The distinct plastid genome structure of Maackia fauriei (Fabaceae: Papilionoideae) and its systematic implications for genistoids and tribe Sophoreae

Ethics statement

Maackia fauriei is not an endangered or protected species in Korea. We did not collect plants from the protected areas that required permission.

Plastid genome sequencing

Leaves of Maackia fauriei were collected from Jeju Island, Korea, and preserved with silica gel. Voucher specimen (J.Y. Lee & I.S. Choi 1208046) were deposited in the Herbarium of Inha University (IUI), Incheon, Korea. Genomic DNA was extracted from the dried tissues by a protocol that utilized the DNeasy Plant Mini Kit (Qiagen, Seoul, Korea). The quantity and quality of the extracted genomic DNA were assessed by gel electrophoresis and spectroscopy with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). The gDNAs were then fragmented into 500-bp segments for library construction per the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA), and were sequenced using the Illumina HiSeq 2000 System (Illumina Inc.) at the BML Co. in Daejeon, Korea.

Plastid genome assembly

The sequencing run produced 25,318,060 paired-end reads (101 bp each). Poor-quality sequences were removed by Trimmomatic 0.32 [36]. To isolate plastid-related reads and assemble the plastome, we largely followed the method described by Wang and Messing [37]. Briefly, the paired-end reads were mapped on the reference genome of Lupinus luteus L. (GenBank Accession Number NC_023090) using Geneious ver. 7.1.3 (Biomatters Ltd., Auckland, NZ). The mapped reads were then reassembled de novo using Geneious and the generated plastome contigs were re-aligned to the plastome of L. luteus to identify gaps among the contigs. This generated four contigs (two for LSC and two each for the SSC and IR regions) with a total length of approximately 154 kb. To fill those gaps, we used purified DNA and performed polymerase chain reactions (PCRs) with primers designed via Primer3 [38].

Gene annotation

The complete plastome for M. fauriei was annotated using DOGMA [39] and Geneious. The tRNAs were confirmed by tRNAscan-SE [40]. Other protein-coding regions were checked based on data from the NCBI (http://blast.ncbi.nlm.nih.gov/), and manual corrections were made for the start and stop codons. Particular gene features of the plastome were illustrated with the Web-based tool OGDraw [41].

Investigation of rps16 among legume plastomes

The loss of rps16 was investigated in complete plastomes from 33 legume species (Table 1). Each rps16 gene was extracted from the available sequenced genomes and sequences were aligned by MUSCLE 3.8.31 [42] using default parameters. We re-analyzed and categorized this gene in four ways, as in Kim et al. [43]: 1) intact gene (full-length and in-frame), 2) putative pseudogene (mutation in start or stop codon or frame shift-inducing indels), 3) truncated gene (significant deletion), and 4) complete deletion.

Whole-genome alignments

Other plastome sequences for relevant legumes (Table 1) were obtained from GenBank. Single IR copies were manually removed. Afterward, the complete plastome of M. fauriei and other sequences [Tamarindus indica L. (KJ468103), Arachis hypogaea L. (KJ468094), and Lupinus luteus (NC_023090)] were aligned using genome alignment software Mauve 2.3.1 [44] to check for inversion events.

Survey of inversion events among genistoids and Sophoreae s.l.

To verify the distribution of 36-kb and 24-kb inversions on the phylogenetic tree, we used methods based on PCR amplifications. In all, 16 representative species were selected that belong to five tribes and 15 genera (Table 2). They roughly cover the genistoids and Sophoreae s.l. group. Those plant materials had been collected from East Asia (China, Korea, and Japan) and deposited at the IUI herbarium. Other DNA samples were obtained from the DNA Banks of the Royal Botanic Gardens, Kew (http://apps.kew.org/dnabank/homepage.html) and the University of Johannesburg (UJ), South Africa (http://acdb.co.za/index.php/dna-bank/introduction-2.html). The remaining materials needed for this study were also collected through KNRRC (Medicinal Plants Resources Bank NRF-2010-0005790), supported by the Korea Research Foundation and the Ministry of Education, Science and Technology in 2014. Vouchers were deposited at the herbarium of Gachon University (GCU). The gDNAs were extracted from silica gel-dried leaves, as described above. All PCRs were conducted with a GeneAmp^® PCR System 2700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) according to a program of initial denaturation that was followed by 30 cycles of 10 s at 98°C, 7 s at 58°C, and 2 min at 72°C; and then a final extension for 5 min at 72°C. Each 50-μL reaction mixture included 1 μL of genomic DNA (~ 20 ng), 1 μL each of forward and reverse primers (10 pMol), and 25 μL of PrimeSTAR HS Premix (TaKaRa, Seoul, Korea). The existence of the 36-kb inversion was tested using primer pairs designed by Martin et al. [32]. The pair of rps4-bef-F and ycf3-bef-R was used for determining its absence while ycf3-inv-F and psbI-int-R were used to detect its presence. Three primers were designed to test for the 24-kb inversion: the pair of FGA-ndhJ-F and FGA-trnF-R for its absence and the pair of FGA-ndhJ-F and FGA-trnC-R for its presence (Table 3). The PCR products were visualized on 2% agarose gels, purified by PCR quick-spin TM (iNtRON Biotechnology, Seongnam, Korea), and sequenced with an ABI 3100 Genetic Analyzer and an ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) at the Macrogen, Seoul, Korea.

Phylogenetic analysis

A phylogenetic tree was constructed using 43 complete or partial plastomes for legumes, as collected from the GenBank database (Table 1). The outgroup included Morus mongolica (Bureau) C.K. Schneid. (KM491711), Fragaria vesca L. (NC_015206), Castanea mollissima Blume (NC_014674), and Cucumis sativus L. (DQ119058), as described by Schwarz et al. [45]. The final data set from these 47 plastomes comprised 71 conserved plastid protein-coding genes: atpA, B, E, F, H, and I; ccsA; cemA; clpP; matK; ndhA, B, C, D, E, F, G, H, I, J, and K; petA, B, D, G, L, and N; psaA, B, C, I, and J; psbA, B, C, D, E, F, H, I, J, K, L, M, N, T, and Z; rbcL; rpl2, 14, 16, 20, 23, 32, 33, and 36; rpoA, B, C1, and C2; rps2, 3, 4, 7, 8, 11, 12, 14, 15, and 19; and ycf3. The alignments were made with MAFFT (v. 7.017) and default parameters. Poorly aligned regions were either refined or deleted using Geneious. A maximum likelihood (ML) analysis of the complete, final alignment of all taxa was conducted with RAxML Blackbox [46], using the gamma model of rate heterogeneity and an ML search.

Plastome sequence of Maackia fauriei and variation in rps16 loss among legumes

The complete plastome of Maackia fauriei (GenBank Accession No. KX388160) is 154,541 bp long and has two IR regions (25,494 bp each) that are separated by LSC and SSC regions of 85,140 bp and 18,413 bp, respectively (Fig 2). Approximately 57.9% of the sequence is composed of protein-coding regions while the remaining 42.1% contains non-coding sequences that include introns and intergenic spacers (IGS). The AT and GC contents are 63.5% and 36.5%, respectively. Among the 135 recognized genic features are four unique rRNAs, 31 tRNAs, and 76 protein-coding genes. The rpl22 loss, which is typical legume plastomes, is shared by Maackia. The rps16 is a putative pseudogene caused by “AAAC” duplication in exon2 that results in a frame shift mutation and internal stop codon.

We also investigated the loss of rps16 among 33 complete legume plastomes (Table 1) and found that the gene was intact in 15 species, a putative pseudogene in four species, truncated pseudogene in eight species, and deleted in six species. All of the Caesalpinioideae and Mimosoideae samples contained intact rps16. The types of losses varied for rps16 and were restricted to Papilionoideae. We considered the rps16s from Lupinus L. species as putative pseudogenes similar to that of M. fauriei due to several indel events on the exons and an approximately 200-bp deletion of intron sequences (Fig 3).

The 36-kb and 24-kb inversion events and their phylogenetic distribution among genistoids and Sophoreae s.l.

The legume species used for our comparisons revealed only subtle differences in their gene content. However, the gene order for Maackia fauriei did not resemble that of any other species. To trace these evolutionary changes, we selected four legume plastomes based on the results of phylogenetic analysis. Our Mauve alignment of the LSC region among M. fauriei and other legumes generated seven locally collinear blocks (LCBs) that represented a homologous region without rearrangement (Fig 4). These LCBs indicated that M. fauriei experienced at least three inversion events: 1) a 50-kb inversion between IGSs near accD and trnK (UUU), which is the major landmark in most papilionoid legume plastomes; 2) a 36-kb inversion situated between the 29-bp identical sequences of trnS (GGA) and trnS (GCU), a feature shared by Lupinus among core genistoids and Robinia L. in the robinioids; and 3) a 24-kb inversion embedded in the 50-kb inversion region, an event that is newly discovered from the M. fauriei plastome and which occurs between IGSs near trnC (GCA) and trnF (GAA).

To verify the distribution of these 36-kb and 24-kb inversions on our phylogenetic tree, we used PCR-screening with 16 representative species belonging to five tribes—14 genera of genistoids and the Sophoreae s.l. group. All sequences were deposited in GenBank (Accessions KX430180 through KX430211). This strategy demonstrated that three genera—Cladrastis Raf., Styphnolobium Schott, and Camoensia Welw. ex Benth.–did not have those inversions (Table 2 and Fig 5). However, the 36-kb inversion was distributed within the five tribes of core genistoids while the 24-kb inversion was shared by eight genera in three genistoid tribes—Euchresteae, Sophoreae s.s., and Thermopsideae. Two other genera from Sophoreae s.s.—Bolusanthus and Dicraeopetalum—did not share the 24-kb inversion and had only 36-kb inversions in their plastomes.

Phylogenetic analysis

Our data set for the phylogenetic analysis contained 71 protein-coding genes from 47 taxa, including 43 legumes and four outgroups. This accounted for 53,307 nucleotide positions. The ML analysis of those 47 taxa resulted in a single best-scoring tree with—lnL = 358979.245. Bootstrap analyses indicated that, except for two nodes outside of Papilionoideae, all nodes were supported by values of 100%. The ML phylogeny indicated that Caesalpinioideae was basal and paraphyletic and that Mimosoideae and Papilionoideae formed monophyletic groups nested within the Caesalpinioideae (Fig 6). We also recognized five sub-clades of papilionoids, i.e., dalbergioid s.l., genistoid, indigoferoid/millettioid, robinioid, and IRLC. The dalbergioid s.l. was the first diverging clade, followed by genistoids. Branch lengths were very short between nodes of dalbergioid s.l. and the genistoids. Maackia fauriei was nested within the genistoids with Thermopsideae (Baptisia) and Genisteae (Lupinus). The remaining three sub-clades formed the Old World clade, with indigoferoid/millettioid being the first to branch, followed by robinioid and IRLC. Among the 22 tribes of legumes examined here, six (Caesalpinieae, Mimoseae, Millettieae, Phaseoleae, Galegeae, and Trifolieae) were deemed non-monophyletic. Overall, the relationships within legumes were in agreement with recently described phylogenies [2–4]. The exception was the genistoids, which were resolved as a sister group to the Old World clade.

Characteristics of the Maackia fauriei plastome and independent rps16 loss in genistoids

The complete plastome of Maackia fauriei includes four rRNA, 31 tRNA, and 76 protein-coding gene species. As with the other legume plastomes [45], this genome lacks rpl22, which is transferred to the nucleus [48]. Moreover, this genome has sequences consistent with the presence of a putative pseudogenized rps16. Using slot blot hybridization with various legume species, Doyle et al. [49] found multiple and independent rps16 losses among papilionoids. Schwarz et al. [45] suggested that these losses have independently occurred at least five times in Papilionoideae, but not in genistoids. However, our analysis of rps16 loss patterns demonstrated that the plastomes of genistoids also experienced independent loss events. We categorized rps16 as an intact gene, putative pseudogene, truncated gene, or complete deletion (Table 2). The plastomes of Caesalpinioideae, Mimosoideae, and some of the Papilionoideae (i.e., Indigofera L., Millettia Wight & Arn., Pachyrhizus Rich. ex DC., Glycine L., and Lotus L.) have intact rps16s that have no internal indels in the exons except at the ends. Truncations occur sporadically in papilionoid legumes and are largely attributed to the severe deletion of exon2. Exon1 of rps16 is relatively conserved in papilionoid legumes, except for complete deletions, as observed with Arachis L., Apios Fabr., and most members of Fabeae. Putative pseudogenes are relatively rare and only observed within Wisteria Nutt. and genistoid species (Fig 3). The rps16 of Lupinus species has previously been annotated as in-frame and is regarded as an intact gene [32, 45]. However, it does not seem to be functional because of severe frame-shifts and deletions in the intron revealed by our investigation. Overall, rps16 losses exist for the five major clades—dalbergioid s.l., genistoid, indigoferoid/millettioid, robinioid, and IRLC—and all feature 50-kb inversions in their plastomes.

Systematic implications of plastome inversions in genistoids and Sophoreae

The genistoid is an informal taxonomic group of legumes that are characterized by quinolizidine alkaloids and a base chromosome number of n = 9 [5, 12, 50]. The tribes Genisteae, Crotalarieae, Euchresteae, Podalyrieae, Sophoreae s.s., and Thermopsideae are consistently resolved as a monophyletic group within genistoids, and are considered the core genistoids [3–4, 12, 14]. We found evidence here that the plastome of Maackia fauriei, belonging to Sophoreae s.s., has undergone three inversion events (50-kb, 36-kb, and 24-kb) based on our Mauve alignment with other legume species (Fig 4). Martin et al. [32] examined the distribution of the 36-kb inversion and their taxon sampling included two species outside of the genistoids [i.e., Cladrastis lutea (Michx.) K. Koch and “Sophora japonica”], plus nine genera of core genistoids belonging to one genus within Thermopsideae (Thermopsis) and eight genera within Genisteae (Argyrolobium Eckl. & Zeyh., Lupinus, Chamaecytisus Link, Laburnum Fabr., Retama Raf., Ulex L., Echinospartum Fourr., and Genista L.). They have argued that the 36-kb inversion is potentially a molecular synapomorphy for core genistoids. In the current study, we examined 16 representative species (Table 2) that included five core genistoid tribes (Crotalarieae, Euchresteae, Genisteae, Sophoreae s.s., and Thermopsideae), and also Sophoreae s.l., that are outside of core genistoid [Cladrastis wilsonii Takeda, Styphnolobium japonicum (L.) Schott (= Sophora japonica L.), and Camoensia brevicalyx Benth.]. The 36-kb inversion is absent from species outside of the core genistoid, including those in its putative sister group, Camoensia. The genus Camoensia was once treated as a core genistoid [3] because it was thought to be a member of Sophoreae s.s. [35]. However, Cardoso et al. [4] resurrected Camoensia as being in the monotypic tribe Camoensieae and treated it as part of a sister group instead. Our findings that differ from those of Martin et al. [32] are the plastome conformation and classification for Styphnolobium japonicum (= Sophora japonica) which is clearly not in the core genistoid group [5, 50–53]. Instead, our research indicates that S. japonicum lacks the 36-kb inversion, similar to its close relative Cladrastis. Three genera—Cladrastis, Pickeringia, and Styphnolobium—are currently known as being free from plastome rearrangements and forming a sister clade to vast papilionoid legume taxa marked by 50-kb inversion [2, 4]. Thus, this contrast with the report by Martin et al. [32] might be an outcome of mistakes made during the earlier experimental process rather than being an independent parallel-inversion event at the intra-species level. Unlike the case of Styphnolobium, the 36-kb inversion has occurred independently for the genus Robinia, which is thought to be distantly related evolutionarily but includes 50-kb inversion in its plastome [45]. Thus, the possible existence of a parallel 36-kb inversion from evolutionarily close taxa means that its value in molecular synapomorphy is questionable among genistoids. Additional plastome sequences from other early-diverging papilionoid legumes are warranted. Nevertheless, the absence of such an inversion in Camoensia but its presence in all core genistoid tribes tested here is distinct evidence that supports the recent research on core genistoids [4].

The Sophoreae s.l. have largely included the taxa with least specialized flowers that are similar to those within Caesalpinioideae [6–7] even though its type genus, Sophora, has more specialized and papilionaceous floral characteristics than other basal papilionoid legumes [33]. Recent molecular phylogenetic studies [3–5, 11–13] have also demonstrated that Sophoreae s.s. is more closely related to tribes of Euchresteae and Thermopsideae, which share specialized papilionaceous flowers. Likewise, our study demonstrated that the Sophoreae s.s. has a more specialized plastome organization (Fig 4) that has resulted from the combination of three inversion events (i.e. 50-kb, 36-kb, and 24-kb). This characteristic is also shared with Euchresteae and Thermopsideae. The new Sophoreae sensu Cardoso et al. [4] includes the merger of Euchresteae and Thermopsideae into Sophoreae and removal of a large number of genera that were traditionally considered members of Sophoreae. The formal taxonomic revision for new Sophoreae remains to be done and a synapomorphic characteristic for this group was lacking. Hence, the distinct plastome organization revealed from our study could be a putative molecular synapomorphy for new Sophoreae. In contrast, it is also conceivable that a 24-kb inversion occurred at distantly related lineages, as was the case for 36-kb parallel inversions [45]. However, the plastome conformation of Sophoreae s.s. is not likely to be a homoplasious characteristic. Researchers have assumed that the 36-kb inversion (39-kb inversion for Robinia) was mediated by flip-flop recombination between the conserved 29-bp repeats of two trnS genes [32, 45]. By comparison, the breakpoints of 24-kb inversion are IGSs near trnC (GCA) and trnF (GAA), which are not conserved sequences. Furthermore, the plastome conformation of Sophoreae s.s. was not formed by a single inversion but, combinationally and sequentially, through three independent inversion events. Hence, a 24-kb inversion seems to be unique to a monophyletic group that comprises Sophoreae s.s. and its closely related tribes Euchresteae and Thermopsideae.

Two genera, Bolusanthus and Dicraeopetalum, that are distributed in the Afro-Madagascan region, have long been included in Sophoreae (whether sensu stricto or lato) [4, 6–7, 35]. However, we found a heterogeneous plastome conformation (i.e., lack of a 24-kb inversion) when those two were compared with other Northern Hemisphere genera (Anagyris, Baptisia, Piptanthus, Thermopsis, Maackia, Salweenia, Sophora, and Euchresta) (Table 2 and Fig 5). The presence of a 36-kb inversion in Bolusanthus and Dicraeopetalum and recent phylogenetic evidence [3–4] indicate that they are members of the core genistoid clade. However, inclusion of either into the new Sophoreae (incl. Euchresteae and Thermopsideae) needs more careful consideration. The ITS phylogeny suggests that inclusion of Bolusanthus and Dicraeopetalum into new Sophoreae could make this tribe polyphyletic because those genera reside within a clade only distantly related to other Sophoreae s.s., and they are grouped with other Afro-Madagascan genera, Neoharmsia R. Vig. and Platycelyphium [54]. Furthermore, some morphologically disparate characteristics of Dicraeopetalum, e.g., radially symmetrical flowers, could make new Sophoreae too complicated to be a natural group. Hence, extensive analyses of morphology and molecular phylogeny based on nuclear as well as plastid data are needed to accomplish formalization of new Sophoreae.

Legume phylogeny with a “plastome-scale data set”

Recent progress in the sequencing of legume plastomes has provided a great deal of valuable data [30, 32, 45, 55–59] that can be used to make phylogenetic inferences based on a “plastome-scale data set” [29]. Those approaches have become more common since the development of next-generation DNA sequencing [28, 30, 60]. Moreover, the usefulness of plastomes in phylogenetic analyses has proven to show good resolution among evolutionarily puzzling taxa [17]. In our study, we examined 43 legumes belonging to 22 tribes, based on 71 conserved protein-coding genes in their plastomes (Fig 6). Previously, the plastome-scale phylogeny that featured the most comprehensive sampling for legumes had been made by Schwarz et al. [45]. They included 32 plastomes belonging to 16 tribes. When one considers the wide diversity inherent to legumes, our sample size was still small. Most tribes (14 of 22) have not been sufficiently sampled to test monophyly. Nevertheless, our ML analysis produced a phylogenetic tree that is very representative of the current understanding about legume phylogeny among higher taxa [2]. This is certainly true for the monophyly of informal groups and the non-monophyly of some legume tribes. For example, large informal taxonomic groups of Papilionoideae (dalbergioid s.l., genistoid, indigoferoid/millettioid, robinioid, and IRLC) are resolved as a monophyletic group with 100% bootstrap support. However, the subfamily Caesalpinioideae and six of the 22 legume tribes are not monophyletic (i.e., Caesalpinieae, Mimoseae, Millettieae Phaseoleae, Galegeae, and Trifolieae). In that sense, we believe it is notable that our phylogenetic analysis supports genistoids as sister to the Old World clade and dalbergioid as an earlier diverging lineage. Although the individual groupings of dalbergioid and genistoids are well-established, their phylogenetic positions and interrelationship are still ambiguous. The topology we describe here may have resulted from taxon sampling bias, due to a lack of enough basal papilionoids. However, the high bootstrap values and overall concordance in topology of Old World clades reported previously in other published studies at least suggest that additional plastome sequences for basal papilionoids will be promising data for investigating the higher-level systematics of legumes.