Chloroplast DNA Restriction Site Data Research Articles

A TIME-CALIBRATED PHYLOGENY OF VERBESINA (HELIANTHEAE – ASTERACEAE) BASED ON NUCLEAR RIBOSOMAL ITS AND ETS SEQUENCES

Verbesina L. is a genus of the tribe Heliantheae, subtribe Verbesininae (Asteraceae), with distribution in the Americas, where Mexico and the Andes harbour the richest concentration of species. The approximately 325 species in the genus are shrubs, subshrubs, trees and rarely herbs. Despite its high species diversity and biogeographical importance, the only available phylogenetic hypothesis for Verbesina was based on chloroplast DNA restriction site data. In the present study, nuclear ITS and ETS DNA sequence data were used with an expanded taxon sampling, particularly among the South American Verbesina species, to improve phylogenetic resolution and support, clarify infrageneric relationships, and resolve biogeographical questions in the genus. The results of our new analysis corroborate the monophyly of Verbesina, but its current classification into 12 taxonomic sections, based on morphological characters such as phyllotaxis, head size, corolla colour, and presence of ray flowers, is not congruent with the molecular phylogeny, in which most sections are polyphyletic. We also show that Verbesina diverged in the late Miocene of North America, about 8 Ma. At least two independent Pleistocene dispersals into South America across the Isthmus of Panama and along the Southern Andes are evident, beginning around 3.23 Ma (1.27–3.23 Ma) in the Middle Pliocene, and resulting in an extra-Amazonian distribution of the genus inSouth America. Diversification in South America began around 2.83 Ma with occupation of the Andes. Colonisation of Brazil is estimated to have occurred around 2.15 Ma, from Andean lineages.

NrDNA external transcribed spacer (ETS) sequence data, reticulate evolution, and the systematics of Machaeranthera (Asteraceae)

Abstract Sequence data from the nrDNA external transcribed spacer (ETS) were generated to help resolve questions about the evolution of Machaeranthera left unanswered by previous analyses of chloroplast DNA (cpDNA) restriction site data and nrDNA ITS sequence data. Combined ITS and ETS (nrDNA) evidence agreed with cpDNA evidence in supporting a close relationship between Oonopsis and M. tanacetifolia. Nuclear rDNA evidence also supported an alliance between M. sections Blepharodon and Sideranthus. The nrDNA evidence provided greater support for the relationships of M. gypsitherma, M. heterophylla, M. stenoloba, and M. viscida than earlier analyses of ITS sequence data, reinforcing earlier conclusions that these species have experienced reticulate evolution. Finally, nrDNA evidence provided strong support for a group consisting of M. bigelovii, M. canescens, M. gypsitherma, and M. riparia. The relationships that nrDNA evidence supported for these four species conflicted substantially with those supported b...

The phylogeny of Lens (Leguminosae): new insight from ITS sequence analysis

Lens includes L.culinaris subsp. culinaris (the cultivated lentil) and several wild species distributed from the Mediterranean region to western Asia. We compared sequence variation in the ITS region among species of Lens in an effort to end persisting uncertainty regarding the phylogeny of the genus. The parsimony analysis revealed a single minimum-length tree with a topology congruent with patterns derived by previous studies of nuclear and chloroplast DNA RFLPs. The basal and highly divergent status of the L. nigricans clade is depicted, and the progenitor-derivative relationship between L. culinaris subsp. orientalis and L. culinaris subsp. culinaris is reaffirmed. Resolution in the tree was improved by combining the ITS data set with a pre-existing set of chloroplast DNA restriction site data obtained from the same group of samples.

Divergent and reticulate species relationships in Leucaena (Fabaceae) inferred from multiple data sources: insights into polyploid origins and nrDNA polymorphism

Previous analyses of species relationships and polyploid origins in the mimosoid legume genus Leucaena have used chloroplast DNA (cpDNA) restriction site data and morphology. Here we present an analysis of a new DNA sequence data set for the nuclear ribosomal DNA (nrDNA) 5.8S subunit and flanking ITS 1 and ITS 2 spacers, a simultaneous analysis of the morphology, ITS and cpDNA data sets for the diploid species, and a detailed comparison of the cpDNA and ITS gene trees, which include multiple accessions of all five tetraploid species. Significant new insights into species relationships and polyploid origins, including that of the economically important tropical forage tree L. leucocephala, are discussed. Heterogeneous ITS copy types, including 26 putative pseudogene sequences, were found within individuals of four of the five tetraploid and one diploid species. Potential pseudogenes were identified using two pairwise comparison approaches as well as a tree-based method that compares observed and expected proportions of total ITS variation contributed by the 5.8S subunit optimized onto branches of one of the ITS gene trees. Inclusion of putative pseudogene sequences in the analysis provided evidence that some pseudogenes in allopolyploid L. leucocephala are not the result of post-allopolyploidization gene silencing, but were inherited from its putative diploid maternal progenitor L. pulverulenta.

Molecular Evidence from Chloroplast and Nuclear Markers for Multiple Colonizations of Lavatera (Malvaceae) in the Canary Islands

A molecular phylogenetic study based on chloroplast DNA restriction site and ITS sequence data shows that the two Macaronesian endemics, Lavatera phoenicea and Lavatera acerifolia, represent two independent introductions into the Canary Islands. The molecular phylogenies, combined with morphological, ecological, and biogeographical data, indicate that Lavatera phoenicea may be a bird-pollinated relict of an ancient laurel forest. Lavatera acerifolia, however, is nested in a derived clade of the Lavatera-Malva taxa from the Mediterranean region, suggesting a more recent introduction into the Canary Islands. Incongruence between chloroplast and nuclear phylogenies suggests that hybridization may have played a role in the evolution of L. acerifolia. Several features of L. phoenicea, such as corolla color and high nectar production, appear to be plesiomorphic and are still present because of historical constraints. In contrast, woodiness is a derived feature that originated as an adaptation to insular conditions.

The correlation between development of atypical bisexual flowers and phylogeny in the Aroideae (Araceae)

In the intermediate zone of the inflorescence of genera of Aroideae one can find flowers with male and female characteristics. Until now, two types of developmental sequences of atypical bisexual flowers (ABFs) have been recognized: the Philodendron type and the Cercestis type. In the Philodendron type, bisexual flowers generally consist of functional carpels and staminodes inserted on the same whorl. In the Cercestis type, the gynoecium and stamens are inserted on two different whorls. These different ontogenetic patterns represent two different pathways in the evolution of unisexual flowers in this subfamily. A molecular phylogenetic analysis of 33 genera of Araceae, based on the chloroplast trnL intron and trnL–F intergenic spacer sequences was carried out. We use this phylogenetic analysis and those published by French et al. (1995) and Mayo et al. (1997) to examine the distribution of the two types of ABFs in selected genera. Our results suggest that the two developmental patterns of ABFs in Aroideae sensu Mayo et al. (1997) do not correspond to two separate evolutionary lineages but rather are more or less consistent within clades. Although this new molecular phylogeny does not include all aroid genera, it corroborates in general, at the subfamily level, the molecular analysis of French et al. (1995) based on chloroplast DNA restriction site data and the analysis of Mayo et al. (1997) based on morphological and anatomical data.

Phylogeny and Biogeography of the Flowering Plant Genus Styrax (Styracaceae) Based on Chloroplast DNA Restriction Sites and DNA Sequences of the Internal Transcribed Spacer Region

Phylogenetic relationships within the flowering plant genus Styrax were investigated with DNA sequence data from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and with chloroplast DNA restriction site data from the genes trnK, rpoC1, and rpoC2. The data sets from each genome were analyzed separately and in combination with parsimony methods. The results strongly support the monophyly of each of the four series of the genus but provide little phylogenetic resolution among them. Reticulate evolution may at least partly explain discordance between the molecular phylogenetic estimates and a prior morphological estimate within series Cyrta. The historical biogeography of the genus was inferred with unweighted parsimony character optimization of trees recovered from a combined ITS and morphological data set, after a series of combinability tests for data set congruence was conducted. The results are consistent with the fossil record in supporting a Eurasian origin of Styrax. The nested phylogenetic position of the South American members of the genus within those from southern North America and Eurasia suggests that the boreotropics hypothesis best explains the amphi-Pacific tropical disjunct distribution occurring within section Valvatae. The pattern of relationship recovered among the species of section Styrax ((western North America + western Eurasia) (eastern North America + eastern Eurasia)) is rare among north-temperate Tertiary forest relicts. The monophyly of the group of species from western North America and western Eurasia provides qualified support for the Madrean–Tethyan hypothesis, which posits a Tertiary floristic connection among the semiarid regions in which these taxa occur. A single vicariance event between eastern Asia and eastern North America accounts for the pattern of relationship among intercontinental disjuncts in series Cyrta.

Phylogenetic relationships and infraspecific variation in Canadian Arctic Poa based on chloroplast DNA restriction site data

Infraspecific variation and phylogenetic relationships of Canadian Arctic species of the genus Poa were studied based on chloroplast DNA (cpDNA) variation. Restriction site analysis of polymerase chain reaction amplified cpDNA was used to reexamine the status of infraspecific taxa, reconstruct phylogenetic relationships, and reexamine previous classification systems and hypotheses of relationships. Infraspecific variation was detected in three species, but only in Poa hartzii Gand. did it correspond to infraspecific taxa where recognition of subspecies ammophila at the species level is supported. Additional variation in P. hartzii ssp. hartzii is hypothesized to be the result of hybridization with Poa glauca in the High Arctic and subsequent introgression resulting in repeated transfer of P. glauca DNA. The variation in Poa pratensis L. had a geographical rather than taxonomic basis, and is hypothesized to correspond to indigenous arctic versus introduced extra-arctic populations. In P. glauca Vahl cpDNA variation was detected only in western Low Arctic and boreal populations and may represent greater variation where the species survived the Pleistocene glaciations. Cladistic parsimony analysis of cpDNA restriction site data mostly confirms recent infrageneric classification systems. Poa alpina L., along with the non-arctic Poa annua L. and Poa sect. Sylvestres, formed the basalmost clades. The remaining taxa group into two main clades: one consisting of Poa sects. Poa, Homalopoa, Madropoa and Diocopoa; the second, of Poa sects. Secundae, Pandemos, Abbreviatae and Stenopoa. Poa sect. Poa, comprising Poa arctica R. Br. and P. pratensis, is a strongly supported monophyletic group, not closely related to P. alpina. Poa hartzii is confirmed as a member of a paraphyletic or weakly supported P. sect. Secundae. Poa glauca and Poa abbreviata R. Br. are distinct members within a generally unresolved Poa. sect. Stenopoa-Abbreviatae complexKey words: Poa, Canadian arctic, chloroplast DNA, restriction site analysis, infraspecific variation, phylogeny.

Phylogenetic relationships and infraspecific variation in Canadian Arctic <i>Poa</i> based on chloroplast DNA restriction site data

Infraspecific variation and phylogenetic relationships of Canadian Arctic species of the genus Poa were stud- ied based on chloroplast DNA (cpDNA) variation. Restriction site analysis of polymerase chain reaction amplified cpDNA was used to reexamine the status of infraspecific taxa, reconstruct phylogenetic relationships, and reexamine previous classification systems and hypotheses of relationships. Infraspecific variation was detected in three species, but only in Poa hartzii Gand. did it correspond to infraspecific taxa where recognition of subspecies ammophila at the spe- cies level is supported. Additional variation in P. hartzii ssp. hartzii is hypothesized to be the result of hybridization with Poa glauca in the High Arctic and subsequent introgression resulting in repeated transfer of P. glauca DNA. The variation in Poa pratensis L. had a geographical rather than taxonomic basis, and is hypothesized to correspond to in- digenous arctic versus introduced extra-arctic populations. In P. glauca Vahl cpDNA variation was detected only in western Low Arctic and boreal populations and may represent greater variation where the species survived the Pleisto- cene glaciations. Cladistic parsimony analysis of cpDNA restriction site data mostly confirms recent infrageneric classi - fication systems. Poa alpina L., along with the non-arctic Poa annua L. and Poa sect. Sylvestres, formed the basalmost clades. The remaining taxa group into two main clades: one consisting of Poa sects. Poa, Homalopoa, Madropoa and Diocopoa; the second, of Poa sects. Secundae, Pandemos, Abbreviatae and Stenopoa. Poa sect. Poa, comprising Poa arctica R. Br. and P. pratensis, is a strongly supported monophyletic group, not closely related to P. alpina. Poa hartzii is confirmed as a member of a paraphyletic or weakly supported P. sect. Secundae. Poa glauca and Poa abbreviata R. Br. are distinct members within a generally unresolved Poa. sect. Stenopoa-Abbreviatae complex

Molecular phylogeny of Old WorldTrifolium (Fabaceae), based on plastid and nuclear markers

Phylogenies of Old WorldTrifolium species were constructed using nucleotide sequence data of the internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast DNA restriction site data from PCR-amplified genes and genic regions (rbcL,trnK, andrpoC1–C2). Biogeography, morphological evolution, and the existing classification forTrifolium were examined. The genusTrifolium is strongly supported as monophyletic, however, only one small section (Chronosemium) is monophyletic, although the data are in conflict regarding its placement. The two largest sections of the genus, Sects.Lotoidea andTrifolium, are not supported as monophyletic, as currently circumscribed. Many members of Sect.Lotoidea are basal within the genus, supporting previously-proposed hypotheses concerning plesiomorphic morphological characters and a Mediterranean-Mideast biogeographic origin of the genus.

Phylogenetic Analysis of the Cultivated and Wild Species of Phaseolus (Fabaceae)

The species of Phaseolus were exhaustively sampled for both ITS/5.8S DNA sequence and non-molecular data. With all related New World genera designated as outgroups, a phylogenetic analysis of combined data reveals a strongly supported monophyletic Phaseolus. Other well supported relationships include nine monophyletic species clades within Phaseolus, designated as the P vulgaris, P filiformis, P lunatus, P polystachios, P leptostachyus, P pauciflorus, P tuerckheimii, and P pedicellatus groups, and P microcarpus. Only the last of these is monotypic and consistently resolved in a sensitivity analysis as the earliest branch in the Phaseolus clade, though with poor bootstrap support. The five most commonly domesticated species in the genus arise from within the P vulgaris and P lunatus groups. The gene pools traditionally recognized for the domesticated species P vulgaris and P lunatus are not detected with ITS sequence variation. This is in spite of a very high degree of interand intra-specific ITS sequence divergence in Phaseolus. Though the genus Phaseolus has a complex taxonomic and nomenclatural history, recent circumscriptions of the genus are narrow (e.g., Verdcourt 1970) and most of the species with uncertain affinities have been relegated to Vigna. Vigna rather than Phaseolus has become the large catch-all pantropcial genus (Marechal 1982; Delgado-Salinas 1985; McVaugh 1987). In this modern circumscription, Phaseolus is strictly New World, concentrated in tropical and warm temperate North America, and diagnosed by foliage bearing hooked hairs, keel petals that are laterally and tightly coiled, and inflorescence nodes that lack extrafloral nectaries (Baudet 1977; Lackey 1978, 1981, 1983). Still retained in the genus are five economically important species: P acutifolius (tepary bean), P coccineus (scarlet runner bean), P lunatus (lima bean), P polyanthus (year bean), and P vulgaris (common bean). Little has been done to test the hypothesis of monophyly for the modern circumscription of Phaseolus. The traits supposedly diagnostic of Phaseolus occur in species of other genera (e.g., Vigna adenantha has laterally coiled keel petals, and species of Dolichos and Dipogon have inflorescence nodes lacking nectaries). A phylogenetic analysis of chloroplast DNA restriction site data suggests that Phaseolus is monophyletic (Delgado-Salinas et al. 1993). Taxon sampling, however, was not addressed, and this is a concern given that only 10 of approximately 50 species in the genus were sampled. Phaseolus is suggested to be part of a weakly supported monophyletic group of about 10 Old and New World genera that have poorly resolved relationships (Doyle and Doyle 1993; Bruneau et al. 1995). However, other studies suggest that the sister group relationships of Phaseolus include only New World genera. Analysis of chloroplast DNA restriction sites (Delgado-Salinas et al. 1993) resolved a New World clade where the genera Macroptilium and Strophostyles were sister to Phaseolus. A close relationship of Phaseolus to other New World genera is also suggested by an additional analysis of chloroplast DNA restriction sites (Vaillancourt et al. 1993-a study that focused on Vigna), a phenetic analysis of morphological data (Marechal et al. 1978, 1981), and a traditional taxonomic classification (Lackey 1981, 1983). A comprehensive phylogeny including both the wild and cultivated species of Phaseolus has been neglected, possibly because of an emphasis on the study of the domesticated species. What is known comes from phenetic and cladistic analysis of isozyme banding profiles (Jaaska 1996), phenetic analysis of morphological characters (Marechal et al. 1978), and cladistic analysis of chloroplast DNA restriction site data (Delgado-Salinas et al. 1993; Llaca et al. 1994). Yet character or taxon sampling has been insufficient to make any definitive conclusions about the infra-generic phylogenetic relationships

Phylogenetic relationships of subtribe Ecliptinae (Asteraceae: Heliantheae) based on chloroplast DNA restriction site data

Phylogenetic analysis of chloroplast DNA restriction site data for 76 of the 302 genera of Heliantheae sensu lato using 16 restriction endonucleases reveals that subtribe Ecliptinae is polyphyletic and that its genera are distributed in four different lineages. The ecliptinous genera Squamopappus, Podachaenium, Verbesina, and Tetrachyron (of the Neurolaeninae), along with other members of subtribe Neurolaeninae are the basalmost clades of the paleaceous Heliantheae. The mostly temperate species of subtribe Ecliptinae (exemplified by Balsamorhiza, Borrichia, Chrysogonum, Engelmannia, Silphium, Vigethia, and Wyethia) are strongly nested in a clade with the Mesoamerican monotypic genus Rojasianthe as basal. The genera characterized by marcescent ray corollas traditionally classified in subtribe Zinniinae constitute a strongly supported group sister to Acmella, Spilanthes, and Salmea. The largest clade of ecliptinous genera is the most recently derived group within Heliantheae sampled. This large group of mostly Neotropical lowland genera (variously characterized by their winged cypselae, foliaceous phyllaries, and opposite phyllotaxy and exemplified by Perymenium, Wedelia, and Zexmenia) has been and continues to be the most challenging group from a taxonomic standpoint. The study provides new insights as to their relationships that will have a positive impact in future monographic studies of the group. The genera of the Espeletiinae form a monophyletic clade and are sister to members of the Milleriinae and Melampodiinae. This result is consistent with their traditional taxonomic placement with genera such as Smallanthus with which they share a tendency for functionally staminate disc flowers. The phylogenetically enigmatic genus Montanoa is sister to Melampodium. Members of subtribe Galinsoginae are clustered in two main lineages that correspond to the traditional division of the subtribe based on pappus characteristics. There is no support for the monophyly of subtribe Galinsoginae, and the same results indicate some of its genera are paraphyletic.

Chloroplast DNA variation inCoreopsis nuecensoides andC. nuecensis (Asteraceae), a presumed progenitor-derivative species pair

Coreopsis nuecensoides andC. nuecensis are narrowly distributed endemics of southeastern Texas. While they overlap in range, they differ in chromosome number, and F1 hybrids exhibit strong sterility barriers. Previous morphological, cytogenetic, and allozyme studies suggested thatC. nuecensoides andC. nuecensis are very closely related members of a progenitor-derivative species pair. The two species differ substantially in terms of their leaf flavonoid chemistry, but the flavonoid data do not address the exact nature of the relationship between them. We have further examined the relationship between the species by analyzing genetic diversity within both species using chloroplast DNA (cpDNA) restriction site data. Sixteen restriction site changes were used to define thirteen distinct cpDNA haplotypes. The pattern of relationships among haplotypes provides some support for the presumed ancestor-descendent relationship, but other interpretations are possible. Only one cpDNA haplotype was shared by the two species; of the remaining twelve, seven were unique to one species and five to the other. This result is consistent with results of flavonoid studies, in which each species exhibited flavonoid compounds not seen in the other, but differs from the allozyme results, in which the vast majority of alleles were shared by both species and only the presumed progenitor exhibited unique alleles. Taken together, the data support a very close relationship between the species, but are equivocal with regard to a progenitor-derivative relationship.

Hybridization and the origin of the arctic grass Poa hartzii (Poaceae): evidence from morphology and chloroplast DNA restriction site data

The hypothesized hybrid origin of Poa hartzii Gand. (Poaceae) was investigated by analysis of morphological and molecular data. This endemic nearctic caespitose grass has been considered to be a hybrid between two of the three sympatric arctic species, Poa glauca M. Vahl, Poa arctica R. Br., and Poa abbreviata R. Br. Field observations and morphological studies indicate that Poa hartzii is a morphologically distinct apomictic species that reproduces and disperses by seed. Restriction enzyme analysis of polymerase chain reaction amplified chloroplast DNA revealed the presence in Poa hartzii of two very different haplotypes. One haplotype is identical to the dominant type found in Poa glauca, while the second is identical to the haplotype of Poa secunda J. Presl., located south of the arctic region. These results are consistent with an hypothesis of ancient hybrid origin involving Poa glauca and Poa secunda, but not Poa arclica nor Poa abbreviata. They are also consistent with an hypothesis of cytoplasmic transfer via hybridization and introgression from Poa glauca to an ancestral Poa hartzii in the Poa secunda complex. Direction of transfer is suggested by the widespread occurrence of the Poa secunda haplotype in Poa hartzii and by closer morphological similarity with Poa secunda than Poa glauca. The origin of Poa hartzii provides an excellent example of reticulate evolution and the importance of hybridization in the speciation of arctic grasses. Key words: Poa hartzii, arctic Poa, chloroplast DNA, restriction enzyme analysis, hybridization, introgression.

Molecular evidence for a Mediterranean origin of the Macaronesian endemic genus Argyranthemum (Asteraceae)

The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced for 52 species from 32 genera and eight subtribes of Anthemideae. Phylogenetic analyses of ITS data generated trees that are largely incongruent with the recent classification of Anthemideae; most of the subtribes examined are not resolved as monophyletic. However, ITS trees are congruent with morphological, isozyme, phytochemical, and chloroplast DNA (cpDNA) restriction site data in supporting a Mediterranean origin for Argyranthemum, the largest endemic genus of the Atlantic oceanic islands. A combined analysis of ITS sequences and cpDNA restriction sites indicates that Argyranthemum is sister to the other three genera of Chrysantheminae (i.e., Chrysanthemum, Heteranthemis, and Ismelia). Times of divergence of Argyranthemum inferred from the ITS sequences ranged between 0.26 and 2.1 million years ago (mya) and are lower than values previously reported from isozyme and cpDNA data (1.5-3.0 mya). It is likely that rate heterogeneity of the ITS sequences in the Anthemideae accounts for the low divergence-time estimates. Comparison of data for 20 species in Argyranthemum and Chrysantheminae indicates that the cpDNA restriction site approach provided much more phylogenetic information than ITS sequences. Thus, restriction site analyses of the entire chloroplast genome remain a valuable approach for studying recently derived island plants.

Reticulate Evolution in Machaeranthera (Asteraceae)

Previous analyses of chloroplast DNA restriction site data from and related genera support many relationships that are incongruent with morphological and cytological data. To find explanations for these disagreements, sequence data from the internal transcribed spacers (ITS) of nuclear rDNA were obtained from the same genera and species for comparison with chloroplast DNA evidence. Both lines of evidence supported a monophyletic Machaeranthera including and 12 additional genera, and both supported a monophyletic group of genera with chromosome numbers based on x = 6. Both lines of evidence placed stenoloba within M. sect. Sideranthus, suggesting that M. stenoloba has undergone significant morphological divergence compared to its closest relatives. Chloroplast DNA and ITS evidence disagreed substantially on the placements of M. viscida, M. heterophylla, and M. gypsitherma. Chloroplast DNA evidence suggests that M. viscida and M. gypsitherma belong within M. sect. Blepharodon and M. heterophylla belongs outside it. However, ITS evidence suggested that M. heterophylla belongs within M. sect. Blepharodon and M. viscida and M. gypsitherma belong outside it; ITS evidence suggested that M. gypsitherma is closely related to M. riparia of M. sect. Arida. The differing placements of these three species supported by chloroplast DNA and ITS evidence suggest that these species have experienced reticulate evolution. Nees, a genus of northern Mexico and the western United States, is generally charac- terized as consisting of tap-rooted annual or

The circumscription ofHoffmannseggia (Fabaceae, Caesalpinioideae, Caesalpinieae) and its allies using morphological and cpDNA restriction site data

The controversial issue of the circumscription ofHoffmannseggia and its relationship toCaesalpinia was examined using morphological characters and chloroplast DNA restriction site data from 32 species. The 53 morphological characters used included several not previously explored, but together they still failed to give unequivocal resolution of the taxa examined. However, morphological characters did provide some evidence for aHoffmannseggia clade, but different from that traditionally circumscribed. The cpDNA restriction site analysis using 16 enzymes yielded 233 informative characters that provided strong evidence for aHoffmannseggia clade. Both data sets supported the circumscription of a distinct “Pomaria” clade consisting in part of species traditionally placed withinHoffmannseggia. Nevertheless, bothHoffmannseggia andPomaria and their allies fall within a larger clade of species traditionally considered to belong toCaesalpinia broadly defined. BecauseCaesalpinia itself appears to be polyphyletic, we advocate here maintainingHoffmannseggia with the expectation that species ofCaesalpinia s. l. will ultimately be reapportioned to a number of smaller, monophyletic genera.

Phylogenetic implications of chloroplast DNA restriction site variation in subtribes Raphaninae and Cakilinae (Brassicaceae, tribe Brassiceae)

Chloroplast DNA restriction site data was used to assess relationships among 41 taxa of the subtribes Raphaninae and Cakilinae (tribe Brassiceae, Brassicaceae). A total of 456 restriction site mutations was observed, with 237 (52%) being phylogenetically informative. Cladistic analysis, based on Wagner parsimony analysis, indicated four major clades: (i) CAKILE, (ii) CRAMBE, (iii) NIGRA lineage, and (iv) RAPA-OLERACEA lineage. The CAKILE clade was divided into two lineages: (i) Cakile, Erucaria (including Reboudia), and Didesmus (previously assigned by Schulz to the Raphaninae) and (ii) Crambella (previously assigned by Schulz to the Raphaninae). The Raphaninae as currently circumscribed is polyphyletic in origin. The cpDNA data supported separate subtribal status for Crambe (Raphaninae) and indicated two major lineages corresponding to sect. Dendrocrambe and the combined sections Crambe and Leptocrambe. Enarthrocarpus arcuatus, Enarthrocarpus lyratus, Morisia monanthos, Raphanus raphanistrum, Raphanus sativus, and Rapistrum perenne were included in the RAPA-OLERACEA lineage of subtribe Brassicinae. Ceratocnemum rapistroides, Cordylocarpus muricatus, Guiraoa arvensis, Hemicrambe fruticulosa, Kremeriella cordylocarpus, Muricaria prostrata, Otocarpus virgatus, Raffenaldia primuloides, and Rapistrum rugosum were included in the NIGRA lineage of subtribe Brassicinae. Key words: Brassicinae, Raphaninae, Cakilinae, chloroplast DNA restriction site variation, molecular systematics.

Phylogenetic analysis of Helianthus (Asteraceae) based on chloroplast DNA restriction site data

Chloroplast DNA (cpDNA) restriction site data were used to analyze phylogenetic relationships within Helianthus. Data from new samples were consistent with previous results and showed the genus to be a well-defined (11 site changes) lineage within subtribe Helianthinae. The affinities of 2 species, Viguiera similis and V. phenax (V. ludens) that have sometimes been included in Helianthus were shown to lie outside the genus. The two species of Phoebanthus, however, formed a phylogenetic ingroup to the perennial Helianthus. Within the genus, cpDNA data indicated that there are four distinct phylogenetic lineages. Two of the lineages each contained a single, annual species (H. agrestis and H. porteri, respectively). The remaining annual species collectively formed a third lineage (sect. Helianthus). The fourth lineage contained all of the perennial species, including those of Phoebanthus. Within the perennial lineage, there was little variation in restriction site characters. The 3 species of series Pumili formed a paraphyletic group at the base of the perennial lineage, and the 3 species of series Ciliares formed a group that was supported by a single restriction site character.

Astragalus molybdenus s.l. (Leguminosae): Higher Taxonomic Relationships and Identity of Constituent Species

The Rocky Mountain Astragalus molybdenuis s.l. comprises A. molybdenus from Colorado, A. shultziorum from Wyoming, and A. lackschewitzii sp. nov. endemic to Montana. These three species are determined to be closely related to the species of Astragalus sect. Polares. Section Minerales, formerly a monotypic taxon that accommodated A. molybdenus in the Phacoid Phalanx next to sect. Astragalus, is now synonymized with sect. Polares. Supporting evidence comes from morphometric and cladistic analysis of morphological and chloroplast DNA restriction site data, and is consistent with findings reported elsewhere on sequence variation of the internal transcribed spacers that flank 5.8S nuclear ribosomal DNA. A previously overlooked attribute of A. molybdenus s.l. (the mature pod, which is deciduous from a marescent, ventrally ruptured calyx) is used to pinpoint the relationships of this species group to the piptoloboid Homalobi subgroup of Neo-astragalus. Additionally, chloroplast DNA sequences are exceptionally divergent among populations, on the order of 1.5-2.0%, and this evidence is used in part to support the recognition of three constituent species within A. molybdenus s.l.