Reconstructing the deep-branching relationships of the papilionoid legumes

Resolving the phylogenetic relationships of the deep nodes of papilionoid legumes (Papilionoideae) is essential to understanding the evolutionary history and diversification of this economically and ecologically important legume subfamily. The early-branching papilionoids include mostly Neotropical trees traditionally circumscribed in the tribes Sophoreae and Swartzieae. They are more highly diverse in floral morphology than other groups of Papilionoideae. For many years, phylogenetic analyses of the Papilionoideae could not clearly resolve the relationships of the early-branching lineages due to limited sampling. In the eight years since the publication of Legumes of the World, we have seen an extraordinary wealth of new molecular data for the study of Papilionoideae phylogeny, enabling increasingly greater resolution and many surprises. This study draws on recent molecular phylogenetic studies and a new comprehensive Bayesian phylogenetic analysis of 668 plastid matK sequences. The present matK phylogeny resolves the deep-branching relationships of the papilionoids with increased support for many clades, and suggests that taxonomic realignments of some genera and of numerous tribes are necessary. The potentially earliest-branching papilionoids fall within an ADA clade, which includes the recircumscribed monophyletic tribes Angylocalyceae, Dipterygeae, and Amburanae. The genera Aldina and Amphimas represent two of the nine main but as yet unresolved lineages comprising the large 50-kb inversion clade. The quinolizidine-alkaloid-accumulating Genistoid s.l. clade is expanded to include Dermatophyllum and a strongly supported and newly circumscribed tribe Ormosieae. Sophoreae and Swartzieae are dramatically reorganized so as to comprise monophyletic groups within the Core Genistoid clade and outside the 50-kb inversion clade, respectively. Acosmium is excluded from the Genistoids s.l. and strongly resolved within the newly circumscribed tribe Dalbergieae. By providing a better resolved phylogeny of the earliest-branching papilionoids, this study, in combination with other recent evidence, will lead to a more stable phylogenetic classification of the Papilionoideae.     

Towards a new classification of Leguminosae: Naming clades using non-Linnaean phylogenetic nomenclature

The past three decades of research have greatly advanced our understanding of phylogenetic relationships in the family Leguminosae. It has become clear in recent years that our classification system is in need of significant updating if it is to reflect our current understanding of the phylogeny of the family and facilitate effective communication of that knowledge. The goal of this paper is to suggest a set of guidelines for formally defining and naming clades, which draws on many of the recommendations embodied in the draft International Code of Phylogenetic Nomenclature or “PhyloCode”. I provide specific examples of phylogenetic nomenclature applied to several well recognized and well-supported, informally named papilionoid clades to serve as a model for standardizing legume clade names by the legume community in the future. For the most part the clades named here are below subfamily and above tribal ranks in the Linnaean system. It is my contention that a new Linnaean classification, designed to reflect phylogeny, and a clade-based system of phylogenetic nomenclature are mutually complementary approaches to achieving a new classification of the legume family.     

The chicken or the egg? Plastome evolution and an independent loss of the inverted repeat in papilionoid legumes

The plastid genome (plastome), while surprisingly constant in gene order and content across most photosynthetic angiosperms, exhibits variability in several unrelated lineages. During the diversification history of the legume family Fabaceae, plastomes have undergone many rearrangements, including inversions, expansion, contraction and loss of the typical inverted repeat (IR), gene loss and repeat accumulation in both shared and independent events. While legume plastomes have been the subject of study for some time, most work has focused on agricultural species in the IR-lacking clade (IRLC) and the plant model Medicago truncatula. The subfamily Papilionoideae, which contains virtually all of the agricultural legume species, also comprises most of the plastome variation detected thus far in the family. In this study three non-papilioniods were included among 34 newly sequenced legume plastomes, along with 33 publicly available sequences, to assess plastome structural evolution in the subfamily. In an effort to examine plastome variation across the subfamily, approximately 20% of the sampling represents the IRLC with the remainder selected to represent the early-branching papilionoid clades. A number of IR-related and repeat-mediated changes were identified and examined in a phylogenetic context. Recombination between direct repeats associated with ycf2 resulted in intraindividual plastome heteroplasmy. Although loss of the IR has not been reported in legumes outside of the IRLC, one genistoid taxon was found to completely lack the typical plastome IR. The role of the IR and non-IR repeats in the progression of plastome change is discussed.     

A phylogeny of legumes (Leguminosae) based on analysis of the plastid matK gene resolves many well-supported subclades within the family

Phylogenetic analysis of 330 plastid matK gene sequences, representing 235 genera from 37 of 39 tribes, and four outgroup taxa from eurosids I supports many well-resolved subclades within the Leguminosae. These results are generally consistent with those derived from other plastid sequence data (rbcL and trnL), but show greater resolution and clade support overall. In particular, the monophyly of subfamily Papilionoideae and at least seven major subclades are well-supported by bootstrap and Bayesian credibility values. These subclades are informally recognized as the Cladrastis clade, genistoid sensu lato, dalbergioid sensu lato, mirbelioid, millettioid, and robinioid clades, and the inverted-repeat-lacking clade (IRLC). The genistoid clade is expanded to include genera such as Poecilanthe, Cyclolobium, Bowdichia, and Diplotropis and thus contains the vast majority of papilionoids known to produce quinolizidine alkaloids. The dalbergioid clade is expanded to include the tribe Amorpheae. The mirbelioids include the tribes Bossiaeeae and Mirbelieae, with Hypocalypteae as its sister group. The millettioids comprise two major subclades that roughly correspond to the tribes Millettieae and Phaseoleae and represent the only major papilionoid clade marked by a macromorphological apomorphy, pseudoracemose inflorescences. The robinioids are expanded to include Sesbania and members of the tribe Loteae. The IRLC, the most species-rich subclade, is sister to the robinioids. Analysis of the matK data consistently resolves but modestly supports a clade comprising papilionoid taxa that accumulate canavanine in the seeds. This suggests a single origin for the biosynthesis of this most commonly produced of the nonprotein amino acids in legumes.     

Phylogenetic relationships and floral evolution in the papilionoid legume clade Amorpheae

Amorpheae (Fabaceae: Papilionoideae) was first considered a natural group by Rupert Barneby in his illustrated monographDaleae Imagines. Amorpheae currently comprise eight genera, ca. 250 spp., and extensive floral diversity, including loss of corolla and addition of a stemonozone. The Amorpheae and many of Barneby’s subtribal groups are supported as monophyletic by previous phylogenetic analysis of nuclear ribosomal and chloroplast sequence data. However, some relationships remain unclear. A nuclear marker derived from a genomic study inMedicago CNGC 4, was sequenced in selected Amorpheae This is one of the first applications of this marker. for phylogenetic study. The new data confirm some relationships inferred usingtrnK and ITS, but also provide evidence for new arrangements. Combined data were used to explore several aspects of Barneby’s taxonomic framework. The phylogeny, in concert, with data on floral morphology, implies that simplification of the complex papilionoid flower has occurred several times in the history of the Amorpheae.     

Cotyledon areoles in Bobgunnia (Fabaceae: Swartzieae)

Cotyledon areoles, a seed character unique to papilionoid legumes, are reported for the first time in the legume tribe Swartzieae, an anomalous group sometimes considered to be intermediate between subfamilies Caesalpinioideae and Papilionoideae. Bobgunnia madagascariensis has a linear, slightly branched cotyledon areole on the abaxial surface of each cotyledon. Previous reports of other papilionoid seed features in this species are confirmed and extended. These highly distinctive papilionoid seed structures, and recent reports from floral ontogeny and molecular systematics which show Bobgunnia to be closely related to Swartzia and several other genera of Swartzieae, add further evidence to assertions that Swartzia seed structures are derived by simplification from a papilionoid seed, and not by retention of less specialized caesalpinioid seed features. The function and evolutionary advantage of cotyledon areoles are unknown and need investigation. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 287–291.     

A detailed investigation of the Pterocarpus clade (Leguminosae: Dalbergieae): Etaballia with radially symmetrical flowers is nested within the papilionoid-flowered Pterocarpus

The pantropical genus Pterocarpus (Leguminosae: Dalbergieae) with papilionoid flowers, and allied genera in the Pterocarpus clade were sampled for the five molecular markers ITS2, trnL-F, ndhF-rpL32, matK, and rbcL, as part of our ongoing systematic studies in the clade. For wider analyses of the Pterocarpus clade the remaining 14 members of this clade were also sampled for matK. Phylogenetic analyses were performed under the maximum likelihood criterion (ML) and Bayesian criteria. In the five-marker analysis of the core Pterocarpus clade (including 106 accessions) two robustly supported clades were resolved. The first clade includes Centrolobium, Etaballia, Inocarpus, Maraniona, Paramachaerium, Pterocarpus, Ramorinoa, and Tipuana. The second includes all species of Pterocarpus (except P. acapulcensis), Etaballia with radially symmetric flowers, and Paramachaerium. Paramachaerium is placed as sister to the several Pterocarpus species from South America, while Etaballia is resolved within the clade containing the African and Asian species of Pterocarpus. The wider sampled matK data set includes 199 accessions. Discolobium and Riedeliella are recovered as sister to the remaining Pterocarpus clade. Platymiscium is strongly supported as sister to the rest of the members of the clade, and Pterocarpus acapulcensis is also here resolved in a separate lineage from the remaining Pterocarpus accessions. We used the phylogenies to investigate patterns of floral evolvability in the Pterocarpus clade, which include four genera with actinomorphic flowers (Acosmium s.s., Etaballia, Inocarpus and Riedelliela). Our results reinforce the hypothesis that flower evolvability is high in early-branching legume lineages, and that actinomorphy has evolved independently four times in the Pterocarpus clade. In light of our results, the taxonomic status of the monospecific genus Etaballia dubia Benth. was revisited, and the species is synonymized as belonging to Pterocarpus, under the name Pterocarpus dubius Spreng., published in 1827, but hiding in synonymy for nearly two centuries.     

Molecular phylogenetic analysis of Euphorbiaceae sensu stricto based on plastid and nuclear DNA sequences and ovule and seed character evolution

A phylogenetic analysis of Euphorbiaceae sensu stricto is presented using sequences from rbcL, atpB, matK and 18S rDNA from 85 species and 83 genera. The combined analysis of four molecular markers resulted in only one most parsimonious tree and also generated new supported clades, which include Euphorbioideae + Acalyphoideae s.s., subclades A2 + A3, subclades A5 + A6 and a clade uniting subclades A2–A8 within Acalyphoideae s.s. A palisadal exotegmen is a possible synapomorphy for all the Euphorbiaceae, except for the subfamily Peroideae. The presence of vascular bundles in the inner integument and a thick inner integument were shown to be synapomorphies for the clade of inaperturate and articulated crotonoids and for the large clade of Euphorbioideae, Acalyphoideae s.s., inaperturate and articulated crotonoids, respectively. Characters of the aril and vascular bundles in the outer integument are discussed. The selected embryological characters were seen to be highly correlated with the molecular phylogeny. When the results of molecular phylogenetic analysis of a previous study and this study were adjusted along with the selected embryological characters, all clades within Euphorbiaceae were supported except for a clade comprising Euphorbioideae + Acalyphoideae s.s. + inaperturate crotonoids + articulated crotonoids + Adenoclineae s.l. and a clade uniting subclades A4–A8 within Acalyphoideae s.s. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plastome phylogeny and lineage diversification of Salicaceae with focus on poplars and willows

Phylogenetic relationships and lineage diversification of the family Salicaceae sensu lato (s.l.) remain poorly understood. In this study, we examined phylogenetic relationships between 42 species from six genera based on the complete plastomes. Phylogenetic analyses of 77 protein coding genes of the plastomes produced good resolution of the interrelationships among most sampled species and the recovered clades. Of the sampled genera from the family, Flacourtia was identified as the most basal and the successive clades comprised both Itoa and Poliothyrsis, Idesia, two genera of the Salicaceae sensu stricto (s.s.) (Populus and Salix). Five major subclades were recovered within the Populus clade. These subclades and their interrelationships are largely inconsistent with morphological classifications and molecular phylogeny based on nuclear internal transcribed spacer sequence variations. Two major subclades were identified for the Salix clade. Molecular dating suggested that species diversification of the major subclades in the Populus and Salix clades occurred mainly within the recent Pliocene. In addition, we found that the rpl32 gene was lost and the rps7 gene evolved into a pseudogene multiple times in the sampled genera of the Salicaceae s.l. Compared with previous studies, our results provide a well‐resolved phylogeny from the perspective of the plastomes.     

Phylogeny reconstruction in the Caesalpinieae grade (Leguminosae) based on duplicated copies of the sucrose synthase gene and plastid markers

The Caesalpinieae grade (Leguminosae) forms a morphologically and ecologically diverse group of mostly tropical tree species with a complex evolutionary history. This grade comprises several distinct lineages, but the exact delimitation of the group relative to subfamily Mimosoideae and other members of subfamily Caesalpinioideae, as well as phylogenetic relationships among the lineages are uncertain. With the aim of better resolving phylogenetic relationships within the Caesalpinieae grade, we investigated the utility of several nuclear markers developed from genomic studies in the Papilionoideae. We cloned and sequenced the low copy nuclear gene sucrose synthase (SUSY) and combined the data with plastid trnL and matK sequences. SUSY has two paralogs in the Caesalpinieae grade and in the Mimosoideae, but occurs as a single copy in all other legumes tested. Bayesian and maximum likelihood phylogenetic analyses suggest the two nuclear markers are congruent with plastid DNA data. The Caesalpinieae grade is divided into four well-supported clades (Cassia, Caesalpinia, Tachigali and Peltophorum clades), a poorly supported clade of Dimorphandra Group genera, and two paraphyletic groups, one with other Dimorphandra Group genera and the other comprising genera previously recognized as the Umtiza clade. A selection analysis of the paralogs, using selection models from PAML, suggests that SUSY genes are subjected to a purifying selection. One of the SUSY paralogs, under slightly stronger positive selection, may be undergoing subfunctionalization. The low copy SUSY gene is useful for phylogeny reconstruction in the Caesalpinieae despite the presence of duplicate copies. This study confirms that the Caesalpinieae grade is an artificial group, and highlights the need for further analyses of lineages at the base of the Mimosoideae.     

Molecular phylogenetic analysis of Podostemaceae: implications for taxonomy of major groups

The river‐weed family Podostemaceae (c. 300 species in c. 54 genera) shows a number of morphological innovations to be adapted to its unusual aquatic habitat, and its unique or rare bauplan features have been reflected in the traditional (i.e. non‐molecular) classification recognizing numerous monotypic or oligospecific genera. The infrasubfamilial relationships of many genera remained unclear. The present study used molecular phylogenetic analysis of matK sequences for 657 samples (c. 132 species/c. 43 genera). The family was traditionally divided into three subfamilies (Podostemoideae, Tristichoideae and Weddellinoideae). American Podostemoideae were shown to be polyphyletic and divided into four clades, i.e. Ceratolacis, Diamantina, Podostemum and all other genera. Among the podostemoid clades, Diamantina was the first branching clade and a clade comprising Mourera and the Apinagia subclade was then sister to the remainder of the New World and Old World Podostemoideae with low statistic supports. The Old World Podostemoideae comprised four monophyletic clades, i.e. two African clades, one Madagascan clade and one Asian clade, although the relationships among these clades and American Ceratolacis and Podostemum were poorly resolved. African Podostemoideae were polyphyletic, with Saxicolella pro parte being weakly supported as sister to the remaining Old World Podostemoideae plus Ceratolacis and Podostemum. In contrast to the American and African clades, monophyly of four Asian subclades was well supported. Plants of Tristicha (Tristichoideae) and of Weddellina (Weddellinoideae), which are currently treated as monospecific, had great matK differentiation equivalent to at least interspecific variation. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 461–492.     

Presence of the anther gland is a key feature in pollination of the early‐branching papilionoids Dipteryx alata and Pterodon pubescens (Leguminosae)

• The presence of glandular appendages in the anthers is a rare condition in angiosperms. In Leguminosae it occurs in species of the Mimosoid clade and in early‐branching clades of papilionoids such as Dipterygeae. In Dipterygeae such appendages surprisingly exhibit a secretory cavity instead of secretory emergences as is the case for the Mimosoid clade. Thus, the objective of this study was to elucidate the function of anther glands in Dipteryx alata and Pterodon pubescens, species in the Dipterygeae clade that exhibit a pollen release mechanism that is intermediate between the explosive and valvular types.
• Flower buds and flowers were processed for surface, anatomical, histochemical and ultrastructural analyses.
• Anther glands consist of a cavity secreting sticky substances (oleoresins and polysaccharides) that play a key role during the flower's lifespan by aggregating pollen grains and attaching them to the floral visitor's body. Other floral features that are important for understanding the pollen release mechanism that is intermediate between the valvular and the explosive types are: (i) keel petals intertwined with tector trichomes; (ii) glandular appendages in the abaxial and lateral sepals and in petals composed of secretory ducts; and (iii) a continuous secretion process of the anther glands followed by an asynchronous dehiscence of anthers.
• The well‐adapted papilionoid flag blossom with anther glands and keel petals intertwined with trichomes provided the foundation for a successful canalisation toward a pollen release mechanism intermediate between the explosive and valvular types inside early‐branching papilionoids.

     

Flora treatment of the Leguminosae–Papilionoideae of Gabon

This short note highlights the work undertaken to prepare the treatment of the Leguminosae subfamily Papilionoideae for the Flore du Gabon. Examples are given in the form of some maps prepared from the BRAHMS database available in Wageningen. Statistics of collection efforts in the country are presented specifically for papilionoids. The results show that the number of new records of papilionoid legumes for the Flora of Gabon is still increasing and that the current knowledge of papilionoids in Gabon is far from complete.     

Molecular Phylogeny of Magnolia (Magnoliaceae) Inferred from cpDNA Sequences and Evolutionary Divergence of the Floral Scents

Magnolia (disjuncts), however, have similar chemical profiles. A molecular phylogeny of Magnoliaceae was constructed to reveal phylogenetic relationships of taxa by sequencing the trnK intron (including the matK coding region), psbA-trnH, and atpB-rbcL intergenic spacer regions of chloroplast DNA from 25 Magnolia, two Michelia, and two Liriodendron taxa. The psbA-trnH spacer region showed twice the sequence divergence (0.0157) of the trnK intron (0.0073) or the matK coding region (0.0077). The strict consensus tree constructed from the combined data set (ca. 3,700 bp) indicated the genus Magnolia was polyphyletic containing Michelia species as ingroup. The clade of Magnolia liliifera var. obovata, M. coco, and M. delavayi formed the first branch. Among the remaining species, two additional large clades were recognized, i.e., one comprised of American evergreen Magnolia species and another of subgenus Yulania. The relationship among sect. Rytidospermum taxa was not clearly resolved. Parsimonious mapping of the floral scent chemical characters was performed onto the molecular phylogenetic tree to discuss evolutionary trends of the floral scent chemistries. Received 7 December 1998/ Accepted in revised form 18 May 1999     

Molecular Phylogeny of the Papilionoideae (Family Leguminosae): RbcL Gene Sequences versus Chemical Taxonomy

Total DNA was extracted from 67 species (30 genera) of the subfamily Papilionoideae (family Leguminosae). The rbcL gene was amplified by polymerase chain reaction (PCR) and sequenced directly. RbcL sequences were evaluated with character-state (maximum parsimony; PAUP) and distance methods (neighbour-joining; MEGA). Morphology-based classifications of tribes and subtribes are mostly congruent with rbcL phylogeny. Differences occur for members of the genus Sophora and for intratribal relationships within the Genista/Cytisus complex: Sophora appears in two clades; one clade with S. japonica at the base of the Papilionoideae and a second more advanced group (with S. davidii, S. jaubertii, and S.flavescens) which represents a sister clade of the Thermopsideae. The Cytisus complex includes the genera Cytisus, Chamaecytisus, Calicotome and Spartocytisus but not Cytisophyllum which appears as a distinct member of the Genista complex. Chamaespartium sagittale is very close to Genista supporting the view that it does not represent an independent genus but should be treated as Genista sagittalis. Close to Genista are Teline and Spartium, whereas Argyrolobium, Retama, Cytisophyllum, Ulex, Petteria, Adenocarpus, Chamaespartium tridentatum and Laburnum can be considered as “outliers” of the Genista-group sensu stricto. RbcL phylogeny is compared with profiles of alkaloids and other natural products. First results indicate that secondary metabolite profiles, if compared with morphology or rbcL sequences, are of limited value as a taxonomic marker.     

Legume phylogeny and the evolution of a unique contractile apparatus that regulates phloem transport

Protein bodies called forisomes undergo Ca(2+)-dependent deformations to occlude sieve tubes reversibly, providing a unique regulatory mechanism of phloem transport. Because forisomes are known exclusively from the Papilionoideae (Leguminosae), the evolution of forisome function may have played a role in the rapid radiation of this huge taxon. The unexpected discovery of a papilionoid species lacking forisomes led us to evaluate a representative set of species covering 33 of the 36 legume tribes traditionally recognized. We found forisomes in Papilionoideae but not in Caesalpinioideae and Mimosoideae. Forisomes were absent from several species of the papilionoid tribe Galegeae. Forisomes with tail-like protrusions occurred less frequently than tailless ones; their distribution correlated with taxonomic units but not sharply enough to render forisome type a reliable criterion for classification. Thus, the distribution of forisome types appeared to reflect physiological variability in the pathways of forisome assembly rather than the evolution of forisome genes. On the other hand, Ca(2+)-dependent forisome deformation and sieve tube plugging occurred in Bobgunnia madagascariensis, a member of the swartzioid clade that presumably is the sister group of all other papilionoids, suggesting that forisomes and their unique mechanism of deformation are a synapomorphy of the Papilionoideae.     

Plastid genome sequences of legumes reveal parallel inversions and multiple losses of rps16 in papilionoids

To date, publicly available plastid genomes of legumes have for the most part been limited to the subfamily Papilionoideae. Here we report 13 new plastid genomes of legumes spanning all three subfamilies. The genomes representing Caesalpinioideae and Mimosoideae are highly conserved in gene content and gene order, similar to the ancestral angiosperm genome organization. Genomes within the Papilionoideae, however, have reduced sizes due to deletions in nine intergenic spacers primarily in the large single copy region. Our study also indicates that rps16 has been independently lost at least five times in legumes, with additional gene and intron losses scattered among the papilionoids. Additionally, genera from two distinct lineages within the papilionoids, Lupinus and Robinia, have a parallel inversion of 36 and 39 kb, respectively. This parallel inversion is novel as it appears to be caused by a 29 bp repeat within two trnS genes. This repeat is present in all available legume plastid genomes indicating that there is the potential for this inversion to be present in more species. This case of a homoplasious inversion is also evidence that some inversion events may not be reliable phylogenetic markers.