Phe to Phe) and GUC to GUU (Val to Val). Exhibiting an editing efficiency of up to 80% was found in thirteen genes including atpA, atpE, atpI, psbZ, rps14, accD, psbJ, psbF, petL, clpP, pdbN, petB, and ndhD. Nevertheless, no RNA editing sites were detected in B. tersa based on the leaf transcriptome set in this study.

3.4. Simple Sequence Repeats and Dispersed Repeats

To identify genetic variation between Brownlowia species and two related species in Brownlowioideae, simple sequence repeats (SSRs) and dispersed repeats were examined (Figure 4, Tables S3 and S4). For SSRs, a total of 85, 83, 82, and 81 SSRs were identified in B. tersa, B. argentata, P. triptera, and D. trichospermus, respectively (Figure 4A and Table S3). The most abundant of SSR types were mononucleotide repeats, accounting for 74.22–79.17%. Remaining SSRs included 9–10 tetranucleotide repeats (8.91–10.30%), 3–8 dinucleotide repeats (3.09–7.92%), 3–8 trinucleotide repeats (3.12–8.24%), 2–4 pentanucleotide repeats (1.98–4.12%), and 1 hexanucleotide repeat in P. triptera. These SSRs were mostly located in the LSC region rather than other regions (Figure 4B). For example, 66, 8, and 11 SSRs were discovered within the LSC, SSC, and IR regions of B. tersa, respectively. The A/T tandem repeat was most frequent (70.10–76.04%) (Figure 4C). Notably, a few unique A/T tandem repeats were detected between B. tersa and B. argentata (Table S3). The second most abundant SSRs were AAAT/ATTT, ranging from five in B. tersa, B. argentata, and P. triptera to six in D. trichospermus. It is noteworthy that AACT/AGTT, AGAT/ATCT, and AAAAT/ATTTT repeats were detected in three species, B. tersa, B. argentata, and P. triptera, whereas AATG/ATTC and AAAAG/CTTTT repeats were detected in only D. trichospermus. The AAAGAT/ATCTTT repeat was detected only in P. triptera.
Furthermore, dispersed repeats (F: forward; R: reverse; P: palindromic; and C: complement) were detected in four chloroplast genomes in Brownlowioideae (Figure 4D and Table S4). Each chloroplast genome contained 49 dispersed repeats. Although the numbers of dispersed repeats were similar, they were different in the four species. There are 17, 17, 13, and 16 forward repeats, 10, 11, 14, and 13 reverse repeats, 22, 21, 19, and 20 palindromic repeats, and 0, 0, 3, and 0 complement repeats for B. tersa, B. argentata, P. triptera, and D. trichospermus, respectively. Among the species, palindromic repeat (38.78–44.90%) was the most common type, followed by forward repeat (26.53–34.69%), and reverse repeat (20.41–28.57%). Complement repeat was detected in only P. triptera. Almost all repeats were in the range of 21–30 bp in length (Figure 4E). The number of dispersed repeats with a unit length of <20 bp in D. trichospermus (13, 26.53%) was higher than those in other species (1–2, 2%). Most dispersed repeats were located in the LSC and IR regions (Figure 4F). In the SSC region, dispersed repeats were found in only D. trichospermus.

3.5. Nucleotide Diversity

In the two datasets, including four species in Brownlowioideae (B. tersa, B. argentata, P. triptera, and D. trichospermus) and two Brownlowia species, the nucleotide diversity (Pi) of 68 protein-coding genes and 123 interspace regions was estimated (Figure 5). The Pi value of the protein-coding genes ranged from 0 to 0.011 in the four species and 0 to 0.010 in the two Brownlowia species (Figure 5A). Most Pi values in the two Brownlowia species were 0. The Pi value of psbM in the LSC region of Brownlowia (Pi = 0.010) showed the highest value, which was higher than the Pi value of psbM in the four species (Pi = 0.005). In addition, the Pi value of the interspace regions ranged from 0 to 0.048 in the four species and 0 to 0.039 in the two Brownlowia species (Figure 5B). Most Pi values in the two Brownlowia species also were 0. Interestingly, the Pi value of rps14-psaB (Pi = 0.039) in two Brownlowia species was higher than the Pi value of rps14-psaB in the four species (0.020). In the four species, there were five interspace regions, psbI-trnS, trnR-atpA, petD-rpoA, rpl16-rps3, and trnN-ndhF, that passed the threshold Pi > 0.25. Four interspace regions, psbI-trnS, trnR-atpA, petD-rpoA, and rpl16-rps3, were located in the LSC region, while trnN-ndhF was located in the IR region. The highest Pi value (Pi = 0.048) was found in the petD-rpoA region, and the second-highest Pi value (Pi = 0.044) was found in the rpl16-rps3 region.

3.6. Phylogenetic Relationships

A maximum likelihood (ML) was constructed based on 68 conserved protein-coding genes among 41 plant species in nine subfamilies in Malvaceae and three outgroup species. (Figure 6). There were nine clades in Malvaceae including representative nine subfamilies. The ML tree showed the existence of two major clades, Malvadendrina and Byttneriina. The Malvadendrina clade contained seven subfamilies including Bombacoideae, Brownlowioideae, Dombeyoideae, Helicteroideae, Malvoideae, Sterculioideae, and Tilioideae. The Byttneriina contained two subfamilies including Byttnerioideae and Grewioideae. In Brownlowioideae, the phylogenetic tree shows that two Brownlowia species and two terrestrial plant species (P. triptera and D. trichospermus) are monophyletic. B. tersa and B. argentata were clustered together and P. triptera was a sister group, with 100% bootstrap support. These Brownlowia species are closely related to the terrestrial species P. triptera than D. trichospermus. In addition, Brownlowioideae formed a sister clade with the clade of Dombeyoideae and Tilioideae, with 88% bootstrap support. Moreover, chloroplast infA gene loss and gain in Malvaceae were examined. The infA pseudogene was found in the Brownlowia species and other most plant species in Malvaceae, while the infA gain was observed in the genus Althaea, Hibiscus, Talipariti, and Tilia.

3.7. Selective Pressure Genes

The Ka/Ks ratios of 68 shared protein-coding genes were calculated between pairwise species of four species in Brownlowioideae (BA—B. argentata; BT—B. tersa; PT—P. triptera; and DT—D. trichospermus) (Figure 7 and Table S5). D. trichospermus was assumed to be an ancestor of Brownlowioideae based on the result of our phylogenetic tree. Among species pairs, the Ka/Ks ratios in the chloroplast genes were around 0.07, indicating strong purifying selections. In particular, rpoC2 had Ka/Ks ratios > 1.0 in two pairwise species, BA-PT (the Ka/Ks ratio = 2.32) and BT-PT (1.11), implying possible positive selection in the Brownlowia species compared to P. triptera. To evaluate positive sites based on Bayes Empirical Bayes (BEB) analysis, rpoC2 (ω > 1) was positively selected in five amino acid sites/changes (80L: Leu (PT) to Phe (BA and BT); 594M: Met to Ile; 697Q: Gln to Pro; 894H: His to Arg; and 1324R: Arg to Trp).

4. Discussion

4.1. Chloroplast Genome Structure and Evolution in the Genus Brownlowia

In this study, the chloroplast genomes of B. tersa and B. argentata were successfully assembled into 159.5 kb based on stLFR data. The chloroplast genome size of the two Brownlowia species is consistent with related land plant species in Brownlowioideae such as Pentace triptera (159.4 kb) and Diplodiscus trichospermus (158.6 kb) [16,18]. It was slightly smaller than the chloroplast genome size of other true mangroves (Heritiera angustata, Heritiera fomes, and Heritiera littoralis) and mangrove associates (Thespesia populnea and Hibiscus/Talipariti tiliaceum) in Malvaceae as well as mangroves in Combretaceae, Euphorbiaceae, and Rhizophoraceae, whose size varied from 160.1 kb to 168.8 kb in length [12,15,17,19,24,25]. On the contrary, the size of Brownlowia genomes was larger than the size of mangrove chloroplast genomes in Acanthaceae (148.3–150.3 kb) [15,24,49]. The chloroplast genome sizes generally varied among different plant species due to the contraction, expansion, and loss of IR regions.
Comparative analyses of the two Brownlowia species and representatives of two other genera of Brownlowioideae (Pentace and Diplodiscus) revealed a similar pattern of structure and gene order as well as an equal number of genes. However, high levels of divergence between Brownlowia and Diplodiscus were observed in several intergenic spacer regions, which is concordance with the phylogenetic result in this study showing that Diplodiscus was less closely related species than Pentace. IR regions had lower divergence and were more conserved compared to LSC and SSC regions, which is consistent with the result of the nucleotide diversity analysis.
Among plant species, the contraction and expansion of IRs is a common event leading to gene gain and loss in IR regions [50,51]. In our study, complete rps19 and ycf1 genes among four species in Brownlowioideae were found adjacent to the junctions, with slight length variations. For instance, the full ycf1 gene crossed the SSC/IRa with short length variations (33 bp in Brownlowia to 109 bp in Diplodiscus) of the 3′ end of this gene in the IRa region, indicating a slight contraction of the IR region in the Brownlowia species compared to other species in Malvaceae. The contraction and expansion of IR regions are main factors for the genome length and number of genes in the chloroplast genomes of each plant species during evolution.

4.2. RNA Editing in Brownlowia Species

Chloroplast RNA editing events in higher plants mainly occur through the conversion of cytidine to uridine (C to U) [52]. RNA editing is essential for various plant developmental processes and evolutionary adaptation such as plant embryogenesis, growth, adaptation to environmental changes, and signal transduction [53,54]. In the present study, no RNA editing site was detected in B. tersa based on one transcriptome dataset. The absence of RNA editing can cause abnormal plant development such as etiolation and yellow leaves [55,56,57]. Therefore, more leaf RNA data should be generated to evaluate whether RNA editing sites exist in the B. tersa chloroplast genome. In B. argentata, most RNA-editing sites were found at the second codon positions, followed by the first and third positions, which is consistent with several terrestrial plant species [58,59,60] and mangrove species in Rhizophoraceae [61]. Our editing events at the second codon positions are affected by amino acid changes, especially Ser-to-Leu, Pro-to-Leu, and Ser-to-Phe. These events were found in several plant species [58,59,60,61]. Notably, ndhB involving cyclic electron flow around photosystem I had the largest number of RNA editing sites in Brownlowia species and other plant species [60,61,62]. In particular, the start codon of ndhD in Brownlowia species was affected by an RNA-editing event from ACG to AUG with 16% editing efficiency, which is consistent with ACG codon rather than AUG codon at a translation initiation site in other plant species [63,64,65]. In fact, the ACG codon could be the initiation codon for translation in ndhD in some species [66]. The ACG-to-AUG editing varies and depends on developmental and environmental factors [63]. In addition, some RNA editing sites were unique to plant species, suggesting adaptive evolution. For example, RNA-editing sites in several genes such as atpA, ndhA, rpl20, rpl23, rpoC1, and rps1 were observed in B. argentata, but not in other mangrove species in Rhizophoraceae [61]. These genes might be candidate genes for studying the adaptive evolution in mangrove Brownlowia species.

4.3. SSRs, Dispersed Repeats, and Nucleotide Diversity

To identify differences in the chloroplast genomes of two Brownlowia species and two land plant species in Brownlowioideae, SSRs and repeats were examined. Mononucleotides have the most SSR repeats that are common in plants [24,25,67]. Different SSRs may be produced as genetic markers for identifying Brownlowia species and related species. Dispersed repeats were observed with the highest number of palindromes, followed by forward and reverse types, which are in concordance with several plants [68,69]. On the contrary, forward repeat is a major type of dispersed repeat in many mangrove chloroplast genomes [24,25,49]. Indeed, both palindromic and forward repeats accounted for a high proportion in chloroplast genomes compared to reverse and complementary repeats [24,25,49,68,69]. The high peak of Pi diversity among four species in Brownlowioideae was observed in non-coding regions (five hotspot regions). High Pi values are generally found in spacer regions between genes [70]. These genetic variations are important as resources of molecular markers and evolution studies.

4.4. Phylogenetic Relationships of Malvaceae

Phylogenetic reconstructions based on 68 chloroplast protein-coding genes among 41 species in nine subfamilies in Malvaceae and three outgroup species have provided insights into the evolutionary history of Brownlowia species. Our ML tree and several previously published trees in Brownlowioideae had highly congruent topologies [2,71,72,73]. For example, the phylogenetic relationships based on a chloroplast ndhF sequence, Brownlowia, Pentace, Berrya, and Carpodiptera were a monophyletic group in the clade Brownlowioideae [71]. The phylogenetic relationships with four chloroplast genes, atpB, matK, ndhF, and rbcL, also showed the placement of Brownlowia together with Pentace and Berrya in the clade Brownlowioideae [72]. The Bayesian phylogenetic tree with eight molecular markers (six chloroplast genes: rbcL, atpB, trnK-matK, trnH-psbA, trnL-trnF, and ndhF; one mitochondrial matR gene; and one nuclear internal transcribed spacer (ITS) sequence) agreed that Brownlowia was closely related to Pentace, followed by Diplodiscus, Jarandersonia, Berrya, Carpodiptera and Chritiana in the clade Brownlowioideae [73]. Thus, the relationships of Brownlowia species employing chloroplast genes are definitively resolved with the best-supported topology.
Resolving relationships among subfamilies in Malvaceae are critical for evaluating their genetic evolution. There were numerous phylogenetic analyses in Malvaceae [2,16,18,62,71,72,73,74]. The phylogenetic positions of subfamilies with Malvaceae were ambiguous. However, the subfamilial relationships in Malvaceae have been resolved recently using whole chloroplast genes or chloroplast genomes [16,18]. Our phylogenetic tree confirmed two major clades of Malvaceae, Byttneriina, and Malvadendrina. The clade of Byttneriina containing two subfamilies, Byttnerioideae and Grewioideae, were sister to the remainder clades of Malvadendrina, with seven subfamilies, including Brownlowioideae, Bombacoideae, Dombeyoideae, Helicterroideae, Malvoideae, Sterculioideae, and Tiliodeae [2,16,18,62,71,72,73,74]. Helicterroideae occupied the earliest branching position within Malvadendrina based on chloroplast genes [16,18,62,71,73]. In contrast, Dombeyoideae based on an ITS sequence [72] or Tilioideae based on chloroplast atpB and rbcL sequences [2] were located at the base of Malvadendrina. The present study showed the clade of Brownlowioideae that was clearly separated from the clade of Tilioideae with a high support value. Dombeyoideae and Tilioideae were grouped together and were sister to Brownlowioideae, which is in concordance with recently previous phylogenetic relationships in Malvaceae based on numerous chloroplast genes [16,18]. Malvoideae and Bombacoideae (Malvatheca) formed a close clade with high confidence (100% bootstrap value), which is in concordance with previous studies [2,16,18,71]. In our newly phylogenetic tree, Sterculioideae and Malvatheca were grouped together with a 54% bootstrap value (no high support). It is consistent with some studies [72,73], while it is contrast to the resolved phylogenetic trees that Sterculioideae formed a clade sister to Malvatheca and Brownlowioideae, Tilioideae, and Dombeyoideae [16,18]. In fact, there was still difficulty over taxonomic divisions based on morphological features within these families [75]. For example, androecial traits shared by Sterculioideae and Malvatheca, such as sessile or subsessile anthers and the lack of staminodes, support their sister group relationships [72]. These results from our phylogenetic relationships and others reveal that numerous chloroplast gene sequences could resolve phylogenetic relationships among subfamilies in Malvaceae that led to reliable phylogenetic positions. Many genetic variations and morphological data should be compared and combined to evaluate phylogenetic relationships among subfamilies in Malvaceae.
Among protein-coding genes, the infA pseudogene was found in Brownlowia and most plants in Malvaceae, except Althaea, Hibiscus, Talipariti, and Tilia species that have a complete infA gene [12]. The infA gene could transfer from chloroplast to nuclear during angiosperm evolution [76].

4.5. Selective Pressure

Challenging environments may impose selective pressure on chloroplast genes that are involved in photosynthesis adaptation to extreme environments. In our study, we found positive selection in rpoC2 between Brownlowia and Pentace species, indicating adaptive evolution in Brownlowia species. It was also under positive selection in various plant species growing in divergent environments such as light intensity, high altitude, and coastal ecotype [77,78,79,80,81]. The rpoC2 gene encodes RNA polymerase subunit that contained the large number of non-synonymous sites compared with other rpo genes (rpoA, rpoB, and rpoC1) and was associated with the structural properties of the respective plastid-encoded polymerase subunits under positive pressure [77]. Thus, the rpoC2 gene in Brownlowia species may be adapted to inland coastal areas.

5. Conclusions

In this study, we newly sequenced and reported the chloroplast genome of two true mangroves in the genus Brownlowia, Brownlowia tersa (a near-threatened species) and Brownlowia argentata (a data deficient species), based on stLFR data. Our study also revealed chloroplast structure, protein-coding genes, RNA editing sites, repeats, and SSRs. Chloroplast genome structure, gene content, and gene organization of the two Brownlowia species were similar. Notably, the chloroplast genomes may not be efficient markers for DNA barcoding in Brownlowia mangrove species due to high genetic similarity. Among B. tersa, B. argentata, Pentace triptera, and Diplodiscus trichospermus in Brownlowioideae, specific SSRs and hotspot regions such as psbI-trnS, trnR-atpA, petD-rpoA, rpl16-rps3, and trnN-ndhF could be utilized as potential markers. Furthermore, a phylogenetic tree based on 68 conserved chloroplast protein-coding genes across plant species in nine subfamilies in Malvaceae showed that B. tersa and B. argentata have closer relationships with P. triptera. Among genes, rpoC2 was subjected to positive selection. These results provide valuable insights into studying evolution and phylogenetic relationships in B. tersa and B. argentata and their related species.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cimb47020074/s1.

Author Contributions

Conceptualization, P.R.-a., S.T. and W.P.; methodology, P.R.-a., D.S. and T.Y.; software, P.R.-a.; validation, P.R.-a.; formal analysis, P.R.-a., D.S., T.Y., W.K. and S.U.-T.; investigation, P.R.-a., D.S., S.U.-T., O.P.N.T., P.C. and P.W.; resources, O.P.N.T., P.C. and P.W.; data curation, P.R.-a.; writing—original draft preparation, P.R.-a.; writing—review and editing, P.R.-a. and W.P.; visualization, P.R.-a. and W.K.; supervision, S.T. and W.P.; project administration, S.T. and W.P.; funding acquisition, S.T. and W.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science and Technology Development Agency, grant number No. P2351537.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/) (accession numbers: PP419968-PP419969).

Acknowledgments

We thank the research team from the Mangrove Forest Research Center for their sample collection.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The circular chloroplast maps of Brownlowia tersa and Brownlowia argentata. Genes located inside and outside the circle are transcribed counter-clockwise and clockwise, respectively. In the inner circle, the gray and lighter gray areas indicate GC and AT contents of the chloroplast genome. LSC is a large single copy. IRA and IRB are two inverted repeats. SSC is a small single copy. Introns in genes are indicated by an asterisk symbol (*). Genes are shown in different colors based on different functional groups.
Figure 1. The circular chloroplast maps of Brownlowia tersa and Brownlowia argentata. Genes located inside and outside the circle are transcribed counter-clockwise and clockwise, respectively. In the inner circle, the gray and lighter gray areas indicate GC and AT contents of the chloroplast genome. LSC is a large single copy. IRA and IRB are two inverted repeats. SSC is a small single copy. Introns in genes are indicated by an asterisk symbol (*). Genes are shown in different colors based on different functional groups.
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Figure 2. Alignment map of eight chloroplast genomes in Malvaceae. Vertical and horizontal axes represent a percentage sequence identity and genome position, respectively.
Figure 2. Alignment map of eight chloroplast genomes in Malvaceae. Vertical and horizontal axes represent a percentage sequence identity and genome position, respectively.
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Figure 3. Comparison of the junctions of LSC, SSC, IRa, and IRb regions among eight chloroplast genomes in Malvaceae.
Figure 3. Comparison of the junctions of LSC, SSC, IRa, and IRb regions among eight chloroplast genomes in Malvaceae.
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Figure 4. Statistical analysis of simple sequence repeats (SSRs) and dispersed repeats in four chloroplast genomes in Brownlowioideae. (A) Sorted by type of SSR. (B) Sorted by SSR region of chloroplast genomes. (C) Frequency by SSR type. (D) Sorted by type of dispersed repeat. (E) Frequency by dispersed repeat size. (F) Sorted by dispersed repeat region of chloroplast genomes.
Figure 4. Statistical analysis of simple sequence repeats (SSRs) and dispersed repeats in four chloroplast genomes in Brownlowioideae. (A) Sorted by type of SSR. (B) Sorted by SSR region of chloroplast genomes. (C) Frequency by SSR type. (D) Sorted by type of dispersed repeat. (E) Frequency by dispersed repeat size. (F) Sorted by dispersed repeat region of chloroplast genomes.
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Figure 5. Nucleotide diversity of chloroplast genome sequences of two Brownlowia species and two related species in Brownlowioideae. The regions are oriented according to their locations in the chloroplast genomes. (A) Coding regions. (B) Non-coding regions. BA, BT, PT, and DT represent Brownlowia argentata, Brownlowia tersa, Pentace triptera, and Diplodiscus trichospermus, respectively.
Figure 5. Nucleotide diversity of chloroplast genome sequences of two Brownlowia species and two related species in Brownlowioideae. The regions are oriented according to their locations in the chloroplast genomes. (A) Coding regions. (B) Non-coding regions. BA, BT, PT, and DT represent Brownlowia argentata, Brownlowia tersa, Pentace triptera, and Diplodiscus trichospermus, respectively.
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Figure 6. Maximum likelihood (ML) tree for 68 chloroplast protein-coding genes in 44 plant species. Bootstrap values with 1000 replicates show above the branches. The Brownlowia species in this study are indicated in red text, while other mangrove species in Malvaceae are indicated in blue text. The Brownlowioideae lineages are indicated in label yellow. Gain, loss, and pseudogene of the infA gene are indicated by blue, orange, and red rectangles, respectively.
Figure 6. Maximum likelihood (ML) tree for 68 chloroplast protein-coding genes in 44 plant species. Bootstrap values with 1000 replicates show above the branches. The Brownlowia species in this study are indicated in red text, while other mangrove species in Malvaceae are indicated in blue text. The Brownlowioideae lineages are indicated in label yellow. Gain, loss, and pseudogene of the infA gene are indicated by blue, orange, and red rectangles, respectively.
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Figure 7. Heatmap of Ka/Ks ratios among six pairwise species in 68 chloroplast genes. The Ka/Ks ratios are shown in the key beside this figure. BA, BT, PT, and DT are Brownlowia argentata, Brownlowia tersa, Pentace triptera, and Diplodiscus trichospermus, respectively.
Figure 7. Heatmap of Ka/Ks ratios among six pairwise species in 68 chloroplast genes. The Ka/Ks ratios are shown in the key beside this figure. BA, BT, PT, and DT are Brownlowia argentata, Brownlowia tersa, Pentace triptera, and Diplodiscus trichospermus, respectively.
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Table 1. Chloroplast genome features of two Brownlowia species in the subfamily Brownlowioideae.
Table 1. Chloroplast genome features of two Brownlowia species in the subfamily Brownlowioideae.
CharacteristicsBrownlowia tersaBrownlowia argentata
Genome size (bp)159,478159,510
   LSC length (bp)88,39488,435
   SSC length (bp)19,98419,985
   IR length (bp)25,55025,545
GC content (%)
Genome
37.05
37.05
   LSC34.8934.89
   SSC 31.3531.36
   IR 43.0143.01
Coding regions
   No. of total genes130130
   No. of protein coding genes8484
   No. of rRNAs88
   No. of tRNAs3737
   No. of duplicated genes in IR1616
   No. of pseudogenes1 (ψinfA)1 (ψinfA)
Table 2. Genes in the chloroplast genome of B. tersa and B. argentata.
Table 2. Genes in the chloroplast genome of B. tersa and B. argentata.
CategoryGene GroupsGene Name
PhotosynthesisSubunits of photosystem IpsaA, psaB, psaC, psaI, psaJ
Subunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ,
psbK, psbL, psbM, psbN, psbT, psbZ
Subunits of NADH dehydrogenasendhA *, ndhB * (×2), ndhC, ndhD, ndhE, ndhF, ndhG,
ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complexpetA, petB *, petD *, petG, petL, petN
Subunits of ATP synthaseatpA, atpB, atpE, atpF *, atpH, atpI
Large subunit of rubiscorbcL
Self-replicationLarge subunit of ribosomerpl2 * (×2), rpl14, rpl16 *, rpl20, rpl22, rpl23 (×2), rpl32,
rpl33, rpl36
Small subunit of ribosomerps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12 ** (×2),
rps14, rps15, rps16 *, rps18, rps19
DNA-dependent RNA polymeraserpoA, rpoB, rpoC1 *, rpoC2
Ribosomal RNAsrrn4.5 (×2), rrn5 (×2), rrn16 (×2), rrn23 (×2)
Transfer RNAstrnA-UGC * (×2), trnC-GCA, trnD-GUC, trnE-UUC,
trnF-GGA, trnG-GCC *, trnH-GUG, trnI-GAC * (×2),
trnK-UUU, trnL-CAA (×2), trnL-UAA *, trnM-CAU (×2),
trnN-GUU (×2), trnP-UGG, trnQ-UUG, trnR-ACG (×2),
trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA,
trnT-GGU, trnT-UGU, trnV-GAC (×2), trnV-UAC,
trnW-CCA, trnP-UGG, trnQ-UUG
Other genesMaturasematK
ProteaseclpP **
Envelope membrane proteincemA
Acetyl-CoA carboxylaseaccD
C-type cytochrome synthesis geneccsA
Translation initiation factorψinfA
Genes of unknownProteins of unknown functionycf1, ycf2 (×2), ycf3 **, ycf4
Notes: * Gene with one intron. ** Gene with two introns.
Table 3. RNA editing sites of B. argentata.
Table 3. RNA editing sites of B. argentata.
Gene NameEditing Position in cp GenomeEditing Position in cp GeneEditing Position in CodonCodon ChangeAmino Acid ChangeEditing Efficiency
matK25605141CAC to UACH to Y47%
30932372UCU to UUUS to F45%
31632141CAU to UAUH to Y32%
33401551CAC to UACH to Y57%
atpA10,5774723UUC to UUUF to F42%
10,8453832UCA to UUAS to L80%
11,0793052UCA to UUAS to L93%
atpE13,321312CCU to CUUP to L93%
atpI15,6162102UCA to UUAS to L98%
rps216,924832UCG to UUGS to L48%
17,038452ACA to AUAT to I50%
rpoC123,3201632UCA to UUAS to L46%
24,534142UCA to UUAS to L34%
rpoB27,2481892UCG to UUGS to L40%
27,2631842UCA to UUAS to L37%
psbZ37,905172UCA to UUAS to L95%
rps1439,067502UCA to UUAS to L92%
39,136272UCA to UUAS to L84%
ndhC53,7161082UCA to UUAS to L74%
accD62,0292662UCG to UUGS to L69%
62,6204632CCA to CUAP to L94%
62,6384692CCU to CUUP to L95%
psaI63,525282UCU to UUUS to F79%
psbJ68,099202CCU to CUUP to L94%
psbF68,484262UCU to UUUS to F86%
petL69,59322CCU to CUUP to L83%
rps1872,056742UCG to UUGS to L16%
rpl2072,4811032UCA to UUAS to L19%
clpP73,8901871CAU to UAUH to Y95%
psbN78,377102UCU to UUUS to F97%
petB79,69343GUC to GUUV to V79%
80,0991401CGG to UGGR to W95%
rpoA82,1802772UCA to UUAS to L45%
82,810672UCU to UUUS to F23%
rpl2390,239 and 157,707302UCA to UUAS to L31%
90,257 and 157,689242UCU to UUUS to F58%
ndhB99,260 and 148,6864942CCA to CUAP to L78%
99,486 and 148,4604191CAU to UAUH to Y68%
99,905 and 148,0412792UCA to UUAS to L43%
99,911 and 148,0352772UCG to UUGS to L73%
100,678 and 147,2682492UCU to UUUS to F39%
100,687 and 147,2592462CCA to CUAP to L35%
100,813 and 147,1332042UCA to UUAS to L51%
100,838 and 147,1081961CAU to UAUH to Y40%
100,882 and 147,0641812ACG to AUGT to M60%
100,957and 146,9891562CCA to CUAP to L67%
101,275 and 146,671502UCA to UUAS to L79%
ndhF115,941972UCA to UUAS to L55%
ndhD120,2084372UCA to UUAS to L76%
120,2204332UCA to UUAS to L93%
120,6402932UCA to UUAS to L82%
121,1351282UCA to UUAS to L63%
121,51612ACG to AUGT to M16%
ndhA124,7461893UCA to UUAS to L36%
126,1071142UCA to UUAS to L79%
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Ruang-areerate, P.; Sangsrakru, D.; Yoocha, T.; Kongkachana, W.; U-Thoomporn, S.; Prathip Na Thalang, O.; Chumriang, P.; Wanthongchai, P.; Tangphatsornruang, S.; Pootakham, W. Comparative Chloroplast Genomes and Phylogenetic Relationships of True Mangrove Species Brownlowia tersa and Brownlowia argentata (Malvaceae). Curr. Issues Mol. Biol. 2025, 47, 74. https://doi.org/10.3390/cimb47020074

AMA Style

Ruang-areerate P, Sangsrakru D, Yoocha T, Kongkachana W, U-Thoomporn S, Prathip Na Thalang O, Chumriang P, Wanthongchai P, Tangphatsornruang S, Pootakham W. Comparative Chloroplast Genomes and Phylogenetic Relationships of True Mangrove Species Brownlowia tersa and Brownlowia argentata (Malvaceae). Current Issues in Molecular Biology. 2025; 47(2):74. https://doi.org/10.3390/cimb47020074

Chicago/Turabian Style

Ruang-areerate, Panthita, Duangjai Sangsrakru, Thippawan Yoocha, Wasitthee Kongkachana, Sonicha U-Thoomporn, Onnitcha Prathip Na Thalang, Pranom Chumriang, Poonsri Wanthongchai, Sithichoke Tangphatsornruang, and Wirulda Pootakham. 2025. "Comparative Chloroplast Genomes and Phylogenetic Relationships of True Mangrove Species Brownlowia tersa and Brownlowia argentata (Malvaceae)" Current Issues in Molecular Biology 47, no. 2: 74. https://doi.org/10.3390/cimb47020074

APA Style

Ruang-areerate, P., Sangsrakru, D., Yoocha, T., Kongkachana, W., U-Thoomporn, S., Prathip Na Thalang, O., Chumriang, P., Wanthongchai, P., Tangphatsornruang, S., & Pootakham, W. (2025). Comparative Chloroplast Genomes and Phylogenetic Relationships of True Mangrove Species Brownlowia tersa and Brownlowia argentata (Malvaceae). Current Issues in Molecular Biology, 47(2), 74. https://doi.org/10.3390/cimb47020074

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