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Jul 04, 2023

Comparative chloroplast genomics and insights into the molecular evolution of Tanaecium (Bignonieae, Bignoniaceae)

Scientific Reports volume 13, Article number: 12469 (2023) Cite this article 270 Accesses 2 Altmetric Metrics details Species of Tanaecium (Bignonieae, Bignoniaceae) are lianas distributed in the

Scientific Reports volume 13, Article number: 12469 (2023) Cite this article

270 Accesses

2 Altmetric

Metrics details

Species of Tanaecium (Bignonieae, Bignoniaceae) are lianas distributed in the Neotropics and centered in the Amazon. Members of the genus exhibit exceptionally diverse flower morphology and pollination systems. Here, we sequenced, assembled, and annotated 12 complete and four partial chloroplast genomes representing 15 Tanaecium species and more than 70% of the known diversity in the genus. Gene content and order were similar in all species of Tanaecium studied, with genome sizes ranging between 158,470 and 160,935 bp. Tanaecium chloroplast genomes have 137 genes, including 80–81 protein-coding genes, 37 tRNA genes, and four rRNA genes. No rearrangements were found in Tanaecium plastomes, but two different patterns of boundaries between regions were recovered. Tanaecium plastomes show nucleotide variability, although only rpoA was hypervariable. Multiple SSRs and repeat regions were detected, and eight genes were found to have signatures of positive selection. Phylogeny reconstruction using 15 Tanaecium plastomes resulted in a strongly supported topology, elucidating several relationships not recovered previously and bringing new insights into the evolution of the genus.

The chloroplast is a circular organelle with a prokaryotic origin in plant cells. This organelle is responsible for photosynthesis and critical for the biosynthesis of starch, fatty acids, pigments, and amino acids1,2. Chloroplast genomes, also known as plastomes, have a predominantly conserved quadripartite structure that consists of a Large Single-Copy (LSC), two Inverted Repeats (IR), and a Small Single-Copy (SCC) region3,4. Despite the constancy in the overall structure, different patterns, rearrangements, structure organization, size, gene content, and order have been documented during the last decade5,6,7.

The structural variation observed in plastomes is due to intergenic region length and gene number, among others9,10. While closely related lineages tend to show lower variation, many cases of closely related species with high variation in plastome sizes have been observed9,10. This is probably associated with parasitism, IR loss, expansions, or contractions7,8,9. The increasing number of studies focusing on various plant clades adds publicly available data, allowing plastome comparisons among different angiosperm clades.

During the past three decades, chloroplast data has been extensively used to reconstruct plant phylogenies at different taxonomic levels11,12,13,14,15,16,17. The broad use of chloroplast data in molecular phylogenetic studies is due to its haploid nature, predominant uniparental inheritance, relatively stable gene structure, and high copy number per cell, which facilitates sequencing. While chloroplast sequencing initially targeted a few genes through Sanger approaches, the development of High-Throughput Sequencing (HTS) technologies allowed for whole plastome sequencing18.

The fast increase of HTS applications in the last couple of decades revolutionized the use of genomic data to understand the evolutionary history of green plants. In the tribe Bignonieae specifically, phylogenies reconstructed using plastome data have led to strongly supported and well-resolved topologies16,19,20. These phylogenies have improved our understanding of phylogenetic relationships at deep taxonomic levels (i.e., phylogeny backbone) and more recent divergences at the infra-generic level16,20.

Tanaecium Sw. emend. L.G. Lohmann (Bignonieae, Bignoniaceae) is a genus of Neotropical lianas that includes 21 species distributed from Mexico and the Antilles to Argentina, and centered in the Amazon21. The genus exhibits exceptionally diverse flower morphology and pollination systems21, seeds that can be winged or wingless and corky, and bromeliad-like prophylls of the axillary buds, a putative vegetative synapomorphy21. The genus was first sampled in a molecular phylogeny reconstructed using the chloroplast gene ndhF and the nuclear pepC12. Subsequent molecular phylogenetic studies with this group used the same molecular markers22,23,24. While representatives of this genus have been sampled in multiple studies, sampling remains limited, even lacking sampling of the type species of the genus. Moreover, the Tanaecium plastome structure has never been explored. Even though a study reported data on the plastome of T. tetragonolobum (Jacq.) L.G.Lohmann25, this plastome turned out to be Callichlamys latifolia (Rich.) K.Schum.26.

In Bignoniaceae, plastomes range from 150,154 bp in Incarvillea compacta Maxim.27 to 183,052 bp in Bignonia magnifica W.Bull, the latter representing the largest Lamiid plastome known to date28. Bignoniaceae plastomes also show structural rearrangements, such as the loss of the ycf4 gene reported for Adenocalymma20, and variation in gene number, ranging from 110 to 157 genes16,19,20,25,27,28,29.

This study aims to increase our knowledge of Bignoniaceae plastome structure and evolution and bring new insights into the evolutionary history of Tanaecium by reporting on the plastome structure of the genus for the first time. To achieve this goal, we (1) sequenced and assembled complete or nearly complete plastomes of 15 species of Tanaecium, representing more than 70% of the known diversity in the genus 21; (2) characterized the overall plastome structure; (3) performed comparative genomic analyses; (4) identified putative repeats; (5) investigated patterns of selection in the chloroplast genes; and (6) reconstructed a phylogeny for Tanaecium using the newly assembled plastomes.

The paired-end raw reads of the 16 Tanaecium plastomes sequenced (Table 1) varied between 3,858,109 and 14,350,498 bp for T. parviflorum and T. tetragonolobum, respectively (Table 2). Of these, 12 plastomes were complete and four were partial. Mapped reads varied from 101,125 to 660,086 bp for T. duckei and T. revillae, respectively (Table 2). The average read depth varied between 85 × for T. tetragonolobum and 679 × for T. dichotomum 2 (Table 2). All plastomes showed the typical quadripartite structure of angiosperms (Fig. 1), with a pair of IR regions that range from 30,284 bp (T. duckei) to 31,089 bp (T. bilabiatum), intercalated by one LSC region that ranges from 83,490 bp (T. crucigerum; nearly complete, but without missing data in the LSC) to 86,213 bp (T. xanthophyllum), and one SSC region that ranges from 12,504 bp (T. tetragonolobum) to 12,920 bp (T. dichotomum 1) (Table 2). The Tanaecium plastomes have an average length of 159,359 bp, with Tanaecium xanthophyllum representing the largest plastome assembled here, with a total length of 160,935 bp (Table 2). The large size of the T. xanthophyllum plastome is due to an expansion in the LSC region (Table 2). The interquartile range (IQR) and median size ratio for Tanaecium was 0.5%; in turn, the IQR reported for Adenocalymma was 0.7%, for Anemopaegma was 0.4%, and for Amphilophium was 4% as expected based on an earlier study9 (Supplementary Table S7). The average GC content is 38% for all Tanaecium species studied (Table 2). All plastomes encode 137 genes, including 80–81 unique coding genes (CDS) (9 duplicated), 37 tRNA genes, and four rRNA genes (Tables 2 and 3). The Mauve analysis retrieved a single synteny block, indicating no rearrangements in Tanaecium plastomes (Supplementary Fig. S1). The boundaries between the chloroplast main regions are similar within Tanaecium, except for the LSC/IRb border, which can be located between the genes rps19 and rpl2 or within the rps19 gene (Fig. 2).

Representation of the plastome of Tanaecium jaroba. Genes drawn below the line are transcribed in a forward direction, while those drawn above the line are transcribed in a reverse direction. Asterisks (*) represent intron-containing genes.

Comparison of the Large Single Copy (LSC), Inverted Repeat a (IRa), Small Single Copy (SSC), and Inverted Repeat b (IRb) boundaries within Tanaecium and among five other Bignoniaceae plastomes. The psi (ψ) indicates pseudogenes within the plastomes sampled. Genes shown below are transcribed reversely and those shown above the lines are transcribed forward. Minimum and maximum sizes for the regions and genes in the plastome boundaries are indicated in base pairs (bp). Numbers in superscript represent the literature from where the plastome boundary information were consulted. Tanaecium type 1 = T. bilabiatum, T. crucigerum, T. cyrtanthum, T. decorticans, T. jaroba, T. parviflorum, T. pyramidatum, T. tetragonolobum, T. tetramerum, and T. truncatum; Tanaecium type 2 = T. dichotomum 1, T. dichotomum 2, T. duckei, T. revillae, T. selloi, and T. xanthophyllum.

The analysis performed using the DnaSP to calculate the nucleotide variability (π) values within 800 bp across plastomes showed that there is intrageneric variability in Tanaecium (Fig. 3A). The π values range from 0 to 0.06, with a mean value of 0.009. The most variable region, the only one containing π > 0.05, was the rpoA gene. Seven regions showed π values between 0.03 and 0.049 (i.e., clpP, psaI-ycf4, petD-rpoA, rps11, rps12-clpP, ycf4, and rpoA), while twelve regions showed π > 0.02 (i.e., ycf2, ycf1, rpl33, clpP-psbB, rpl33-rps18, rpl32-trnL, rpl32, clpP, ycf4, rpl20-rps12, rps11, and rps18) (Fig. 3A). The non-coding regions are more variable (7.65% of the intergenic regions (IGS) and 6.05% of the introns) than the coding regions (5.75%; Supplementary Table S1). Among all plastome regions, the 15 regions with the highest percentage of variable sites are: rps12-clpP, clpP intron, trnN-ycf1, rpoA, clpP, accD, psaI-ycf4, rps18, accD-psaI, trnH-psbA, ycf4, trnL-ccsA, rpoA-rps11, rpl33-rps18, and rbcL-accD (Fig. 3B; Supplementary Table S1). The 15 most variables regions in absolute numbers are: accD, ycf1, clpP intron, rpoA, rps18, trnN-ycf1, ycf2, rpl33-rps18, rps12-clpP, ndhF, clpP, rpoC2, psaA-ycf3, rpl32-trnL, and psaI-ycf4 (Fig. 3C; Supplementary Table S1).

(A) Sliding window analysis of the complete plastomes of 15 Tanaecium species (window length: 800 bp, step size: 200 bp). X-axis, the position of the midpoint of each window. Y-axis, nucleotide diversity (π) of each window. (B,C) Fifteen most variable genes within the assembled Tanaecium plastomes. (B) Percentage of variable sites according to gene length. (C) Number of variable sites per gene.

The total number of SSRs (i.e., tandem repeats of short motifs of DNA with lengths varying from 1 to 6 bp) in Tanaecium range from 44 to 59 SSRs, distributed along the three regions (Fig. 4A–C; Supplementary Table S2). Most SSRs found are A or T mononucleotide repeats, accounting for 54–73% of the total repeats. Out of the total number of SSRs detected, 26–44 (56.5–74.6%) are mono-repeats, 1–5 (1.8–10.9%) are di-repeats, 4–6 (7.4–13%) are tri-repeats, 4–9 (8.2–17.6%) are tetra-repeats, 0–4 (0–6.8%) are penta-repeats, while 0–5 (0–10.2%) are hexa-repeats (Fig. 4B; Supplementary Table S2). In addition, most of the SSRs in Tanaecium are located in the LSC region (71–82.4%). The IR regions include between 1.9 and 15.2%, while the SSC region includes between 4.3 and 27% of the SSRs (Fig. 4A; Supplementary Table S2). The coding regions contain 20.3–30.4% of the SSRs, while the introns contain 4.5–21.7%, and the intergenic spacers contain 54.3–72.7% of the SSRs (Fig. 4C; Supplementary Table S2).

Distribution of SSRs in the Tanaecium plastomes. (A) Distribution of SSRs (IRa omitted). (B) Number of SSRs by type. (C) Distribution of SSR by coding and non-coding regions.

We identified tandem repeat sequences longer than 30 bp throughout the Tanaecium plastomes (Fig. 5A; Supplementary Table S3). Most of these tandem repeats are found in the LSC regions, followed by the IR, with only a few tandem repeats found in the SSC (Fig. 5B; Supplementary Table S3). The most frequent repeats were 30–39 bp in length (Fig. 5C; Supplementary Table S3). Most of the tandem repeats are located in the IGS, followed by the CDS, while few repeats were found in introns (Fig. 5D; Supplementary Table S3). The plastomes of Tanaecium contain 20–67 forward repeats, up to two reverse repeats, and single palindromic repeats, leading to a total of 22–67 repeats (Supplementary Table S3). The longest repeats vary between 79 bp in T. parviflorum and 418 bp in T. pyramidatum (Supplementary Table S3). The longest repeats are located in eight regions: accD, rpoA, ycf1, and rps18 genes, or the rpl23/trnI-CAU, rpl33/rps18, psaA/ycf3, and trnN-GUU/ycf1 intergenic regions (Supplementary Table S3). A shared repeat with 41 bp showed the first repeat in the intergenic region rps2/trnV-GAC, the second in the ndhA intron for all Tanaecium species, and four additional Bignonieae plastomes included in this study (Fig. 5A; Supplementary Table S4).

Distribution of tandem repeats, 30 bp or longer in the Tanaecium plastomes. (A) Distribution of the repeats (IRa omitted). (B) Distribution and size of the repeats along the unique regions of the plastome: Large Single Copy (LSC), Small Single Copy (SSC), and Inverted Repeat (IR). (C) Distribution of the repeats by size. (D) Distribution of the repeats by size and coding and non-coding regions.

The 81 protein-coding genes of the Tanaecium plastomes encoded 22,686 codons averaged over all taxa (Supplementary Table S5). The most abundant codons encoded leucine (10.5%), followed by isoleucine (8.3%); whereas the least abundant codons encoded cysteine (1.07%), followed by the stop codons (0.35%) (Fig. 6). Thirty-two codons showed codon usage bias (RSCU < 1), of which only three are not G- and C-ending. Thirty codons were used more frequently than expected at equilibrium (RSCU > 1), with one not representing an A/U-end codon. Codon bias was not detected (RSCU = 1) in the frequency of use for the start codon AUG (methionine) and UGG (tryptophan) (Supplementary Table S5). None of the 81 genes were found to be under positive selection in Tanaecium using HyPhy30 in MEGA 731. However, signals of positive selection were detected using the codon models BUSTED32 and FUBAR33 in eight coding regions: accD (29 sites), clpP (15 sites), rpoA (39 sites), rps18 (15 sites), rps7 (2 sites), ycf1 (37 sites), ycf2 (71 sites), and ycf4 (9 sites) (Supplementary Table S6).

Codon content of amino acids encoding proteins in the chloroplast genomes of Tanaecium. All frequencies are averages over all taxa.

The phylogeny of Tanaecium plus one outgroup was inferred using all 16 plastomes, removing one of the IRs and the poorly aligned regions. The final alignment included a total of 121,710 bp (86% of the original 140,117 positions), where 7,051 bp were variable and 2168 bp were parsimony informative. The best-fit model of substitution was the GTR + F + I + G4. The phylogeny recovered a monophyletic Tanaecium, with maximum support value (bootstrap support (BS) = 100; Fig. 7). Most nodes showed maximum support, with low to moderate values observed for only one node (BS = 77; Fig. 7). Tanaecium xanthophyllum emerged as sister-group to the remaining species, all of which are divided in two main clades: Clades A and B. Clade A comprises Clade I (i.e., T. bilabiatum, T. crucigerum, T. jaroba, and T. cyrtanthum) and Clade II (i.e., T. selloi, T. dichotomum 1, T. revillae, and T. dichotomum 2). In turn, Clade B is composed of Clade III (i.e., T. tetragonolobum, T. truncatum, and T. duckei), sister to Clade IV (i.e., T. pyramidatum and T. decorticans), both of which are sister to Clade V (i.e., T. tetramerum and T. parviflorum) (Fig. 7).

Maximum likelihood phylogeny inferred using IQ-TREE 1.5.5. The species highlighted in bold is the species type of the genus, Tanaecium jaroba.

In this study, we sequenced and assembled for the first time 16 plastomes representing 15 of the 21 Tanaecium species currently recognized21. These plastomes were compared with previously published Bignoniaceae plastomes, providing novel insights into chloroplast evolution in the family. The newly assembled plastomes were used as a basis to reconstruct the most comprehensive phylogeny of Tanaecium to date. The phylogenetic placement of Tanaecium jaroba, the type species of the genus, was inferred for the first time, corroborating the current generic classification21.

The quadripartite plastome structure found in Tanaecium is the most common among angiosperms3,7,8,34. Some exceptions for this structure have been reported in the papilionoid legumes35, saguaro cactus36, and Geraniaceae37. Although plastome structural changes have been reported for angiosperms6,8, including tribe Bignonieae20, no rearrangement had ever been documented for Tanaecium. The two different patterns of boundaries between the four main regions found in Tanaecium plastomes are similar to that found in Adenocalymma peregrinum20 (Fig. 2). Contractions and expansions of IRs were detected multiple times during land plant evolution38, including other Bignoniaceae16,19,20,28. Within this plant family, the plastomes of Bignonia magnifica bear exceptionally large IR regions, representing the largest plastome among all Lamiids known to date28.

The obtained Tanaecium plastomes show a pattern of size range variation that matches that of the LSC expansions/contractions (Table 2). This is a typical pattern among seed plants, although the number of genes and intergenic region length is more commonly used to explain plastome size variation10. In other Bignonieae, the LSC size variation is relatively common16,19,20, and the variation in gene number seems less frequent for the group16,19,20.

When the Bignonieae IQR and median size variation ratio are compared with those expected for other angiosperms9, Tanaecium, Adenocalymma, and Anemopaegma show less than 1% variation at the genus level as reported for other groups9 (Supplementary Table S7), while Amphilophium shows variation greater than 4%9 (Supplementary Table S7). Even though the high variation found in Amphilophium was previously attributed to polyphyly9, this interpretation was based on an outdated classification system. Amphilophium monophyly has been shown repeatedly39,40. In this context, we attribute the high IQR and median size variation ratio found in Amphilophium to the gene number and LSC length variation10,16.

The total number of genes found in Tanaecium plastomes is similar to those found in other Bignoniaceae16,20. While ycf15 and ycf68 genes are lacking in some Bignoniaceae genera16,19,20, those genes were found in Tanaecium, Callichlamys latifolia (Rich.) K.Schum.25, and Crescentia cujete L.29. Partial ycf15 genes were also recorded in the Convolvulaceae41. The complete or partial loss of genes is common in land plants6,9,10, including the Bignoniaceae20.

The most variable locus in Tanaecium is rpoA, which contains hypervariable sites with π > 0.05. This gene is frequently listed among the most variable regions in other plant clades42 and has been shown to represent one of the most hypervariable genes for Amphilophium (Bignoniaceae)16. In turn, the accD gene is the most variable in terms of absolute numbers in Tanaecium (Fig. 3), and the second most variable in Amphilophium, followed by the ycf1 gene16. The accD gene is highly variable in other Bignoniaceae species and angiosperm clades such as Artemisia (Asteraceae)43 and Lamprocapnos (Papaveraceae)8. The rps18 gene is among the most variable in absolute numbers in Tanaecium, Stemonaceae44, Bromeliaceae45, and Campanulaceae17. Interestingly, the rps18 gene shows low evolutionary rates in Anemopaegma (Bignoniaceae)19, indicating that chloroplast genes can hold different levels of variation in distinct lineages and at different taxonomic levels. This aspect complicates the selection of candidate barcode genes for the angiosperms as a whole, emphasizing the importance of studies aiming to characterize plastomes of entire clades.

Single Sequence Repeats (SSRs) are commonly detected in plastomes, often showing interspecific polymorphism, and high variation at lower taxonomic levels, representing useful tools for population-level studies46. The SSRs identified in Tanaecium vary in location, type, and number. Most SSRs are located in the LSC region, with the mononucleotide A/T repeats representing the most abundant type (Fig. 4). The higher frequency of mononucleotides is a common trend among land plants47. Most of the long repeats of Tanaecium are located in the LSC, followed by IR regions, with only a few located in the SSC. This pattern differs from that found in other Bignoniaceae species, where most of the larger than 30 bp repeats are located in the IR, with only a few cases showing a pattern that is similar to that found here16,48. The chloroplast SSRs detected in Tanaecium will likely be helpful for future population genetics and microevolutionary studies, as well as for community-level studies of potential barcode designs, given the presence of shared repeats.

Plastomes have a synonymous codon usage bias in the protein-coding genes, which affects gene expression and plays an essential role in the evolution of these genomes49. Our results showed that amino acids that have A- and U-ending codons are more common in Tanaecium, consistent with codon usage bias in most of the angiosperm plastomes, including Bignoniaceae representatives48,50. In plants, the main evolutionary driving force acting on codon use are natural selection and mutation pressure51,52,53. Thus, the patterns observed in Tanaecium bring important information not only about the nature of plastome mutations, but also about putative environmental impact. More expressed genes might display higher codon bias54, which can be seen in plastomes due to the photosynthetic machinery associated with the chloroplast function. Our results also showed a preference for using the amino acid leucine, which has a high RSCU (Fig. 6; Supplementary Table S5), suggesting a potential impact of selection pressure on codon usage51,54.

Adaptive evolution or positive selection is generally estimated using the synonymous/non-synonymous substitutions ratio55. Even though our analyses using a maximum likelihood approach in HyPhy have failed to detect any signal of positive selection, evidence for positive selection was recovered through the analyses conducted with BUSTED and FUBAR. This result likely reflects the fact that a relatively high fraction of sites (5–10%) needs to be under positive selection for accurate detection in BUSTED32, while FUBAR assumes that the selection pressure for each site is constant throughout the phylogeny33. Thus, it is likely that the genes really have evidence for selection. For the eight genes under positive selection in Tanaecium, seven of them were also shown to be under positive selection in Amphilophium (except ycf4)16, while three were shown to be under positive selection in Handroanthus impetiginosus (Mart. ex DC.) Mattos (i.e., rps7, ycf1, and ycf4)48. The genes found under selection are associated with different plant cell functions. They are associated explicitly with ribosome biogenesis and protein synthesis56, RNA polymerase biogenesis57, assembly and stability of the photosystem I58, environmental stress and plant growth59, among other important components of cell function and survival60,61.

The ML phylogeny reconstructed here sampled 15 out of the 21 currently accepted species of Tanaecium, representing the most comprehensive phylogeny of the genus to date, regarding the number of characters and taxa. A previous topology was inferred to investigate the relationship of a recently described Tanaecium species, sampling 11 species of the genus and using only the nuclear marker pepC and the chloroplast gene ndhF21. The sampling used here is different, making comparisons among the resulting topologies difficult. In addition, some relationships were not clearly solved in the previously published tree reconstructed with two markers, with several nodes showing low/moderate support21. Yet, the placement of the newly described species in that study was similar to the one inferred here (i.e., T. decorticans + T. pyramidatum). Moreover, the phylogeny inferred here is the first to include the type species of the genus (i.e., T. jaroba), confirming the monophyly of the genus hypothesized earlier12. Our results indicate that the variation found among plastomes is sufficient to reconstruct robust phylogenetic relationships of the 16 Tanaecium taxa sampled here with good support. Additional studies will be released soon, further investigating the phylogenetic relationships among Tanaecium species, their morphological evolution, and biogeographical history.

We sequenced, assembled, and annotated the plastomes of 15 out of 21 species of Tanaecium currently recognized21, namely: Tanaecium bilabiatum (Sprague) L.G.Lohmann, Tanaecium crucigerum Seem., Tanaecium cyrtanthum (Mart. ex DC.) Bureau & K.Schum., Tanaecium decorticans Frazão & L.G.Lohmann, Tanaecium dichotomum (Jacq.) Kaehler & L.G.Lohmann, Tanaecium duckei A.Samp., Tanaecium jaroba Sw., Tanaecium parviflorum (Mart. ex DC.) Kaehler & L.G.Lohmann, Tanaecium pyramidatum (Rich.) L.G.Lohmann, Tanaecium revillae (A.H.Gentry) L.G.Lohmann, Tanaecium selloi (Spreng.) L.G.Lohmann, Tanaecium tetragonolobum (Jacq.) L.G.Lohmann, Tanaecium tetramerum (A.H.Gentry) Zuntini & L.G.Lohmann, Tanaecium truncatum (A.Samp.) L.G.Lohmann, and Tanaecium xanthophyllum (DC.) L.G.Lohmann. We sampled two individuals of T. dichotomum, representing different morphotypes of this species (i.e., Tanaecium dichotomum 1 and Tanaecium dichotomum 2). All sampled taxa, vouchers, and respective GenBank accession numbers are summarized in Table 1.

Leaf tissue was pulverized with Tissuelyzer® (Qiagen, Duesseldorf, Germany) for 5 min at 50 Hz and DNA was subsequently extracted following the CTAB protocol62. The protocol was adapted by adding 2-Mercaptoethanol and polyvinylpyrrolidone (PVP). DNA was quantified using the Qubit® Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). A total of 5 μg of DNA was fragmented using a Covaris S-series sonicator, generating DNA fragments of approximately 300 bp. Libraries for Illumina platform sequencing were prepared following Nazareno et al.25 Sequencing was conducted in an Illumina HiSeq 2500 Genome Analyzer (Illumina, San Diego, California, USA) as paired-read, with 22 samples per lane, at USP-Esalq (Piracicaba, Brazil).

Plastomes were assembled using the Fast-Plast pipeline (McKain and Wilson, unpubl.; https://github.com/mrmckain/Fast-Plast). This pipeline uses Trimmomatic 0.3563 to remove the adaptors and low-quality sequences. The trimmed reads were mapped against a database that included the published plastomes of Adenocalymma peregrinum (MG008314.1), Olea europaea L. (NC_013707.2), Sesamum indicum L. (NC_016433.2), Salvia miltiorhiza Bunge (NC_020431.1), and C. latifolia (KR534325) using Bowtie 2.1.064. Mapped reads were assembled into contigs using SPAdes 3.1.065. Resulting contigs were assembled with the software afin (https://bitbucket.org/afinit/afin), using the parameters -l 50, -f 0.1, -d 100, -× 100, and -i 2. For species for which it was harder to obtain comprehensive contigs, we tested values between 10 and 20 for minimum contig (-p) parameter overlap. The final assembly from Fast-Plast or afin was checked, and edited with Geneious 9.0.266. The plastome assembly was verified through a coverage analysis conducted in Jellyfish 2.1.367 using a 25-bp sliding window of coverage across the plastome of each species. Only sites with a depth higher than two were kept.

Plastome annotation was initially conducted in Geneious 9.0.266 using the Adenocalymma peregrinum plastome as a reference20. The annotated loci were verified using BLAST68,69, with correct start and stop codons of the Open Reading Frames (ORFs) checked manually in Geneious 9.0.266. The boundaries between the LSC, IRs, and SSC regions were verified using the online IRscope70 and confirmed manually in Geneious 9.0.266. The graphical representation of the annotated Tanaecium plastomes was created using OGDRAW71.

We performed comparative analyses using the 16 Tanaecium plastomes sequenced (Table 1). We removed one of the IR regions from all plastomes to avoid data duplication, except for the analyses to determine synteny and identify possible rearrangements which were conducted for the complete plastomes using Mauve 2.4.072. These analyses utilized mauveAligner as alignment algorithm, MUSCLE 3.673 as the internal aligner, with full alignment and minimum locally collinear block (LCB) score automatically calculated. Genomes were not assumed to be collinear. We used the online IRscope70 to compare Tanaecium plastome borders between the four main regions (i.e., LSC, IRs, and SSC) within the genus and with other five previously published Bignonieae plastomes: Adenocalymma peregrinum (MG008314.1), Amphilophium steyermarkii (MK163626), Anemopaegma arvense (MF460829), Callichlamys latifolia (KR534325.1), and Crescentia cujete (KT182634) (Table 1). To compare the length variation of Tanaecium plastomes and other Bignonieae genera with previously published plastomes, we used the box-plot approach proposed by Turudić et al.9.

Tanaecium plastomes were aligned in MAFFT 7 online version74 where analyses of intrageneric variability were conducted. The poorly aligned regions were removed using Gblocks 0.91b75, assuming the least stringent settings. We calculated nucleotide variability values (π) within the assembled Tanaecium plastomes using DnaSP 6.1076 through a sliding window analysis with a 200 bp step size and 800 bp window length. We used R77 to plot the DnaSP results. We extracted annotated coding and non-coding regions using Geneious 9.0.266 to evaluate the number of variable sites (V) using the software MEGA 731. The protein-coding regions were previously re-aligned individually with the translation alignment tool in Geneious 9.0.266 using the ClustalW plugin78.

To identify and locate microsatellites or Simple Sequence Repeats (SSRs) in Tanaecium plastomes, we used MISA79 with the following parameters: motif length of SSR between one and six nucleotides, a minimum repetition number set as 10 units for mono-, five for di-, and four for trinucleotide SSRs, and three units for each tetra-, penta-, and hexanucleotide SSRs. We used REPuter80 to identify tandem repetitions, allowing forward, palindrome, and reverse repeated elements with a minimum repeat size ≥ 30 bp and Hamming distance of 0.

To investigate the codon usage and the role of selection on Tanaecium plastomes, we extracted 81 protein-coding genes from the 16 genomes aligned and annotated. Each coding region was re-aligned separately in Geneious66, using the translation alignment tool ClustalW plugin. Codon usage bias occurs when some codons are used more often than other synonymous codons during gene translation between different taxa81. We assessed the relative synonymous codon usage (RSCU) from the 81 protein-coding genes using MEGA 731, with default parameters.

In addition, we investigated synonymous (Ks) and non-synonymous (Ka) substitutions and their ratio (Ka/Ks) in the 81 coding regions using the package HyPhy30 in MEGA 731. We also used other codon models to further analyze the selective pressure on the protein-coding genes using HyPhy30 in the Datamonkey server82: i.e., BUSTED (branch-site unrestricted statistical test for episodic diversification; Murrell et al.32) was used to investigate diversifying selection on the selected genes, while FUBAR (fast unconstrained Bayesian AppRoximation; Murrell et al.33) was used to identify episodic/diversifying selection on codon sites with posterior probability of > 0.9.

The 16 plastomes of the 15 Tanaecium species assembled here were aligned using the Adenocalymma peregrinum (MG008314) plastome as an outgroup and the online version of MAFFT 774. The Ira regions were excluded from the alignment to avoid data duplication. We used Gblocks to remove poorly aligned regions with the least stringent settings75. The number of variable and parsimony informative sites for the resulting alignment was calculated in MEGA 731. The final alignment was used to perform maximum likelihood (ML) analyses in IQ-TREE 1.5.583, including model selection and 1000 bootstrap (BS) replicates in a single run84.

The assembled plastomes of Tanaecium are available in GenBank (NCBI) with the accession numbers OL782596, OP169019–OP169021, and OP218850–OP218861.

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The authors thank the curator of the SPF herbarium for allowing us to examine specimens and sample leaf tissue for DNA extraction. We are indebted to Adenor Lima, Aécio Santos, Alcides Frazão, Alison Nazareno, Ananias Reis, Augusto Giaretta, Beatriz Gomes, Carolina Siniscalchi, Celma A. Nunes, Eric Kataoka, Hélcio de Souza, Jéssica Francisco, José M. Assis, Leandro Giacomim, Leila Meyer, Maila Bayer, Martin Acosta, Osmar Ferreira, Ricardo Ribeiro, and Thais Almeida for assistance during fieldwork. We thank the staff of the Instituto Nacional de Pesquisas da Amazônia (INPA), Universidade Federal do Oeste do Pará (UFOPA), Embrapa Amazônia Oriental (IAN), Embrapa Recursos Genéticos e Biotecnologia (CENARGEN), Universidade Federal do Acre (UFAC), Fundação de Tecnologia do Estado do Acre (FUNTAC), Herbário da Amazônia Meridional (HERBAM), and Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for collecting permits, general assistance during fieldwork, and for hosting us on two different visits. We also thank Alison Nazareno, Eric Kataoka, Luiz H. Fonseca, and Marcelo Reginato for assistance during library preparation and plastome assembly, and the Core Facility for Scientific Research from the Universidade de São Paulo (CEFAP-USP/GENIAL) for allowing us to use the Covaris S2 sonicator, Qubit, and the SEAL server. This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 142224/2015-4, 310871/2017-4, 151133/2021-2), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2011/50859-2, 2015/10914-5, 2018/23899-2), a collaborative FAPESP-NSF-NASA grant (2012/50260-6), International Association for Plant Taxonomy (IAPT 2016), Systematic Research Fund (SRF 2016), Society of the Systematic Biologists (SSB 2017), and American Society of Plant Taxonomists (ASPT 2019).

Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil

Annelise Frazão & Lúcia G. Lohmann

Departamento de Biodiversidade e Bioestatística, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brazil

Annelise Frazão

Programa de Pós-Graduação em Botânica, Departamento de Botânica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Verônica A. Thode

Department of Integrative Biology, University and Jepson Herbaria, University of California, Berkeley, Berkeley, CA, USA

Lúcia G. Lohmann

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A.F. and L.G.L. designed the study, defined sampling, obtained samples, and obtained funding. A.F. and V.A.T. annotated plastomes and performed comparative and phylogenetic analyses. A.F. assembled Illumina sequences. A.F., V.A.T., and L.G.L. interpreted the results and co-wrote the manuscript.

Correspondence to Annelise Frazão or Lúcia G. Lohmann.

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Frazão, A., Thode, V.A. & Lohmann, L.G. Comparative chloroplast genomics and insights into the molecular evolution of Tanaecium (Bignonieae, Bignoniaceae). Sci Rep 13, 12469 (2023). https://doi.org/10.1038/s41598-023-39403-z

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Received: 06 April 2023

Accepted: 25 July 2023

Published: 01 August 2023

DOI: https://doi.org/10.1038/s41598-023-39403-z

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