Comparative Chloroplast Genomics at Low Taxonomic Levels: A Case Study Using Amphilophium (Bignonieae, Bignoniaceae)

Chloroplast (cp) genome organization, gene order, and content have long been considered conserved among land plants. Despite that, the generation of thousands of complete plastomes through next-generation sequencing (NGS) has challenged their conserved nature. In this study, we analyze 11 new complete plastomes of Amphilophium (Bignonieae, Bignoniaceae), a diverse genus of Neotropical lianas, and that of Anemopaegma prostratum. We explored the structure and content of the assembled plastomes and performed comparative analyses within Amphilophium and among other plastomes available for Bignoniaceae. The overall gene content and orientation of plastomes is similar in all species studied. Plastomes are not conserved among Amphilophium, showing significant differences in length (155,262–164,786 bp), number of genes duplicated in the IRs (eight, 18, or 19), and location of the SC/IR boundaries (i.e., LSC/IRa junction between rps19 and rpl2 genes, within petD, or within petB). Length differences reflect expansions of the IRs and contractions of the LSC regions. The plastome of A. prostratum is 168,172 bp, includes 19 duplicated genes, and has the LSC/IRa boundary located within the petB gene. Amphilophium plastomes show high nucleotide diversity, with many hypervariable regions, and 16 genes with signatures of positive selection. Multiple SSRs and repeat regions were identified for Amphilophium and Anemopaegma prostratum. The differences in structure detected within Amphilophium plastomes in terms of LSC/IR and IR/SSC boundaries, number of duplicated genes, and genome sizes are mostly shared between taxa that belong to the same clade. Our results bring new insights into the evolution of plastomes at low taxonomic levels.


INTRODUCTION
Chloroplasts are photosynthetic organelles that have an important role in plant carbon fixation, as well as in the biosynthesis of starch, fatty acids, amino acids, and pigments (Jansen and Ruhlman, 2012;Zhao et al., 2015;Daniell et al., 2016). In angiosperms, the chloroplast genome (plastome) generally has a circular structure that ranges from 120 to 180 kb in size and contains a quadripartite structure, composed of two Inverted Repeat (IR) regions, a Large Single Copy (LSC), and a Small Single Copy (SSC) region (Palmer, 1985;Green, 2011). While plastome organization, gene order, and content has been thought to be conserved among land plants (Odintsova and Yurina, 2003;Wicke et al., 2011;Cai et al., 2015;Smith and Keeling, 2015;Reginato et al., 2016), distinct patterns, rearrangements, differences in structure, size, gene content and order have been documented (Chumley et al., 2006;Haberle et al., 2008;Guisinger et al., 2011;Weng et al., 2014;Firetti et al., 2017;Fonseca and Lohmann, 2017). Furthermore, expansions and contractions of IRs with different orders of magnitude have occurred multiple times during land plant evolution (Zhu et al., 2016;. These shifts result in gene gains or losses attributed to the transfer of genes from SC regions into the IRs or otherwise, leading to plastome size variation among plant lineages (Goulding et al., 1996;Chumley et al., 2006;Raubeson et al., 2007;Wang et al., 2008;Dong et al., 2013;Sun et al., 2013;Zhu et al., 2016;Firetti et al., 2017).
The lianescent genus Amphilophium Kunth emend L.G. Lohmann includes 47 species and represents the third largest genus of the Neotropical tribe Bignonieae (Bignoniaceae, Lamiales) (Lohmann and Taylor, 2014). Species of Amphilophium occur from Mexico and the Antilles to northern Argentina, southern Brazil, and Uruguay, where they grow in wet and dry forests, or are restricted to savannas or the Amazonian "campinas" (Lohmann and Taylor, 2014). Species of the genus have attractive flowers and interesting fruit morphology, being commonly cultivated through South-Western United States (Lohmann, in review), Latin America, and Asia (Pool, 2007a,b). Corolla shape and fruit morphology can be highly variable (Alcantara and Lohmann, 2010). The first molecular phylogenetic studies to sample Amphilophium were based on the plastid gene ndhF and the nuclear pepC and included 11 species (Lohmann, 2006;Lohmann et al., 2013). These studies aimed at re-evaluating generic limits (Lohmann, 2006) and studying broad-scale biogeographical patterns (Lohmann et al., 2013) within the whole tribe Bignonieae. A subsequent phylogenomic study of the genus used sequences of 78 plastid-coding genes of 32 species of Amphilophium to reconstruct specieslevel relationships and the fine-scale biogeographic history of the genus . Thode et al. (2019) recovered a strongly supported phylogeny of Amphilophium, corroborating the monophyly of the genus and its division into five main clades (Lohmann, 2006;Lohmann et al., 2013). These five clades differ morphologically from each other and generally correspond to genera recognized in the past (Gentry, 1973;Pool, 2007aPool, ,b, 2009. Despite the existence of phylogenetic and biogeographic information for Amphilophium (Lohmann, 2006;Lohmann et al., 2013;Thode et al., 2019), the plastome structure for the members of this genus remains unknown.
The first complete Bignoniaceae plastome reported in the literature was that of Tanaecium tetragonolobum (Jacq.) L.G. Lohmann (tribe Bignonieae) (Nazareno et al., 2015). This plastome is 153,776 base pairs (bp) long, with a typical quadripartite structure, including 142 genes. Plastomes of eight Anemopaegma species (Firetti et al., 2017), and ten species from the "Adenocalymma-Neojobertia" clade (Fonseca and Lohmann, 2017) were published subsequently. Among all Lamiales plastomes published to date, those from Anemopaegma are the largest (Firetti et al., 2017). The plastomes of the Anemopaegma species range from 167,413 to 168,987 bp and include 141 genes (Firetti et al., 2017). The large size of the Anemopaegma plastomes is associated with the large amount of repetitive sequences and expansion of the IRs (Firetti et al., 2017). On the other hand, the plastomes of the "Adenocalymma-Neojobertia" clade range from 157,027 to 159,725 bp, and generally include 132 genes, although the ycf4 gene was lost in two species sampled (Fonseca and Lohmann, 2017). Plastomes of the "Adenocalymma-Neojobertia" clade also show a series of genomic translocations (Fonseca and Lohmann, 2017). Apart from the Bignonieae plastomes, the plastome of Crescentia cujete L., a member of the Tabebuia alliance (sensu Olmstead et al., 2009), was also sequenced (Moreira et al., 2016). This plastome is 154,662 bp in length and includes 132 genes (Moreira et al., 2016).
In this study, we assembled the complete plastomes of 11 species of Amphilophium (Bignonieae, Bignoniaceae) representing the breath of the morphological diversity of the genus and the five main clades recovered previously (Lohmann, 2006;Lohmann et al., 2013;Thode et al., 2019), plus that of Anemopaegma prostratum DC., an outgroup. This study aims to improve our understanding of plastome characteristics, structural diversity, and evolution within tribe Bignonieae. For that, we: (i) characterized the overall plastome structure; (ii) performed comparative genomic analyses within Amphilophium, and among Amphilophium and other Bignonieae genera; (iii) documented selection patterns within Amphilophium plastid genes; and (iv) identified putative repeated regions.  Nazareno et al., 2015 KR534325 verified the boundaries between the LSC, the IRs, and the SSC iteratively using the software afin 1 and by searching the specific motifs from each junction in the original read pool using the UNIX "grep" function for all plastomes assembled. The reads found with the sequences of the junctions between the plastome regions were later assembled in Sequencher 5.3.2 (Genecodes, Ann Arbor, MI, United States). Plastome annotations were performed in Geneious 9.1.5 (Kearse et al., 2012), DOGMA (Wyman et al., 2004), and BLAST (Altschul et al., 1990(Altschul et al., , 1997, with Open Reading Frames (ORFs) checked manually by searching for the start and stop codons. The graphical representations of each plastome with annotations were created in OGDRAW (Lohse et al., 2013). In addition, the junction sites between the LSC/IRa/SSC/IRb regions with full annotations for the adjacent genes were manually analyzed in Geneious, examined, and plotted in IRscope (Amiryousefi et al., 2018) 2 .
The 11 Amphilophium plastome sequences were aligned in MAFFT 7 (Katoh and Standley, 2013) using the FFT-NS-2 method (Katoh et al., 2002). To identify variable regions and intra-generic variations within the genus, we visualized the alignment using mVISTA (Frazer et al., 2004) in Shuffle-LAGAN mode (Brudno et al., 2003), using the annotated plastome of A. paniculatum as reference. The same alignment was used to calculate the nucleotide variability values (π) within Amphilophium plastomes. The sliding window analysis was performed in DnaSP 6.10 (Rozas et al., 2017) with step size of 200 bp and window length of 800 bp. We plotted the π values using R (R Development Core Team, 2017).

Selection on Plastid Genes
To evaluate the role of selection on the plastid-coding regions, we used the CODEML application in PAML 4.8 (Yang, 2007) performing a Bayesian identification of codon sites under positive selection. This analysis infers the omega values (ω) in codon alignments of protein-coding sequences and tests for positive selection. The omega value measures the ratios of the nonsynonymous and synonymous substitution (ω = dN/dS) (Nielsen and Yang, 1998). Sites are considered to be under negative selection (deleterious or purifying selection) when ω < 1; under neutrality (when the substitution does not change the amino acid) when ω = 1; and under positive selection (adaptive selection) when ω > 1. The fixation of advantageous mutations (adaptive evolution) may be related to evolutionary innovations and species divergence. The 78 protein-coding genes (see above) of the 11 Amphilophium plastomes and that of Anemopaegma prostratum were aligned in Geneious, using the translation alignment tool ClustalW plugin. The CODEML analysis for each gene was performed using as the constraint topology the ML tree from Thode et al. (2019). The terminal and corresponding internal branches of the taxa that were not sampled in this study were removed from the tree in the R package "ape" (Paradis and Schliep, 2018) using the function "drop.tip." Anemopaegma prostratum was designated as outgroup. Parameters were: runmode = 0, seqtype = 1, CodonFreq = 2, and model = 0, and NSsites = 2 (modeling three classes of sites: 0 < = ω < 1, ω = 1, and ω > 1). Results were considered significant when the posterior probability (Pr) >0.95.

Repeat Analyses
We used MISA (Beier et al., 2017) to identify and locate microsatellites or Simple Sequence Repeats (SSRs; i.e., tandemly arranged repeats of short DNA motifs of 1-6 bp in length) in the plastomes of the Amphilophium species and Anemopaegma prostratum. The following criteria were used while searching for SSRs: SSR motif length between one and six nucleotides, with a minimum number of repetitions set as 10, 5, and 4 units for mono-, di-, and trinucleotide SSRs, respectively, and three units for each tetra-, penta-, and hexanucleotide SSRs. We used REPuter (Kurtz et al., 2001) to identify forward, palindrome, reverse, and complement repeated elements with a minimum repeat size ≥30 bp and a sequence identity ≥90% (Hamming distance = 3).

Assembly and Characteristics of the Chloroplast Genomes
The eleven Amphilophium plastomes range in length from 155,262 (A. gnatophalantum) to 164,749 bp (A. steyermarkii) (Table 2, Figure 1, and Supplementary Figures S1, S2). A minimum of 8,102,426 paired-end raw reads, and a maximum of 23,885,903 reads, with average read depths between 54.5 and 248x for A. cuneifolium and A. dolichoides were obtained, respectively (Supplementary Table S1). All plastomes show the typical quadripartite structure of angiosperms, which consists of a LSC, with length between 75,206 (A. steyermarkii) and 84,697 bp (A. chocoensis); a SSC with length between 12,595 (A. dusenianum) and 12,852 bp (A. chocoensis); and a pair of IRs with length between 29,701 (A. chocoensis) and 38,390 bp (A. steyermarkii) (  Figure 2A). The IR is expanded at the LSC/IRa and IRb/LSC boundaries in some Amphilophium species and in Anemopaegma relative to Adenocalymma peregrinum (Fonseca and Lohmann, 2017) and Tanaecium tetragonolobum (Nazareno et al., 2015; Table 2 and Figures 1, 2). The coding regions of the 11 Amphilophium plastomes range from 83,262 (A. chocoensis) to 88,536 bp (A. steyermarkii). The noncoding regions vary from 71,907 (A. gnatophalantum) to 76,284 bp (A. paniculatum). In Anemopaegma prostratum the coding  regions are 89,640 bp in length, while the noncoding regions are 78,532 bp ( Table 2 and Supplementary Figure S2B). The average GC content is 37.8% for all species studied ( Table 2), similar to other Bignoniaceae plastomes sequenced to date (Nazareno et al., 2015;Moreira et al., 2016;Firetti et al., 2017;Fonseca and Lohmann, 2017). The 12 plastomes assembled here encode 113 unique genes, including 79 protein-coding genes (CDS), 30 tRNA genes, and four rRNA genes (Tables 2, 3 and Supplementary Table S2). The number of duplicated CDS in the IRs varies depending on the degree of IR expansion and contraction of the LSC regions. While some species show eight duplicated CDS in the IRs (i.e., ndhB, rpl2, rpl23, rps12, rps7, ycf1, ycf2, and ycf15), others show 18 (i.e., the previous eight regions plus infA, rpl14, rpl16, rpl22, rpl36, rpoA, rps11, rps19, rps3, and rps8), or 19 duplications (the previous 18 regions plus petD) (Tables 2-4 and Figure 1). All species include seven tRNA and all four rRNA genes duplicated in the IR regions. The total number of genes ranges from 132 to 143 (Tables 2, 4 and Figure 1). The plastomes assembled in this study include 18 intron-containing genes, of which 15
In all Amphilophium studied, one copy of the duplicated rps15 is a pseudogene (ψ) that is 141-240 bp long and is located within the boundary between IRa/SSC, while the functional rps15 gene is 270-279 bp long and located within the SSC/IRb border. In Anemopaegma prostratum, the ψrps15 is 261 bp long, while the rps15 gene is 279 bp (Figure 2). The IRb/LSC junction in A. chocoensis, A. cuneifolium, A. gnatophalantum, and A. lactiflorum is between rpl2 and the trnH-GUG genes (Figure 2A); in A. carolinae, A. dolichoides, A. dusenianum, A. ecuadorense, A. paniculatum, and A. pilosum it is between †petD and trnH-GUG (Figures 2B,C); and in A. steyermarkii, Anemopaegma prostratum, and Anemopaegma arvense (Firetti et al., 2017) it is between †petB and trnH-GUG (Table 4 and Figures 2D,E). The structure found in the IRa/SSC/IRb borders of the A. steyermarkii and Anemopaegma prostratum is similar to that found in Anemopaegma arvense (Firetti et al., 2017; Figures 2D,E), and in the plastomes of seven other Anemopaegma species (Firetti et al., 2017). In Adenocalymma peregrinum (Fonseca and Lohmann, 2017), all boundaries are similar to those found in A. chocoensis, A. cuneifolium, A. gnatophalantum, and A. lactiflorum (Figures 2A,F). The boundaries between all regions are different in the plastome of Tanaecium tetragonolobum (Nazareno et al., 2015; Figure 2G) when compared to those from Amphilophium, Adenocalymma, and Anemopaegma (Figure 2). In T. tetragonolobum, the LSC/IRa boundary is located between the rpl22 and rps19 genes, while the IRa/SSC border is located between ψycf1 and the ndhF gene, and the SSC/IRb border is within the ycf1 gene (Nazareno et al., 2015; Table 4 and Figure 2G). The plastomes of Amphilophium, Adenocalymma, and Anemopaegma include an entire duplication of the ycf1 gene in the IRs (Firetti et al., 2017;Fonseca and Lohmann, 2017 ; Figures 1, 2).

Identification of Variable Regions
The structural analysis performed in Mauve retrieve five synteny blocks (Supplementary Figure S3). Amphilophium and Adenocalymma peregrinum plastomes (Fonseca and Lohmann, 2017) show the same structure and linear order and are similar to those observed in Anemopaegma arvense (Firetti et al., 2017), Anemopaegma prostratum, and Tanaecium tetragonolobum (Nazareno et al., 2015), except for two local changes. The first is a large inversion of approximately 8 kb, located in the IR regions of both Anemopaegma plastomes, comprising the genes rpl23, trnL-CAA, ycf2, and trnI-AAU (Supplementary Figure S3: yellow block). The second is a smaller inversion (∼1,800 bp) observed within the ycf1 gene in the plastome of Tanaecium tetragonolobum (Supplementary Figure S3: blue block). No major inversions are found within the Amphilophium and Adenocalymma peregrinum plastomes (Supplementary Figure S3).

SSR and Tandem Repeat Analyses
We screened and identified six kinds of repeat patterns using MISA. In Amphilophium plastomes, the total number of SSRs range from 44 (A. paniculatum) to 57 SSRs (A. dusenianum), while 42 SSRs are recovered in Anemopaegma FIGURE 3 | Comparison of the assembled Amphilophium plastomes using mVISTA. Complete plastomes of Amphilophium species are compared using A. paniculatum as reference. Blue blocks indicate conserved genes, while red blocks indicate conserved noncoding sequences (CNS). White blocks represent regions with sequence variation among the 11 Amphilophium species. Gray arrows indicate the direction of gene transcription. prostratum (Figure 5). The most abundant SSRs are A or T mononucleotide repeats, which account for 54-69.6% of the total SSRs; G or C repeats, on the other hand, are rare ( Figure 5A and Supplementary Table S3). The total number of SSR motifs in Amphilophium is as follows: 29-39 (58-74%) mono-, 2-4 (3.6-8%) di-, 3-7 (6.5-15%) tri-, 4-9 (7-17%) tetra-, 0-5 (0-9.6%) penta-, and 0-2 (0-4.8%) hexanucleotides ( Figure 5A and Supplementary Table S3). Furthermore, most of the SSRs in the Amphilophium species are located in the LSC region and range between 71.2 and 86.4%. In Amphilophium, the IR regions include between 8.5 and 22% of the SSRs, while the SSC region include between 2 and 8.8% (Figure 5B and  Table S3). SSRs are found mainly in intergenic regions. The plastomes of the Amphilophium species contain between 57.4 and 82% of the SSRs in the intergenic spacers, between 14.6 and 24% in the coding regions, and between 12 and 20.8% in the introns (Figure 5C and Supplementary Table S3). In Anemopaegma prostratum, 69% of the SSRs are located in the LSC, 23.8% in the IRs, and 7.1% in the SSC region. Of the total number of SSRs found in A. prostratum, 66.7% are in the intergenic regions, 23.8% in the coding regions, and 9.5% in the exons (Figures 5B,C and Supplementary Table S3).
We also used REPuter to identify the tandem repeat sequences of ≥30 bp of the Amphilophium and Anemopaegma prostratum plastomes. The total number of repeats in Amphilophium range between 38 (A. lactiflorum) and 56 (A. dusenianum), all located in the LSC and IR regions, with maximum sizes ranging from 50 to 150 bp (Figures 5D,E and Supplementary Tables S4, S5). The Amphilophium plastomes contain between 33 and 50 forward repeats, and 1 to 6 palindrome repeats, with reverse repeats being rare, ranging from 0 to 3 (Supplementary Table S4). In most Amphilophium plastomes, repeats with 30-39 bp are the most common, except in A. carolinae, A. dolichoides, and A. steyermarkii, all of which have a large number of repeats ranging from 40 to 49 bp ( Figure 5D and Supplementary  Table S4). These repeats are found predominantly in intergenic regions (14-36 bp) and exons (12-33 bp), with a few repeats located in the introns (0-8 bp) (Figure 5F and Supplementary Table S4). In Anemopaegma prostratum plastomes, the total number of repeats is 50, three of which are located in the LSC and 47 located in the IR regions; 24 are located in the intergenic regions and 26 in the exons; 49 are forward repeats and one palindrome with a maximum size of 165 bp. Different from the Amphilophium plastomes, most of the repeats in A. prostratum range between 60 and 69 bp (Figures 5D-F and  Supplementary Tables S4, S5). The locations of the repetitive sequences vary among Amphilophium species, although some regions show repeats on all 11 species (e.g., accD, rbcL/accD, ycf1, and ycf2), while some locations show repeats on most species (e.g., rps12/trnV-GAC, trnN-GUU/ycf1, ycf3, psbT/psbN, rps11, rpl23/trnI-CAU) (Supplementary Table S5).

Plastome Features
In this study, we assembled 11 complete plastomes of Amphilophium species and the plastome of Anemopaegma prostratum, another species from tribe Bignonieae. The organization of Amphilophium plastomes is similar among the species studied and other angiosperm plastomes. Amphilophium plastomes show expansions of the IRs and contractions on the LSC in some species. The overall genomic structure among Amphilophium plastomes is not conserved though, including differences in length, boundaries between the SC/IR regions, number of duplicated genes in the IRs, and total length (Tables 2, 4 and Figures 1-4). We detected a difference of nearly 9.5 kb between the smallest (A. gnatophalantum) and largest (A. steyermarkii) genomes, respectively (Table 2 and Figure 2F). Expansions of the IRs of ca. 8.7 kb and LSC contractions of ca. 9.5 kb are observed (Table 2, Figures 1, 2 and Supplementary  Figure S1). The Anemopaegma prostratum plastome also shows an IR expansion and a LSC contraction, similar to the plastomes of eight other Anemopaegma species sequenced in a previous study (Firetti et al., 2017). Nonetheless, the IR expansion of Anemopaegma plastomes is even larger than those found in Amphilophium plastomes, with a ca. 10 kb expansion, when the IRs of A. gnatophalantum and Anemopaegma prostratum are compared. On the other hand, the LSC in the Anemopaegma prostratum plastome is ca. 9.4 kb smaller than that of A. chocoensis ( Table 2). The SSC show a small variation in size within Amphilophium plastomes, with a difference of 257 bp between the smallest (A. dusenianum) and largest regions (A. chocoensis) (Table 2 and Figures 1, 2). According to the IR expansion toward the LSC, the Amphilophium plastomes exhibit different junctions between regions (i.e., between rps19 and rpl2, within petD, and within petB), as well as a different number of completely duplicated protein-coding genes (i.e., eight, 18, or 19). Besides the expansion of the IRs and differences in the boundaries of the regions within the Amphilophium plastomes, no rearrangements or major inversions are detected. An inversion of ∼8 kb that includes the genes rpl23, trnL-CAA, ycf2, and trnI-AAU is observed in Anemopaegma prostratum and in the plastomes of other eight Anemopaegma species. However, these inversions were not observed in any other Lamiales (Firetti et al., 2017;Supplementary Figure S3). The boundary positions observed in Anemopaegma prostratum were conserved among eight other Anemopaegma plastomes (Firetti et al., 2017). The Anemopaegma plastomes are the largest described to date for Lamiales, with 19 completely duplicated CDS in the IRs (Firetti et al., 2017). PCR amplifications were performed to check the boundary positions and the inversion of the ycf2 gene in Anemopaegma (Firetti et al., 2017).
The IR/SC boundaries are conserved in ten plastomes of the "Adenocalymma-Neojobertia" clade (Fonseca and Lohmann, 2017). Despite that, the genome structure is quite variable within the "Adenocalymma-Neojobertia" clade, with rearrangements in the LSC and IR regions and a complete loss of the ycf4 gene in two species (Fonseca and Lohmann, 2017). Furthermore, plastomes of the "Adenocalymma-Neojobertia" clade show eight duplicated CDS in the IRs (Fonseca and Lohmann, 2017). All the boundaries between plastome regions of Tanaecium tetragonolobum (Nazareno et al., 2015) and Crescentia cujete (Moreira et al., 2016) are located in positions that are different from those of Amphilophium, Adenocalymma, and Anemopaegma. In these two species, the LSC/IRb boundary is located between the rpl22 and rps19 genes, the IRb/SSC border is located between the ψycf1 and the ndhF gene, and the SSC/IRa border is located within the ycf1 gene (Nazareno et al., 2015;Moreira et al., 2016 ; Figure 2). The plastomes of these two species also show a partial duplication of the ycf1 (ψycf1) and a duplication of the complete copy of the rps15 gene. Differently, the plastomes of Amphilophium, Adenocalymma, and Anemopaegma show a complete duplication of the ycf1 gene as well as a partial duplication of the ycf15 in the IRs (Firetti et al., 2017;Fonseca and Lohmann, 2017 ; Figures 1, 2). Part of the ycf1 and ycf15 genes are included in the SSC region in other angiosperm groups (Dugas et al., 2015). The shift of the IRs/SSC junctions in Amphilophium, Anemopaegma, and the "Adenocalymma-Neojobertia" clade result in the expansion of the IRs and contraction of the SSC (Firetti et al., 2017;Fonseca and Lohmann, 2017). The expansion of the IRs toward the SSC has also been reported in Pelargonium (Chumley et al., 2006), members of Apiales (Downie and Jansen, 2015), in some Leguminosae genera (Dugas et al., 2015), and in Lamprocapnos spectabilis (Papaveraceae) . Multiple instances of IR expansion and/or contraction occurred during land plant evolution, with movement of entire genes from the SC regions into the IR or vice-versa (Zhu et al., 2016). The terminal IR gene adjacent to the SSC region is usually more conserved across land plants, however the IR/LSC boundary has changed more dynamically during the evolution of plant lineages (Raubeson et al., 2007;Wang et al., 2008;Dong et al., 2013;Zhu et al., 2016). While most shifts are small, others have expanded or contracted the IR by several kb, resulting in gene gains or losses as a consequence of the relocation of genes into or out of the IR (Goulding et al., 1996;Chumley et al., 2006;Wang et al., 2008;Sun et al., 2013;Zhu et al., 2016;Firetti et al., 2017;. Notable examples of size variation in the IRs due to boundary shifts are found, for example, in Monsonia speciosa (7 kb) (Guisinger et al., 2011), Lamprocapnos spectabilis (51 kb) , and Pelargonium transvaalense (88 kb) (Chumley et al., 2006), though the angiosperm IR is typically 25 kb . IR expansions and contractions often result in variation of genome size among different plant groups and are important for plastome evolution (Kim and Lee, 2005;Wang et al., 2008;Asaf et al., 2016;Dong et al., 2016;Yang et al., 2016;Zhang et al., 2016;Zhu et al., 2016;Xu et al., 2017;Li and Zheng, 2018).
The different patterns observed in the Amphilophium plastomes in terms of LSC/IR and IR/SSC boundaries, number of duplicated genes, and genome sizes are mostly shared among taxa that belong to the same clade . Anemopaegma, used here as outgroup, showed a plastome structure that is similar to that found in A. steyermarkii (Figures 1, 2). Nonetheless, Anemopaegma is not necessarily the closest relative of Amphilophium, as the genus is sister to a clade comprising Anemopaegma Mart. ex Meisn., Bignonia L., Mansoa DC, and Pyrostegia C. Presl (Lohmann, 2006;Lohmann et al., 2013). A larger sampling within Amphilophium is necessary to further investigate the evolution of plastomes within the genus. Broader scale studies within tribe Bignonieae as a whole would certainly provide novel insights into the high diversity found in the structure, composition, and organization of plastomes in Adenocalymma (Fonseca and Lohmann, 2017), Amphilophium (this study), Anemopaegma (Firetti et al., 2017), and Tanaecium (Nazareno et al., 2015).
While the conservation of plastome structure and low levels of nucleotide diversity have been observed in several groups (Odintsova and Yurina, 2003;Wicke et al., 2011;Cai et al., 2015;Smith and Keeling, 2015;Reginato et al., 2016), our results show that plastomes may be variable within closely related lineages. Plastome rearrangements, differences in structure, size, gene content, and order were documented in many other angiosperm groups (Goulding et al., 1996;Chumley et al., 2006;Raubeson et al., 2007;Haberle et al., 2008;Wang et al., 2008;Guisinger et al., 2011;Dong et al., 2013;Weng et al., 2014;Zhu et al., 2016;Firetti et al., 2017;Fonseca and Lohmann, 2017;. Altogether, these results bring new insights into the evolution of plastomes, suggesting that plastomes may be highly conserved or highly variable in different plant groups. The analyses of complete Bignonieae plastomes indicate that genomes are variable at both the genus and species level within this tribe (Nazareno et al., 2015;Firetti et al., 2017;Fonseca and Lohmann, 2017).

Signature of Positive Selection on Plastid Genes
Our study shows that among the 78 protein-coding genes within Amphilophium, 16 are significantly under positive selection (ω > 1) (i.e., ycf1, ycf2, rpoA, accD, rps18, rps7, ycf4, clpP, rbcL, rpoC1, rps2, rpoC2, infA, atpA, rps8, and rps16). Three of these genes (namely ycf1, accD, and rbcL) have been reported to be putatively under positive selection in Brassicaceae out of 10 genes identified with ω > 1 for the family (Hu et al., 2015). Within six species of Ipomoea, the genes accD, cemA, and ycf2 were under positive selection (Park I. et al., 2018). Within eight Anemopaegma, on the other hand, four genes (i.e., atpB, ndhA, petA, and psaB) out of 70 protein-coding genes were shown to be under positive selection (Firetti et al., 2017). Positive selection on the clpP gene has been also observed in Geranium (Park et al., 2017), legume (Dugas et al., 2015), Silene (Erixon and Oxelman, 2008), and Lamprocapnos  species. The chloroplast genes ndhF and matK also showed positive selection in previous studies. The matK gene is often used in phylogenetic studies (Carbonell-Caballero et al., 2015;Daniell et al., 2016) and showed to be positively selected in more than 30 plant groups, suggesting that this gene is subject to distinct ecological selective pressures (Chen and Xiao, 2010;Daniell et al., 2016). The positive selection signatures found on a high number of plastid genes within Amphilophium, suggest that these genes might be undergoing adaptative evolution in response to the environment (Kimura, 1989;Hu et al., 2015;Raman and Park, 2016;Ivanova et al., 2017). These results might be also associated with the remarkable morphological and ecological variation found among members of the genus. Amphilophium species show extremely diverse flower morphologies, occur in various environments, and show significant variation in diversification rates . Nonetheless, while plastid genes have been suggested to show signatures of positive selection (e.g., Erixon and Oxelman, 2008;Chen and Xiao, 2010;Carbonell-Caballero et al., 2015;Dugas et al., 2015;Hu et al., 2015;Daniell et al., 2016;Firetti et al., 2017;Park et al., 2017;Park I. et al., 2018), further studies that integrate field experiments, physiology, and molecular evolutionary biology are needed to understand this topic and the significance of adaptative evolution in plastid genes (Bock et al., 2014). Plastomes are shaped by the selective forces that act on the fundamental cellular functions that they code for and are, thus, expected to display signatures of the adaptive path undertaken by different plant species during evolution (Hu et al., 2015). Understanding the patterns of adaptation and divergence among the representatives of specific phylogenetic clades may provide important insights about the forces driving evolution (Wicke et al., 2014;Hu et al., 2015).

SSRs in Amphilophium Plastomes
Single Sequence Repeats (SSRs) are repeats of 1-6 bp frequently observed in plastomes that are important markers for evolutionary studies, population genetics, and for the study of genome polymorphisms (Avise, 1994;Ebert and Peakall, 2009;Qi et al., 2016;Yu et al., 2017). In this study, the number of SSRs found within Amphilophium plastomes ranged from 44 to 57, while 42 SSRs are documented in Anemopaegma prostratum. These results are similar to the 36-47 SSRs documented previously for Anemopaegma plastomes (Firetti et al., 2017), but significantly lower than the 347 chloroplast SSRs found for Tanaecium tetragonolobum (Nazareno et al., 2015). In these two studies (Nazareno et al., 2015;Firetti et al., 2017), the SSRs were identified with a less stringent threshold than the one used here (i.e., seven to mononucleotide repeats, four to di-and three to, tri-, tetra-, penta-, and hexa-). As in Anemopaegma and Tanaecium, mononucleotide repeats are the most common SSRs found in noncoding regions of Amphilophium plastomes. Most SSRs contain A or T motifs, contributing to the overall plastome AT richness (Qian et al., 2013;Cauz-Santos et al., 2017;Park et al., 2017;Li and Zheng, 2018). The largest amount of SSRs is located in the LSC. These SSRs will be useful for future population genetic studies involving Amphilophium (Figures 5A-C). Dispersed repeats represent a major component of plastomes and influence genome structure in terms of genome size, genome recombination and rearrangements, and gene duplication (Cavalier-Smith, 2002;Nie et al., 2012). In this study, the number of repeats in Amphilophium plastomes found by REPuter range from 38 to 56, with 50 repeats being found in Anemopaegma prostratum. This finding was similar in Tanaecium tetragonolobum, which included 47 repeats (Nazareno et al., 2015), but different to eight other Anemopaegma species studied that showed between 88 and 169 dispersed repetitive sequences, the highest number documented within Lamiales to date (Firetti et al., 2017). Most repeat sequences within Amphilophium are 30-39 bp long, except from the repeats found in A. carolinae, A. dolichoides, and A. steyermarkii ( Figure 5D). These three species show multiple repeats with 40-49 bp and the largest number of repeats >80 bp. Most dispersed repetitive sequences are found in noncoding regions ( Figure 5F).

CONCLUSION AND FUTURE DIRECTIONS
The comparative analyses involving 11 Amphilophium plastomes and the plastome of Anemopaegma prostratum provided important new insights into Bignoniaceae plastome structure and evolution. Within Amphilophium, plastomes show different boundaries between the IR/SC regions, lengths, and number of duplicated genes in the IRs as well as high nucleotide variability and signature of positive selection. Our results show that plastomes may be highly variable, even at low taxonomic levels, indicating that differences in plastome structure, gene content, and nucleotide diversity vary among different plant groups. A larger sampling of taxa, including complete plastomes for a higher number of representatives of Amphilophium and other genera of tribe Bignonieae is necessary to further investigate the evolution of plastome structure in the genus and in the tribe as a whole.