Phylogeny of Saxifragales (angiosperms,eudicots): analysis of a rapid,ancient radiation

Rapid, ancient radiations pose one of the most difficult challenges for phylogenetic estimation. We used DNA sequence data of 9,006 aligned base pairs from five genes (chloroplast atpB, matK, rbcL, and 18S and 26S nrDNA) to elucidate relationships among major lineages of Saxifragales (angiosperms, eudicots). These relationships were poorly supported in previous studies, apparently because the lineages originated in rapid succession. Using an array of methods that explicitly incorporate assumptions about evolutionary process (weighted maximum parsimony, maximum likelihood, LogDet/paralinear transformed distances), we show that the initial diversification of Saxifragales was indeed rapid. We suggest that the poor resolution of our best phylogenetic estimate is not due to violations of assumptions or to combining data partitions having conflicting histories or processes. We show that estimated branch lengths during the initial diversification are exceedingly short, and we estimate that acquiring sufficient sequence data to resolve these relationships would require an extraordinary effort (approximately 10(7) bp), assuming a linear increase in branch support with branch length. However, our simulation of much larger data sets containing a distribution of phylogenetic signal similar to that of the five sampled gene sequences suggests a limit to achievable branch support. Using statistical tests of differences in the likelihoods of topologies, we evaluated whether the initial radiation of Saxifragales involved the simultaneous origin of major lineages. Our results are consistent with predictions that resolving the branching order of rapid, ancient radiations requires sampling characters that evolved rapidly at the time of the radiation but have since experienced a slower evolutionary rate.     

Sectional relationships in the genus Rhododendron (Ericaceae): evidence from matK and trnK intron sequences

 Phylogenetic relationships among all eight subgenera and 12 sections in Rhododendron as well as its related genera were inferred from matK and trnK intron sequences. The results of this study provided the following insights: (1) Rhododendron is paraphyletic because Menziesia is nested within Rhododendron. (2) Subgenus Therorhodion forms a basal lineage of tribe Rhodoreae. (3) Subgenera Hymenanthes and Tsutsusi are monophyletic. (4) Subgenera Azaleastrum and Pentanthera are polyphyletic. (5) Subgenus Rhododendron is monophyletic, if section Rhododendron subsection Ledum is excluded. Received January 8, 1999, in definite form December 22, 2000 Accepted April 12, 2001     

Phylogenetic systematics of the tribe Millettieae (Leguminosae) based on chloroplast trnK/matK sequences and its implications for evolutionary patterns in Papilionoideae

Phylogenetic relationships in the tribe Millettieae and allies in the subfamily Papilionoideae (Leguminosae) were reconstructed from chloroplast trnK/matK sequences. Sixty-two accessions representing 57 traditionally recognized genera of Papilionoideae were sampled, including 27 samples from Millettieae. Phylogenies were constructed using maximum parsimony and are well resolved and supported by high bootstrap values. A well-supported "core Millettieae" clade is recognized, comprising the four large genera Millettia, Lonchocarpus, Derris, and Tephrosia. Several other small genera of Millettieae are not in the core Millettieae clade. Platycyamus is grouped with Phaseoleae (in part). Ostryocarpus, Austrosteenisia, and Dalbergiella are neither in the core Millettieae or Phaseoleae clade. These taxa, along with core Millettieae and Phaseoleae, form a monophyletic sister group to Indigofereae. Cyclolobium and Poecilanthe are close to Brongniartieae. Callerya and Wisteria belong to a large clade that includes all the legumes that lack the inverted repeat in their chloroplast genome, which confirms previous rbcL and phytochrome gene family phylogenies. The evolutionary history of four characters was examined in Millettieae and allies: the presence of canavanine, inflorescence types, the dehiscence of pods, and the presence of winged pods. trnK/matK sequence analysis suggests that the presence of a pseudoraceme or pseudopanicle and the accumulation of nonprotein amino acids are phylogenetically informative for Millettieae and allies with only a few exceptions.     

Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.     

Phylogenetic relationships and evolution in Chrysosplenium (Saxifragaceae) based on matK sequence data

Chrysosplenium (Saxifragaceae) consists of 57 species widely distributed in temperate and arctic regions of the Northern Hemisphere, with two species restricted to the southern part of South America. Species relationships within the genus are highly problematic. The genus has traditionally been divided into two groups, sometimes recognized as sections (Oppositifolia and Alternifolia), based on leaf arrangement, or, alternatively, into 17 series. Based on morphological features, Hara suggested that the genus originated in South America and then subsequently migrated to the Northern Hemisphere. We conducted phylogenetic analyses of DNA sequences of the chloroplast gene matK for species of Chrysosplenium to elucidate relationships, test Hara's biogeographic hypothesis for the genus, and examine chromosomal and gynoecial diversification. These analyses revealed that both sections Oppositifolia and Alternifolia are monophyletic and form two large sister clades. Hence, leaf arrangement is a good indicator of relationships within this genus. Hara's series Pilosa and Macrostemon are each also monophyletic; however, series Oppositifolia, Alternifolia, and Nepalensia are clearly not monophyletic. MacClade reconstructions suggest that the genus arose in Eastern Asia, rather than in South America, with several independent migration events from Asia to the New World. In one well-defined subclade, species from eastern and western North America form a discrete clade, with Old World species as their sister group, suggesting that the eastern and western North American taxa diverged following migration to that continent. The South American species forms a clade with species from eastern Asia; this disjunction may be the result of ancient long-distance dispersal. Character mapping demonstrated that gynoecial diversification is dynamic, with reversals from inferior to half-inferior ovaries, as well as to ovaries that appear superior. Chromosomal evolution also appears to be labile with several independent origins of n = 12 (from an original number of n = 11) and multiple episodes of aneuploidy.     

Nucleotide sequence variation of the chloroplast trnK/matK region in two wild Fagopyrum (Polygonaceae) species, F. leptopodum and F. statice

Nucleotide sequence polymorphisms of the intron of the chloroplast trnK (UUU) gene, including a matK gene, were investigated within two wild Fagopyrum species, F. leptopodum and F. statice, to assess the degree and pattern of the inter- and intraspecific differences in coding and noncoding chloroplast DNA regions in higher plants. Ten and five accessions were used for F. leptopodum and F. statice, respectively. The length of the trnK intron region in these species ranged from 2494 to 2506 bp. In the trnK intron, the net nucleotide substitution number per site (Da) between the two species was 0.00109, lower than the nucleotide diversity (pi), 0.00195 for F. leptopodum and 0.00144 for F. statice, suggesting a low level of interspecific divergence. This result seems to be due to the phylogenetic pattern that both species are interspersed with each other, which was revealed by the phylogenetic analyses based on the nucleotide substitutions and indels. In the matK gene region (1524 bp), seven and two nucleotide substitutions were found within F. leptopodum and F. statice, respectively. All of the nine nucleotide substitutions (eight of which were nonsynonymous) within and between F. leptopodum and F. statice were clustered in the 5' part of the matK gene region, and no variation was found in the 3' part. This suggests that most of the 3' part is occupied by the conserved domains that are important for the binding activity of the gene product to the precursor mRNA, and therefore implies that the 3' part is more functionally constrained than the 5' part.     

Phylogeny and character evolution in Medicago (Leguminosae): Evidence from analyses of plastid trnK/matK and nuclear GA3ox1 sequences

? Premise of the study: The genus Medicago, with about 87 species, includes the model legume species M. truncatula, and a number of important forage species such as M. sativa (alfalfa), M. scutellata (snail medic), and M. lupulina (black medic). Relationships within the genus are not yet sufficiently resolved, contributing to difficulty in understanding the evolution of a number of distinguishing characteristics such as aneuploidy and polyploidy, life history, structure of cotyledons, and number of seeds per fruit. ? Methods: Phylogenetic relationships of 70-73 species of Medicago and its sister genus Trigonella (including Melilotus) were reconstructed from nucleotide sequences of the plastid trnK/matK region and the nuclear-encoded GA3ox1 gene (gibberellin 3-β-hydroxylase) using maximum parsimony and Bayesian inference methods. ? Key results: Our results support certain currently recognized taxonomic groups, e.g., sect. Medicago (with M. sativa) and sect. Buceras. However, other strongly supported clades-the "reduced subsection Leptospireae clade" that includes M. lupulina, the "polymorpha clade" that includes M. murex and M. polymorpha and the "subsection Pachyspireae clade" that includes M. truncatula-each of which includes species presently in different subsections of sect. Spirocarpos, contradict the current classification. ? Conclusions: These results support the hypothesis that some characters considered important in existing taxonomies, for example, single-seeded fruits that have arisen more than once in both Medicago and Trigonella, are indeed homoplastic. Others, such as the 2n = 14 chromosome number, have also arisen independently within the genus. In addition, we demonstrate support for the utility of GA3ox1 sequences for phylogenetic analysis among and within closely related genera of legumes.     

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.     

Xylem of early angiosperms: Nuphar (Nymphaeaceae) has novel tracheid microstructure1

SEM studies of xylem of stems of Nuphar reveal a novel feature, not previously reported for any angiosperm. Pit membranes of tracheid end walls are composed of coarse fibrils, densest on the distal (outside surface, facing the pit of an adjacent cell) surface of the pit membrane of a tracheid, thinner, and disposed at various levels on the lumen side of a pit membrane. The fibrils tend to be randomly oriented on the distal face of the pit membrane; the innermost fibrils facing the lumen take the form of longitudinally oriented strands. Where most abundantly present, the fibrils tend to be disposed in a spongiform, three-dimensional pattern. Pores that interconnect tracheids are present within the fibrillar meshwork. Pit membranes on lateral walls of stem tracheids bear variously diminished versions of this pattern. Pits of root tracheids are unlike those of stems in that the lumen side of pit membranes bears a reticulum revealed on the outer surface of the tracheid after most of the thickness of a pit membrane is shaved away by the sectioning process. No fibrillar texturing is visible on the root tracheid pits when they are viewed from the inside of a tracheid. Tracheid end walls of roots do contain pores of various sizes in pit membranes. These root and stem patterns were seen in six species representing the two sections of Nuphar, plus one intersectional hybrid, as well as in one collection of Nymphaea, included for purposes of comparison. Differences between root and stem tracheids with respect to microstructure are consistent in all species studied. Microstructural patterns reported here for stem tracheid pits of Nymphaeaceae are not like those of Chloranthaceae, Illiciaceae, or other basal angiosperms. They are not referable to any of the patterns reported for early vascular plants. The adaptational nature of the pit membrane structure in these tracheids is not apparent; microstructure of pit membranes in basal angiosperms is more diverse than thought prior to study with SEM.     

Phylogenetic utility of MORE AXILLARY GROWTH4 (MAX4)-like genes: a case study in Digitalis/Isoplexis (Plantaginaceae)

We present the first assessment of phylogenetic utility of a potential novel low-copy nuclear gene region in flowering plants. A fragment of the MORE AXILLARY GROWTH 4 gene (MAX4, also known as RAMOSUS1 and DECREASED APICAL DOMINANCE1), predicted to span two introns, was isolated from members of Digitalis/Isoplexis. Phylogenetic analyses, under both maximum parsimony and Bayesian inference, were performed and revealed evidence of putative MAX4-like paralogues. The MAX4-like trees were compared with those obtained for Digitalis/Isoplexis using ITS and trnL-F, revealing a high degree of incongruence between these different DNA regions. Network analyses indicate complex patterns of evolution between the MAX4 sequences, which cannot be adequately represented on bifurcating trees. The incidence of paralogy restricts the use of MAX4 in phylogenetic inference within the study group, although MAX4 could potentially be used in combination with other DNA regions for resolving species relationships in cases where paralogues can be clearly identified.     

Phylogenetic analysis of general bacterial porins: a phylogenomic case study

Bacterial porin proteins allow for the selective movement of hydrophilic solutes through the outer membrane of Gram-negative bacteria. The purpose of this study was to clarify the evolutionary relationships among the Type 1 general bacterial porins (GBPs), a porin protein subfamily that includes outer membrane proteins ompC and ompF among others. Specifically, we investigated the potential utility of phylogenetic analysis for refining poorly annotated or mis-annotated protein sequences in databases, and for characterizing new functionally distinct groups of porin proteins. Preliminary phylogenetic analysis of sequences obtained from GenBank indicated that many of these sequences were incompletely or even incorrectly annotated. Using a well-curated set of porins classified via comparative genomics, we applied recently developed bayesian phylogenetic methods for protein sequence analysis to determine the relationships among the Type 1 GBPs. Our analysis found that the major GBP classes (ompC, phoE, nmpC and ompN) formed strongly supported monophyletic groups, with the exception of ompF, which split into two distinct clades. The relationships of the GBP groups to one another had less statistical support, except for the relationships of ompC and ompN sequences, which were strongly supported as sister groups. A phylogenetic analysis comparing the relationships of the GenBank GBP sequences to the correctly annotated set of GBPs identified a large number of previously unclassified and mis-annotated GBPs. Given these promising results, we developed a tree-parsing algorithm for automated phylogenetic annotation and tested it with GenBank sequences. Our algorithm was able to automatically classify 30 unidentified and 15 mis-annotated GBPs out of 78 sequences. Altogether, our results support the potential for phylogenomics to increase the accuracy of sequence annotations.     

Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota)

The resolving power and statistical support provided by two protein-coding (RPB1 and RPB2) and three ribosomal RNA-coding (nucSSU, nucLSU, and mitSSU) genes individually and in various combinations were investigated based on maximum likelihood bootstrap analyses on lichen-forming fungi from the class Lecanoromycetes (Ascomycota). Our results indicate that the optimal loci (single and combined) to use for molecular systematics of lichen-forming Ascomycota are protein-coding genes (RPB1 and RPB2). RPB1 and RPB2 genes individually were phylogenetically more efficient than all two- and three-locus combinations of ribosomal loci. The 3rd codon position of each of these two loci provided the most characters in support of phylogenetic relationships within the Lecanoromycetes. Of the three ribosomal loci we used in this study, mitSSU contributed the most to phylogenetic analyses when combined with RPB1 and RPB2. Except for the mitSSU, ribosomal genes were the most difficult to recover because they often contain many introns, resulting in PCR bias toward numerous and intronless co-extracted contaminant fungi (mainly Dothideomycetes, Chaetothyriomycetes, and Sordariomycetes in the Ascomycota, and members of the Basidiomycota), which inhabit lichen thalli. Maximum likelihood analysis on the combined five-locus data set for 82 members of the Lecanoromycetes provided a well resolved and well supported tree compared to existing phylogenies. We confirmed the monophyly of three recognized subclasses in the Lecanoromycetes, the Acarosporomycetidae, Ostropomycetidae, and Lecanoromycetideae; the latter delimited as monophyletic for the first time, with the exclusion of the family Umbilicariaceae and Hypocenomyce scalaris. The genus Candelariella (formerly in the Candelariaceae, currently a member of the Lecanoraceae) represents the first evolutionary split within the Lecanoromycetes, before the divergence of the Acarosporomycetidae. This study provides a foundation necessary to guide the selection of loci for future multilocus phylogenetic studies on lichen-forming and allied ascomycetes.     

Xylem of early angiosperms: Novel microstructure in stem tracheids of Barclaya (Nymphaeaceae)

Pit membranes of stem tracheids of all recognized species of Barclaya, an Indomalaysian genus of Nymphaeaceae, were studied with scanning electron microscopy (SEM). Pit membranes of the tracheids are composed of two thick layers, both constructed of fibrils much larger than those of tracheary elements of angiosperms other than Nymphaeaceae. The outer (distal) layer, which comprises the continuous primary wall around the tracheids, is spongiform, perforated by porosities of relatively uniform size, and confined to or most prominent on end walls of stem tracheids. The second layer consists of thick widely spaced fibrils that are oriented axially and are laid down proximally (facing the cell lumen) to the first (outer) layer, although continuous with it. These axial fibrils are attached at their ends to the pit cavities. This peculiar microstructure is not known outside Nymphaeaceae except in Brasenia and Cabomba (Cabombaceae, Nymphaeales), and has not been previously described for Barclaya. The longitudinally oriented threads and strands in perforation plates of secondary xylem of wood and stems of a variety of primitive woody angiosperms (e.g., Illicium) are not homologous to the pit membrane structure observed in stem tracheids of Barclaya, which, like other Nymphaeaceae, has only primary xylem and no perforation plates. The tracheid microstructure reported here is different from pit structures observed in any other group of vascular plants, living or fossil. The tracheid stem microstructures of Barclaya and other Nymphaeaceae appear to be a synapomorphy of Nymphaeaceae and Cabombaceae, and need further study with respect to ultrastructure and function.     

Isolated generative cells in some angiosperms: a further study

Summary Generative cells were isolated from the pollen grains of three angiosperm species by a method similar to that previously reported for Haemanthus katherinae (Baker). Both the external appearance and the internal structure of the isolated generative cells were observed by light and scanning electron microscopy. The dynamic changes occurring in the cells after they had been liberated from the pollen grains were recorded by video-enhanced microscopy. The distribution of microtubules in the isolated cells was revealed by immunofluorescence.This work was done during the senior author's sabbatical leave at the Department of Biological Sciences, Dartmouth College, Hanover, N.H. (USA) and this paper is dedicated to Professor R.D. Allen, who did not live to see this work completed but who was directly involved in supporting this project.     

Floral scent composition in early diverging taxa of Asclepiadoideae,and Secamonoideae (Apocynaceae)

The Apocynaceae–Asclepiadoideae are well known for their specialized floral morphologies and pollination systems and many species have distinct floral aromas. However, our knowledge on the chemistry of floral volatiles in this plant family is relatively limited although it has been suspected that floral scent plays a key function for pollinator attraction. This is the third paper in a series of papers reporting on the floral odours of Asclepiadoideae. Floral odours of eleven species from seven genera (Cibirhiza, Fockea, Gymnema, Hoya, Marsdenia, Stephanotis and Telosma) of early diverging taxa of Apocynaceae–Asclepiadoideae, and two species of Secamone (Apocynaceae–Secamonoideae) were collected using headspace sampling and then analyzed via GC–MS. We detected 151 compounds, of which 103 were identified. The vast majority of chemicals identified are common components in flower odour bouquets of angiosperms. However, striking was the high relative amount of acetoin (97.6%) in the flower scent of Cibirhiza albersiana. This compound has rarely been reported as a flower scent component and is more commonly found in fermentation odours. Bray–Curtis similarities and Nonmetric-Multidiminsional Scaling (NMDS) analyses showed that each of the species has a distinct odour pattern. This is mostly due to only twelve compounds which singly or in different combinations dominated the scent of the species: the benzenoids benzyl acetate, benzaldehyde, methyl benzoate, and 2-phenylethyl alcohol; the monoterpenoids (E)-ocimene, (Z)-ocimene, linalool, and eucalyptol; and the aliphatic compounds acetoin, and (E,Z)-2,6-nonadienal. The floral scent compositions are discussed in relation to tribal affiliations and their potential role for pollinator attraction, and are compared with the scent data available from other Asclepiadoideae species.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).