Molecular phylogenetics reveals Leontodon (Asteraceae, Lactuceae) to be diphyletic

The plastid matK gene, trnL/F spacer, and nuclear rDNA ITS were sequenced for 36 species of Leontodon and 29 taxa of related genera of tribe Lactuceae. Phylogenetic relationships inferred from the independent and combined data are largely congruent and reveal that Leontodon sensu lato (s.l.) as presently defined is diphyletic: L. subgenus Leontodon forms a clade with Helminthotheca, Picris and Hypochaeris as sister genera, whereas L. subgenus Oporinia appears as a separate clade with strong bootstrap support and is thus better treated as a separate genus. Previous sectional classifications of Leontodon s.l. are considered in the light of DNA and additional morphological and karyological data. Support is presented for a core group of Hypochaeridinae sensu stricto (s.s.) with the two clades of Leontodon s.l., Helminthotheca, Picris, and Hypochaeris, whereas Urospermum, Hyoseris, Aposeris, and Rhagadiolus appear to be positioned more distantly.     

Preliminary phylogeny of Valerianaceae (Dipsacales) inferred from nuclear and chloroplast DNA sequence data

Valerianaceae is a relatively small (ca. 350 species), but morphologically diverse angiosperm clade. Sequence data from the entire ndhF gene, the trnL-F intergenic spacer region, the trnL intron, the matK region, the rbcL-atpB intergenic spacer region and internal transcribed spacer (ITS) region of nuclear ribosomal DNA were collected for 21 taxa within Dipsacaceae and Valerianaceae (1 and 20, respectively). These data were included in several phylogenetic analyses with previously published sequences from Dipsacales. Results from these analyses (maximum parsimony, maximum likelihood, and Bayesian analysis) are in strong agreement with many of the conclusions from previous studies, most importantly: (1) Valerianaceae is sister to Dipsacaceae; (2) Triplostegia is more closely related to species of Dipsacaceae than to Valerianaceae; and (3) Valeriana appears not to be monophyletic, with Valeriana celtica falling outside the remainder of the species of Valeriana sampled here (with very strong support). With the exception of V. celtica, these data support two major clades within Valeriana; one that is exclusively New World and another that is distributed in both the Old and New World. Although the species of Valerianaceae and its sister group Dipsacaceae plus Triplostegia, are widely distributed in the Northern Hemisphere, and the data imply that Valerianaceae diversified initially in Asia (the Himalayan Patrinia and Nardostachys falling at the base of the clade), the center of modern species diversity for the group is in the Andes of South America with as many as 175 species restricted to that region. Although the exclusively South American taxa form a clade in the chloroplast and combined ITS and chloroplast analyses, support values tend to be low. Future studies will need to include additional data, in the form of both characters and taxa, before any strong conclusions about the character evolution, diversification, and biogeography of the South American valerians can be made.     

Nuclear ribosomal DNA and karyotypes indicate a NW African origin of South American Hypochaeris (Asteraceae, Cichorieae)

Hypochaeris has a disjunct distribution, with more than 15 species in the Mediterranean region, the Canary Islands, Europe, and Asia, and more than 40 species in South America. Previous studies have suggested that the New World taxa have evolved from ancestors similar to the central European H. maculata. Based on internal transcribed spacer (ITS) sequences and fluorescence in situ hybridization (FISH) with 5S and 18S-25S rDNA of the previously overlooked Hypochaeris angustifolia from Moyen Atlas, Morocco, we show that it is sister to the entire South American group. A biogeographic analysis supports the hypothesis of long-distance dispersal from NW Africa across the Atlantic Ocean for the origin of the South American taxa rather than migration from North America, through the Panamian land bridge, followed by subsequent extinction in North America. With the assumption of a molecular clock, the trans-Atlantic dispersal from NW Africa to South America is roughly estimated to have taken place during Pliocene or Pleistocene.     

Phylogeny and systematics of the genus Lophozia s. str. (Dumort.) Dumort. (Hepaticae) and related taxa from nuclear ITS1-2 and chloroplast trnL-F sequences

Nuclear ITS1-2 and chloroplast trnL-F were sequenced for 21 taxa of Lophozia s. str., two species of Protolophozia, five species of Schistochilopsis, three species of Barbilophozia and Obtusifolium obtusum. The topologies of phylogenetic trees for 49 taxa constructed from combined sequences of these regions by maximum parsimony, maximum likelihood and Bayesian methods are similar. The species of Lophozia s. str., excluding Lophozia sudetica, combine into two main clades and these contradict subdivisions of Lophozia s. str. based on morphology. The species status of Lophozia lantratoviae is confirmed, whereas Lophozia austro-sibirica is almost identical to Lophozia ventricosa var. guttulata. The genus Schistochilopsis is paraphyletic and occupies basal position to Lophozia s. str., while O. obtusum is clearly separated from Schistochilopsis. A low level of divergence was found between L. sudetica and Protolophozia debiliformes, which are closer to Barbilophozia than to Lophozia s. str. Molecular divergence between geographically remote populations of L. sudetica, Lophozia silvicoloides and Protolophozia debiliformis are low as opposed to those of Lophozia polaris, Lophozia pellucida or Lophozia excisa. Consideration of the trnL intron P8 region indels alone can adequately assign some clades revealed by tree building. A consensus secondary structure of the trnL intron P8 region could not be inferred for taxa studied mainly due to high sequence length diversity originated from deletions.     

Evolution of Caulanthus amplexicaulis var. barbarae (Brassicaceae), a rare serpentine endemic plant: a molecular phylogenetic perspective

Intra- and interspecific phylogenetic relationships of the rare serpentine endemic taxon Caulanthus amplexicaulus var. barbarae and related taxa in the "Streptanthoid Complex" of genera (Streptanthus, Caulanthus, Guillenia) were examined using nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL intron sequences. Phylogenetic hypotheses generated from 81 variable ITS nucleotide sites and six variable trnL nucleotide sites indicate that Streptanthus and Caulanthus are nonmonophyletic groups. Caulanthus amplexicaulis var. barbarae and its more widespread nonserpentine sister taxon Caulanthus amplexicaulis var. amplexicaulis formed a distinct monophyletic group. Among the taxa in our study, C. amplexicaulis was most closely related to Streptanthus tortuosus. The ITS sequences supported monophyly of subgenus Euclesia, which includes the bulk of the serpentine endemics in the Streptanthoid Complex. The serpentine taxa were nonmonophyletic, occurring in at least three distinct clades, suggesting that tolerance to serpentine may be gained or lost through relatively few genetic changes. Intraspecific ITS1 and ITS2 sequence divergence within C. amplexicaulis (1.3-1.8%) was higher than in comparable species (0.0-0.3%); implications of this genetic differentiation for the conservation status of C. amplexicaulis var. barbarae are discussed. Evidence is presented that supports a "biotype depletion" model for the origin of this rare endemic taxon.     

Molecular phylogenetics of Houstonia (Rubiaceae): descending aneuploidy and breeding system evolution in the radiation of the lineage across North America

Nuclear and chloroplast DNA sequence variation was used to infer evolutionary relationships within and among members of Houstonia (Rubiaceae) and other closely related genera in North America. Sequences from the internal transcribed spacer (ITS) of the nrDNA and a cpDNA intron in the trnL gene were used to reconstruct phylogenetic relationships of 30 species of Houstonia and closely related genera. The data suggest that the North American species of Houstonia are not monophyletic, but belong to the same lineage as Stenaria. The radiation of this lineage has been accompanied by changes in the basic chromosome number of the major clades through descending aneuploidy. This loss of chromosomes was also associated with northward colonization of North America. However, other characters, such as an annual versus perennial habit, heterostylous and homostylous breeding systems, and the evolution of self fertilization, seem to be labile throughout the lineage, originating multiple times throughout the evolutionary history of the lineage.     

Discordance of chloroplast and nuclear ribosomal DNA data in Osmorhiza (Apiaceae)

Phylogenetic studies were conducted to evaluate interspecific relationships in Osmorhiza (Apiaceae: Apioideae) using sequences of the ITS regions of nuclear ribosomal DNA, the chloroplast ndhF gene, and two noncoding regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer). All data sets suggest the monophyly of the New World taxa and showed that Osmorhiza aristata from Asia is relatively divergent from other members of the genus, even though it is morphologically similar to the eastern North American O. claytonii and O. longistylis. The ITS and chloroplast DNA trees differ in the relationships among the New World taxa, especially the phylogenetic position of O. occidentalis, O. glabrata, and O. depauperata. The lack of congruence between the two data sets may be a result of hybridization or introgression. Although there is high discordance between nrITS and two chloroplast DNA data sets, the latter two show similar topologies.     

Interspecific and progeny relationships in the genus Stylosanthes inferred from chloroplast DNA sequence variation.

The chloroplast trnL (UAA) intron and trnL (UAA)-trnF (GAA) intergenic spacer region have been sequenced from 37 samples, 36 of which representing 19 Stylosanthes species and one from the related genus Zornia. The DNA sequences were used to study phylogenetic relationships in the tropical forage legume genus Stylosanthes, by means of parsimony analysis using the heuristic search method of the computer program PAUP. The resulting cladograms divide Stylosanthes into four separate clades. Within the clades, species are poorly resolved owing to low sequence divergence. Small intra-specific chloroplast DNA variation is observed in S. humilis, S. scabra and the species complex S. guianensis. Variation between S. humilis populations is considered to be geographically structured. The overall results agree well with previously established inter-specific relationships and provide evidence for the genetic origin of the alloploid species S. hamata, S. scabra, S. ingrata, S. sympodialis, S. subsericea, S. capitata and S. fruticosa. This understanding of evolutionary relationships in Stylosanthes, in combination with biogeographical concepts provides a way of discerning isolated habitats in Central and South America, which may therefore contribute to strategies of plant collecting.     

Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences

Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.     

Northern hemisphere biogeography of Cerastium (Caryophyllaceae): insights from phylogenetic analysis of noncoding plastidnucleotide sequences

Phylogenetic relationships and biogeography of the genus Cerastium were studied using sequences of three noncoding plastid DNA regions (trnL intron, trnL-trnF spacer, and psbA-trnH spacer). A total of 57 Cerastium taxa was analyzed using two species of the putative sister genus Stellaria as outgroups. Maximum parsimony analyses identified four clades that largely corresponded to previously recognized infrageneric groups. The results suggest an Old World origin and at least two migration events into North America from the Old World. The first event possibly took place across the Bering land bridge during the Miocene. Subsequent colonization of South America occurred after the North and South American continents joined during the Pliocene. A more recent migration event into North America probably across the northern Atlantic took place during the Quaternary, resulting in the current circumpolar distribution of the Arctic species. Molecular clock dating of major biogeographic events was internally consistent on the phylogenetic trees. The arctic high-polyploid species form a polytomy together with some boreal and temperate species of the C. tomentosum group and the C. arvense group. Lack of genetic variation among the arctic species probably indicates a recent origin. The annual life form is shown to be of polyphyletic origin.     

Nuclear and cpDNA sequences combined provide strong inference of higher phylogenetic relationships in the phlox family (Polemoniaceae)

Members of the phlox family (Polemoniaceae) serve as useful models for studying various evolutionary and biological processes. Despite its biological importance, no family-wide phylogenetic estimate based on multiple DNA regions with complete generic sampling is available. Here, we analyze one nuclear and five chloroplast DNA sequence regions (nuclear ITS, chloroplast matK, trnL intron plus trnL-trnF intergeneric spacer, and the trnS-trnG, trnD-trnT, and psbM-trnD intergenic spacers) using parsimony and Bayesian methods, as well as assessments of congruence and long branch attraction, to explore phylogenetic relationships among 84 ingroup species representing all currently recognized Polemoniaceae genera. Relationships inferred from the ITS and concatenated chloroplast regions are similar overall. A combined analysis provides strong support for the monophyly of Polemoniaceae and subfamilies Acanthogilioideae, Cobaeoideae, and Polemonioideae. Relationships among subfamilies, and thus for the precise root of Polemoniaceae, remain poorly supported. Within the largest subfamily, Polemonioideae, four clades corresponding to tribes Polemonieae, Phlocideae, Gilieae, and Loeselieae receive strong support. The monogeneric Polemonieae appears sister to Phlocideae. Relationships within Polemonieae, Phlocideae, and Gilieae are mostly consistent between analyses and data permutations. Many relationships within Loeselieae remain uncertain. Overall, inferred phylogenetic relationships support a higher-level classification for Polemoniaceae proposed in 2000.     

Generic delimitation and phylogenetic relationships within the subtribe Chironiinae (Chironieae: Gentianaceae), with special reference to Centaurium: evidence from nrDNA and cpDNA sequences

To better understand the evolutionary history of the genus Centaurium and its relationship to other genera of the subtribe Chironiinae (Gentianaceae: Chironieae), molecular analyses were performed using 80 nuclear ribosomal ITS and 76 chloroplast trnLF (both the trnL UAA intron and the trnL-F spacer) sequences. In addition, morphological, palynological, and phytochemical characters were included to a combined data matrix to detect possible non-molecular synapomorphies. Phylogenetic reconstructions support the monophyly of the Chironiinae and an age estimate of ca. 22 million years for the subtribe. Conversely, both molecular data sets reveal a polyphyletic Centaurium, with four well-supported main clades hereafter treated as separate genera. The primarily Mediterranean Centaurium s.s. is closely related to southern African endemics Chironia and Orphium, and to the Chilean species Centaurium cachanlahuen. The resurrected Mexican and Central American genus Gyrandra is closely related to Sabatia (from eastern North America). Lastly, the monospecific genus Exaculum (Mediterranean) forms a monophyletic group together with the two new genera: Schenkia (Mediterranean and Australian species) and Zeltnera (all other indigenous American centauries). Several biogeographical patterns can be inferred for this group, supporting a Mediterranean origin followed by dispersals to (1) North America, Central America, and South America, (2) southern Africa (including the Cape region), and (3) Australia and Pacific Islands.     

Addressing the "hardest puzzle in American pomology:" Phylogeny of Prunus sect. Prunocerasus (Rosaceae) based on seven noncoding chloroplast DNA regions

Prunus subg. Prunus sect. Prunocerasus (Rosaceae) is a North American taxon with 17 commonly recognized taxa. To test the hypothesis of monophyly for the section we sequenced the trnG and rpL16 introns and the trnH-psbA and trnS-trnG intergenic spacers for at least two representatives of each of the five subgenera in Prunus. Additionally we sampled heavily among Prunus subg. Prunus sections Prunus and Armeniaca and Prunus subg. Amygdalus because these groups are putatively most closely related to Prunocerasus. Once monophyly of sect. Prunocerasus was shown we added the sequences of trnL and rpS16 introns and the trnL-trnF spacer in an attempt to increase resolution within the section. The species of sect. Prunocerasus showed an initial split with P. subcordata, the only species from western North America, sister to the rest of the group. The remaining species fell into three primary clades. Within each of the three primary clades there was little phylogenetic resolution. Lastly, we present evidence that P. texana, previously classified in subg. Amygdalus, may be a plum or at least contain a Prunocerasus chloroplast. This is the first phylogenetic hypothesis presented for sect. Prunocerasus, and the clades recovered contrast sharply with previously defined groups based on morphological characters.     

Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences

To evaluate the monophyly of subtribe Pleurothallidinae (Epidendreae: Orchidaceae) and the component genera and to reveal evolutionary relationships and trends, we sequenced the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S gene for 185 taxa. In addition, to improve the overall assessments along the spine of the topology, we added plastid sequences from matK, the trnL intron, and the trnL-F intergenic spacer for a representative subset of those taxa in the ITS study. All results were highly congruent, and so we then combined the sequence data from all three data sets in a separate analysis of 58 representative taxa. There is strong support in most analyses for the monophyly of Pleurothallidinae and in some for inclusion of Dilomilis and Neocognauxia of Laeliinae. Although most genera in the nine clades identified in the analyses are monophyletic, all data sets are highly congruent in revealing the polyphyly of Pleurothallis and its constitutent subgenera as presently understood. The high degree of homoplasy in morphological characters, especially floral characters, limits their usefulness in phylogenetic reconstruction of the subtribe.     

Phylogeny of taxaceae and cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA ITS region

Phylogeny of the Taxaceae genera and the monotypic family Cephalotaxaceae has been extraordinarily controversial. In this paper chloroplast matK genes and nuclear ITS sequences were determined for all six genera of the two families and representatives of other conifer families. Analysis using either the nonsynonymous sites or the deduced amino acid sequences of matK genes strongly indicates that taxad genera and Cephalotaxaceae are monophyletic, with the Taxodiaceae/Cupressaceae clade as their sister group. Cephalotaxus is basal to the taxad genera, among which two clades, Torreya/Amentotaxus and Taxus/Pseudotaxus/Austrotaxus, are resolved. They correspond to Janchen's two tribes, Torreyeae and Taxeae. In Taxeae, Austrotaxus is the first to branch off. Analyses of the nuclear ITS sequence data corroborated the topology of the matK gene tree. These results refute the views that Cephalotaxaceae has no alliance with Taxaceae and that Austrotaxus and Amentotaxus should be excluded from the Taxaceae. We estimated the divergence time between the Taxodiaceae/Cupressaceae and the Cephalotaxaceae/Taxaceae clades to be 192-230 Myr ago and the divergence time between taxads and Cephalotaxus to be 149-179 Myr ago. Soon after the latter divergence event, within 6-8 Myr, the two taxad tribes originated. In conclusion, our data do not support Florin's claim that taxads could be traced to Devonian psilophytes (359-395 Myr ago).     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

Patterns of evolution in western North American Mimulus (Phrymaceae)

A well-supported phylogeny is presented from both chloroplast DNA (the trnL/F region) and two regions of nuclear rDNA (ITS [internal transcribed spacer] and ETS [external transcribed spacer]) with nearly complete sampling for Mimulus (Phrymaceae) in western North America. Three separate genera are derived from within the clade that contains all the Mimulus species in western North America. The taxonomic status of the proposed sections of Mimulus and the relationships of many taxonomically difficult species are considered with observations on morphological evolution. Discordance between data sources provides support for the hypothesis that M. evanescens is a hybrid between M. latidens and M. breviflorus. In two major clades (Eunanus and Diplacus), patterns of genetic variation do not match the current taxonomy. The clustering of taxa in Eunanus is strongly associated with geographic distributions. Mimulus aurantiacus sensu Thompson, M. nanus, and M. floribundus are found to be progenitor species to other species that appear to be derived from within them. Polyploidy and aneuploidy events are clustered near the tips of the phylogeny. Thus, these two mechanisms are concluded to have played a relatively small role in the evolution of persistent lineages in Mimulus. The phylogenetic distribution of rare taxa is also examined.     

Low variability of internal transcribed spacer rDNA and trnL (UAA) intron sequences of several taxa in the Festuca ovina aggregate (POACEAE)

Identification and classification of numerous Festuca species is still a difficult problem due to the close morphological resemblance. The most difficult fine fescues to identify belong to the Festuca ovina aggregate, which is the largest group in the genus Festuca. Many taxons are considered to be separate species based on quantitative taxonomic characters, differences in ploidy level or the structure of sclerenchyma cells. In order to evaluate the taxonomic value of DNA-based markers, sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and the chloroplast trnL (UAA) intron was performed in the ten most problematic fine fescues belonging to the Festuca ovina aggregate. Intraspecific ITS variants were found in a single case while in other cases only intragenomic ITS polymorphisms were detected with 1-2 ambiguous positions. Among the sequences of the trnL (UAA) intron even intragenomic polymorphisms were not detected in any of the Festuca species studied. Thus, the results do not support the species status of these ten taxa.     

Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions

For Nicotiana, with 75 naturally occurring species (40 diploids and 35 allopolyploids), we produced 4656bp of plastid DNA sequence for 87 accessions and various outgroups. The loci sequenced were trnL intron and trnL-F spacer, trnS-G spacer and two genes, ndhF and matK. Parsimony and Bayesian analyses yielded identical relationships for the diploids, and these are consistent with other data, producing the best-supported phylogenetic assessment currently available for the genus. For the allopolyploids, the line of maternal inheritance is traced via the plastid tree. Nicotiana and the Australian endemic tribe Anthocercideae form a sister pair. Symonanthus is sister to the rest of Anthocercideae. Nicotiana sect. Tomentosae is sister to the rest of the genus. The maternal parent of the allopolyploid species of N. sect. Polydicliae were ancestors of the same species, but the allopolyploids were produced at different times, thus making such sections paraphyletic to their extant diploid relatives. Nicotiana is likely to have evolved in southern South America east of the Andes and later dispersed to Africa, Australia, and southwestern North America.     

Potential phylogenetic utility of the low-copy nuclear gene pistillata in dicotyledonous plants: comparison to nrDNA ITS and trnL intron in Sphaerocardamum and other Brassicaceae.

We report the potential phylogenetic utility of DNA sequence data from the last 700 bp of a ca. 1-kb intron of the MADS-box gene pistillata from a sampling of Sphaerocardamum species and other Brassicaceae. These results are compared with nrDNA ITS and the chloroplast trnL intron for the same taxa to demonstrate the potential phylogenetic utility of this pistillata intron and to identify potential historically independent sequences for an ongoing study of relationships within Sphaerocardamum. Analyses of the DNA sequence data for Brassicaceae indicated that pairwise divergences and potentially informative characters were higher in the pistillata intron (0.6-30.8%, 284 characters) and ITS (0-24%, 94 characters) than in the chloroplast trnL intron (0-4.2%, 17 characters). A comparison of Sphaerocardamum sequences identified low divergences and numbers of informative characters for trnL intron (0-2.4%, 1 character) and nrDNA ITS (0-2.5%, 2 characters) and substantially more variation among the pistillata sequences (0.15-3.7%, 19 characters). Phylogenetic analyses of these pistillata sequences fully resolve ingroup relationships without character conflict. Results of pistillata PCR amplifications from a broader dicot sample showed that some primers may be useful in amplifying orthologous pistillata sequences. Ultimately this pistillata intron may be a valuable source of phylogenetic characters at lower taxonomic levels.