Martin F. Wojciechowski

Evolutionary Rates Analysis of Leguminosae Implicates a Rapid Diversification of Lineages during the Tertiary

Tertiary macrofossils of the flowering plant family Leguminosae (legumes) were used as time constraints to estimate ages of the earliest branching clades identified in separate plastid matK and rbcL gene phylogenies. Penalized likelihood rate smoothing was performed on sets of Bayesian likelihood trees generated with the AIC-selected GTR+ Gamma +I substitution model. Unequivocal legume fossils dating from the Recent continuously back to about 56 million years ago were used to fix the family stem clade at 60 million years (Ma), and at 1-Ma intervals back to 70 Ma. Specific fossils that showed distinctive combinations of apomorphic traits were used to constrain the minimum age of 12 specific internal nodes. These constraints were placed on stem rather than respective crown clades in order to bias for younger age estimates. Regardless, the mean age of the legume crown clade differs by only 1.0 to 2.5 Ma from the fixed age of the legume stem clade. Additionally, the oldest caesalpinioid, mimosoid, and papilionoid crown clades show approximately the same age range of 39 to 59 Ma. These findings all point to a rapid family-wide diversification, and predict few if any legume fossils prior to the Cenozoic. The range of the matK substitution rate, 2.1-24.6 x 10(-10) substitutions per site per year, is higher than that of rbcL, 1.6- 8.6 x 10(-10), and is accompanied by more uniform rate variation among codon positions. The matK and rbcL substitution rates are highly correlated across the legume family. For example, both loci have the slowest substitution rates among the mimosoids and the fastest rates among the millettioid legumes. This explains why groups such as the millettioids are amenable to species-level phylogenetic analysis with these loci, whereas other legume groups are not.

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.

Improved bootstrap confidence limits in large-scale phylogenies, with an example from Neo-Astragalus (Leguminosae)

Phylogenetic analyses of large data sets pose special challenges, including the apparent tendency for the bootstrap support for a clade to decline with increased taxon sampling of that clade. We document this decline in data sets with increasing numbers of taxa in Astragalus, the most species-rich angiosperm genus. Support for one subclade, Neo-Astragalus, declined monotonically with increased sampling of taxa inside Neo-Astragalus, irrespective of whether parsimony or neighbor-joining methods were used or of which particular heuristic search algorithm was used (although more stringent algorithms tended to yield higher support). Three possible explanations for this decline were examined, including (1) mistaken assignment of the most recent common ancestor of the taxon sample (and its bootstrap support) with the most recent common ancestor of the clade from which it was sampled; (2) computational limitations of heuristic search strategies; and (3) statistical bias in bootstrap proportions, especially that from random homoplasy distributed among taxa. The best explanation appears to be (3), although computational shortcomings (2) may explain some of the problem. The bootstrap proportion, as currently used in phylogenetic analysis, does not accurately capture the classical notion of confidence assessments on the null hypothesis of nonmonophyly, especially in large data sets. More accurate assessments of confidence as type I error levels (relying on iterated bootstrap methods) remove most of the monotonic decline in confidence with increasing numbers of taxa.

Compound Leaf Development and Evolution in the Legumes

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat–lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.

A new subfamily classification of the leguminosae based on a taxonomically comprehensive phylogeny

The classification of the legume family proposed here addresses the long-known non-monophyly of the traditionally recognised subfamily Caesalpinioideae, by recognising six robustly supported monophyletic subfamilies. This new classification uses as its framework the most comprehensive phylogenetic analyses of legumes to date, based on plastid matK gene sequences, and including near-complete sampling of genera (698 of the currently recognised 765 genera) and ca. 20% (3696) of known species. The matK gene region has been the most widely sequenced across the legumes, and in most legume lineages, this gene region is sufficiently variable to yield well-supported clades. This analysis resolves the same major clades as in other phylogenies of whole plastid and nuclear gene sets (with much sparser taxon sampling). Our analysis improves upon previous studies that have used large phylogenies of the Leguminosae for addressing evolutionary questions, because it maximises generic sampling and provides a phylogenetic tree that is based on a fully curated set of sequences that are vouchered and taxonomically validated. The phylogenetic trees obtained and the underlying data are available to browse and download, facilitating subsequent analyses that require evolutionary trees. Here we propose a new community-endorsed classification of the family that reflects the phylogenetic structure that is consistently resolved and recognises six subfamilies in Leguminosae: a recircumscribed Caesalpinioideae DC., Cercidoideae Legume Phylogeny Working Group (stat. nov.), Detarioideae Burmeist., Dialioideae Legume Phylogeny Working Group (stat. nov.), Duparquetioideae Legume Phylogeny Working Group (stat. nov.), and Papilionoideae DC. The traditionally recognised subfamily Mimosoideae is a distinct clade nested within the recircumscribed Caesalpinioideae and is referred to informally as the mimosoid clade pending a forthcoming formal tribal and/or cladebased classification of the new Caesalpinioideae. We provide a key for subfamily identification, descriptions with diagnostic charactertistics for the subfamilies, figures illustrating their floral and fruit diversity, and lists of genera by subfamily. This new classification of Leguminosae represents a consensus view of the international legume systematics community; it invokes both compromise and practicality of use.

PHYLOGENY OF THE CONJUGATING GREEN ALGAE (ZYGNEMOPHYCEAE) BASED ON rbc L SEQUENCES

Sequences of the gene encoding the large subunit of RUBISCO (rbcL) for 30 genera in the six currently recognized families of conjugating green algae (Desmidiaceae, Gonatozygaceae, Mesotaeniaceae, Peniaceae, and Zygnemataceae) were analyzed using maximum parsimony and maximum likelihood; bootstrap replications were performed as a measure of support for clades. Other Charophyceae sensu Mattox and Stewart and representative land plants were used as outgroups. All analyses supported the monophyly of the conjugating green algae. The Desmidiales, or placoderm desmids, constitute a monophyletic group, with moderate to strong support for the four component families of this assemblage (Closteriaceae, Desmidiaceae, Gonatozygaceae, and Peniaceae). The analyses showed that the two families of Zygnematales (Mesotaeniaceae, Zygnemataceae), which have plesiomorphic, unornamented and unsegmented cell walls, are not monophyletic. However, combined taxa of these two traditional families may constitute a monophyletic group. Partitioning the data by codon position revealed no significant differences across all positions or between partitions of positions one and two versus position three. The trees resulting from parsimony analyses using first plus second positions versus third position differed only in topology of branches with poor bootstrap support. The tree derived from third positions only was more resolved than the tree derived from first and second positions. The rbcL‐based phylogeny is largely congruent with published analyses of small subunit rDNA sequences for the Zygnematales. The molecular data do not support hypotheses of monophyly for groups of extant unicellular and filamentous or colonial desmid genera exhibiting a common cell shape. A trend is evident from simple omniradiate cell shapes to taxa with lobed cell and plastid shapes, which supports the hypothesis that chloroplast shape evolved generally from simple to complex. The data imply that multicellular placoderm desmids are monophyletic. Several anomalous placements of genera were found, including the saccoderm desmid Roya in the Gonatozygaceae and the zygnematacean Entransia in the Coleochaetales. The former is strongly supported, although the latter is not, and Entransias phylogenetic position warrants further study.

Phylogenetic Analysis of Nuclear Ribosomal ITS/5.8S Sequences in the Tribe Millettieae (Fabaceae): Poecilanthe-Cyclolobium, the core Millettieae, and the Callerya Group

Abstract The taxonomic composition of three principal and distantly related groups of the former tribe Millettieae, which were first identified from nuclear phytochrome and chloroplast trnK/matK sequences, was more extensively investigated with a phylogenetic analysis of nuclear ribosomal DNA ITS/5.8S sequences. The first of these groups includes the neotropical genera Poecilanthe and Cyclolobium, which are resolved as basal lineages in a clade that otherwise includes the neotropical genera Brongniartia and Harpalyce and the Australian Templetonia and Hovea. The second group includes the large millettioid genera, Millettia, Lonchocarpus, Derris, and Tephrosia, which are referred to as the “core Millettieae” group. Phylogenetic analysis of nuclear ribosomal DNA ITS/5.8S sequences reveals that Millettia is polyphyletic, and that subclades of the core Millettieae group, such as the New World Lonchocarpus or the pantropical Tephrosia and segregate genera (e.g., Chadsia and Mundulea), each form well supported monophyletic subgroups. The third lineage includes the genera Afgekia, Callerya, and Wisteria. These genera are resolved as a basal subclade in the inverted-repeat-lacking clade, which is a large legume group that includes the many well known temperate and herbaceous legumes, such as Astragalus, Medicago and Pisum, but not any other Millettieae. Communicating Editor: Jerrold I. Davis

Revisiting the phylogeny of papilionoid legumes: New insights from comprehensively sampled early-branching lineages

UNLABELLED PREMISE OF STUDY Phylogenetic relationships of the papilionoid legumes (Papilionoideae) reveal that the early branches are more highly diverse in floral morphology than are other clades of Papilionoideae. This study attempts for the first time to comprehensively sample the early-branching clades of this economically and ecologically important legume subfamily and thus to resolve relationships among them. • METHODS Parsimony and Bayesian phylogenetic analyses of the plastid matK and trnL intron sequences included 29 genera not yet sampled in matK phylogenies of the Papilionoideae, 11 of which were sampled for DNA sequence data for the first time. • KEY RESULTS The comprehensively sampled matK phylogeny better resolved the deep-branching relationships and increased support for many clades within Papilionoideae. The potentially earliest-branching papilionoid clade does not include any genus traditionally assigned to tribe Swartzieae. Dipterygeae is monophyletic with the inclusion of Monopteryx. The genera Aldina and Amphimas represent two of the nine main but as yet unresolved lineages comprising the large 50-kb inversion clade within papilionoids. The quinolizidine-alkaloid-accumulating genistoid clade is expanded to include a strongly supported subclade containing Ormosia and the previously unplaced Clathrotropis s.s., Panurea, and Spirotropis. Camoensia is the first-branching genus of the core genistoids. • CONCLUSIONS The well-resolved phylogeny of the earliest-branching papilionoids generated in this study will greatly facilitate the efforts to redefine and stabilize the classification of this legume subfamily. Many key floral traits did not often predict phylogenetic relationships, so comparative studies on floral evolution and plant-animal interactions, for example, should also benefit from this study.

Phylogeny of Robinioid Legumes (Fabaceae) Revisited: Coursetia and Gliricidia Recircumscribed, and a Biogeographical Appraisal of the Caribbean Endemics

Abstract Morphological data and sequences from the nuclear ribosomal ITS region, and the chloroplast trnL intron and matK locus were sampled from robinioid legumes to infer phylogenetic relationships. The monophyletic robinioid clade includes 11 genetically and often morphologically distinct subclades ranked as genera with the following well supported higher level relationships: ((Hebestigma, Lennea), ((Gliricidia, Poitea), (Olneya, Robinia, Poissonia, Coursetia, Peteria, Genistidium, and Sphinctospermum))). In order to render all 11 robinioid genera monophyletic, the genus Hybosema is synonymized with Gliricidia, and the genus Poissonia is resurrected to accommodate four morphologically disparate species previously classified in Coursetia. Three new combinations are required to accommodate these two generic recircumscriptions: Gliricidia robustum, Poissonia heterantha, and Poissonia weberbaueri. Ages of clades and evolutionary substitution rates are derived from a rate-smoothed Bayesian likelihood approach on sequences from the ITS region and the matK locus. Time constraints are derived from the Tertiary fossil wood species Robinia zirkelii, which shares apomorphic wood characters with the Robinia stem clade. The Cuban endemic Hebestigma is estimated to have diverged at least 38 Ma from its Mesoamerican sister genus Lennea, whereas the Greater Antillean Poitea is estimated to have diverged at least 16 Ma from its continental sister Gliricidia. This study reveals that sequences from the ITS region are amenable to exhaustive taxon sampling because of the high levels of variation at and below the species level. The evolutionary substitution rate for the ITS region is estimated at 3.1–3.5 x 10–9 substitutions/site/year, approximately an order of magnitude faster than that estimated for the matK locus.

Astragalus (Fabaceae): A molecular phylogenetic perspective

Nucleotide sequences of the plastidmatK gene and nuclear rDNA internal transcribed spacer region were sampled fromAstragalus L. (Fabaceae), and its closest relatives within tribe Galegeae, to infer phylogenetic relationships and estimate ages of diversification. Consistent with previous studies that emphasized sampling for nrDNA ITS primarily within either New World or Old World species groups,Astragalus, with the exception of a few morphologically distinct species, is strongly supported as monophyletic based on maximum parsimony and Bayesian analyses ofmatK sequences as well as a combined sequence dataset. ThematK data provides better resolution and stronger clade support for relationships amongAstragalus and traditionally related genera than nrDNA ITS.Astragalus sensu stricto plus the genusOxytropis are strongly supported as sister to a clade composed of strictly Old World (African, Australasian) genera such asColutea. Sutherlandia, Lessertia, Swainsona, andCarmichaelia, plus several morphologically distinct segregates of EurasianAstragalus. Ages of these clades and rates of nucleotide substitution estimated from a fossil-constrained, rate-smoothed, Bayesian analysis ofmatK sequences sampled from Hologalegina indicateAstragalus diverged from its sister group,Oxtropis, 12–16 Ma, with divergence of Neo-Astragalus beginning ca 4.4. Ma. Estimates of absolute rates of nucleotide substitution forAstragalus and sister groups, which range from 8.9 to 10.2×10−10 substitutions per site per year, are not unusual when compared to those estimated for other, mainly temperate groups of papilionoid legumes. The results of previously published work and other recent developments on the phylogenetic relationships and diversification ofAstragalus are reviewed.