Relationships of Droseraceae: a cladistic analysis of rbcL sequence and morphological data

Molecular support for the monophyly of Droseraceae and its phylogenetic relationships to other dicot families was investigated using parsimony analysis of nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). Analysis of 100 species of plants including families of subclasses Rosidae, Hamamelidae, Dilleniidae, and Caryophyllidae (sensu Cronquist) placed monophyletic Droseraceae in the same clade as Caryophyllidae and Nepenthaceae (Dilleniidae). In a second analysis of 14 species of Droseraceae, 15 caryophyllids, one Nepenthaceae, and three Santalales, a single most-parsimonious tree was found in which Droseraceae are monophyletic, although the position of Drosophyllum as a member of Droseraceae is only weakly supported. The rbcL tree identified four major lineages within genus Drosera: 1) Dionaea; 2) the regia-clade that contains only Drosera regia; 3) the capensis-clade that contains the South African and temperate species outside of Australia; and 4) the peltata-clade that consists of principally Australian endemics. A separate analysis of 14 morphological and phytochemical characters is in general agreement with the rbcL tree except for the placement of Nepenthes, Drosophyllum, and Drosera burmanni. A combined analysis of both data sets places Drosophyllum in a clade with Triphyophyllum (Dioncophyllaceae).     

Naphthalene glucoside and other phenolics from the shoot and callus cultures of Drosophyllum lusitanicum

The callus and, for the first time established, shoot cultures of Drosophyllum lusitanicum Link. (Droseraceae) yielded new naphthalene glucoside-5-hydroxy-4-methoxy-2-naphthalenecarboxylic acid 5-O-beta-glucoside (drosophylloside) and 5-hydroxy-4-methoxy-2-naphthalenecarboxylic acid methyl ester besides other phenolics like naphthalenes-5-hydroxy-4-methoxy-2-naphthalenecarboxylic acid (ancistronaphthoic acid B), hydroplumbagin 4-O-glucoside, naphthoquinones-plumbagin and 3-chloroplumbagin, C-glycosylflavones- vitexin, isovitexin, orientin and isoorientin. The pattern of phenolics found supports affinity of Drosophyllum to the families-Droseraceae, Ancistrocladaceae and Dioncophyllaceae.     

Relationships in the Caryophyllales as suggested by phylogenetic analyses of partial chloroplast DNA ORF2280 homolog sequences

Phylogenetic relationships within the angiosperm order Caryophyllales were investigated by comparative sequencing of two portions of the highly conserved inverted repeat (totaling some 1100 base pairs) coinciding with the region occupied by ORF2280 in Nicotiana, the largest gene in the plastid genomes of most land plants. Data were obtained for 33 species in 11 families within the order and for one species each of Plumbaginaceae, Polygonaceae, and Nepenthaceae. These data, when analyzed along with previously published ORF (open reading frame) sequences from Nicotiana. Spinacia. Epifagus, and Pelargonium using parsimony, neighbor-joining, and maximum likelihood methods, reveal that: (1) Amaranthus, Celosia, and Froelichia (all Amaranthaceae) do not comprise a monophyletic group; (2) Amaranthus may be nested within a paraphyletic Chenopodiaceae; (3) Sarcobatus (Chenopodiaceae) is allied with Nyctaginaceae + Phytolaccaceae (the latter family excluding Stegnosperma but including Petiveria); and (4) Caryophyllaceae (with Corrigiola basal within the clade) are sister group to Chenopodiaceae + Amaranthaceae. Basal relations within the order remain obscure. Sequence divergence values in pairwise comparisons across all Caryophyllales taxa ranged from 0.1 to 5% of nucleotides. However, despite these low values, 23 insertion and deletion events were apparent, of which five were informative phylogenetically and bolstered several of the relationships listed above. A polymerase chain reaction (PCR) survey for ORF homolog length variants in representatives from 70 additional angiosperm families revealed major deletions, of 100 to 1400 base pairs, in 19 of these families. Although the ORF is located within the mutationally retarded inverted repeat region of most angiosperm chloroplast DNAs, this gene appears particularly prone to length mutation.     

Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences

To study the inter- and infrafamilial phylogenetic relationships in the order Caryophyllales sensu lato (s.l.), ～930 base pairs of the matK plastid gene have been sequenced and analyzed for 127 taxa. In addition, these sequences have been combined with the rbcL plastid gene for 53 taxa and with the rbcL and atpB plastid genes as well as the nuclear 18S rDNA for 26 taxa to provide increased support for deeper branches. The red pigments of Corbichonia, Lophiocarpus, and Sarcobatus have been tested and shown to belong to the betacyanin class of compounds. Most taxa of the order are clearly grouped into two main clades (i.e., "core" and "noncore" Caryophyllales) which are, in turn, divided into well-defined subunits. Phytolaccaceae and Molluginaceae are polyphyletic, and Portulacaceae are paraphyletic, whereas Agdestidaceae, Barbeuiaceae, Petiveriaceae, and Sarcobataceae should be given familial recognition. Two additional lineages are potentially appropriate to be elevated to the family level in the future: the genera Lophiocarpus and Corbichonia form a well-supported clade on the basis of molecular and chemical evidence, and Limeum appears to be separated from other Molluginaceae based on both molecular and ultrastructural data.     

Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae

A group II intron containing the matK gene, which encodes a splicing-associated maturase, was found in the trnK (lysine tRNA) exon in the chloroplast genome of the six extant genera of green algae in the family Characeae, which among green algae are the sister group to embryophytes (land plants). The characean trnK intron (～2.5 kilobases [kb]) and matK ORF (～1.5 kb) are comparable in size to the intron and ORF of land plants, in which they are similarly found inserted in the trnK exon. Domain X, a sequence of conserved amino acid residues within matK, occurs in the Characeae. Phylogenetic analysis using maximum likelihood (GTR + I + gamma likelihood model) and parsimony (branch and bound search) yielded one tree with high bootstrap support for all branches. The matK tree was congruent with the rbcL tree for the same taxa. The number and proportion of informative sites was higher in matK (501, 31% of matK sequence) compared to rbcL (122, 10%). Characeae branch lengths were on average more than five times longer for matK compared to rbcL and provided better resolution within the Characeae. These findings along with recent genomic analyses demonstrate that the intron and matK invaded the chloroplast genome of green algae prior to the evolution of land plants.     

Intraspecific cpDNA variations of diploid and tetraploid perennial buckwheat, Fagopyrum cymosum (Polygonaceae)

We investigated the phylogenetic and biogeographic relationships of natural populations of diploid and tetraploid Fagopyrum cymosum (Polygonaceae). Intraspecific variation of chloroplast DNA sequences was detected in three regions approximately 5 kb long in total: the 3' end of rbcL, accD and associated intergenic spacer region, the trnC (GCA)-rpoB spacer region, the trnK (UUU) intron, and the matK region. The accessions of F. cymosum were divided into two major groups, a Tibet-Himalayan clade and a Yunnan-Sichuan clade, with a high bootstrap probability. It was estimated that these two clades diverged about 0.7 million years ago. The geographical and climatic interruption by the Hengduanshan mountains might have caused the genetic divergence in F. cymosum. Autotetraploid populations of F. cymosum have arisen allopatrically from a diploid progenitor at least twice, once in the Tibet-Himalayan area and once in the Yunnan-Sichuan area. This conclusion reinforces a previous study based on allozyme variation. We also found that F. tataricum, a close relative of F. cymosum, was completely included within the Tibet-Himalayan clade in the phylogenetic tree. This suggests that F. tataricum speciated from F. cymosum in the Tibet-Himalayan area.     

Phylogenetic relationships of Cornaceae and close relatives inferred from matK and rbcL sequences

Phylogenetic relationships were inferred using nucleotide sequences of the chloroplast gene matK for members of Cornales, a well-supported monophyletic group comprising Cornaceae and close relatives. The shortest trees resulting from this analysis were highly concordant with those based on previous phylogenetic analysis of rbcL sequences. Analysis of a combined matK and rbcL sequence data set (a total of 2652 bp [base pairs]) provided greater resolution of relationships and higher internal support for clades compared to the individual data sets. Four major clades (most inclusive monophyletic groups) of Cornales are indicated by both sets of genes: (1) Cornus-Alangium, (2) nyssoids (Nyssa-Davidia-Camptotheca)- mastixioids (Mastixia, Diplopanax), (3) Curtisia, and (4) Hydrangeaceae-Loasaceae. The combined evidence indicates that clades 2 and 3 are sisters, with clade 4 sister to the remainder of Cornales. These relationships are also supported by other lines of evidence, including synapomorphies in fruit and pollen morphology and gynoecial vasculature. Comparisons of matK and rbcL sequences based on one of the most parsimonious rbcL-matK trees indicate that matK has a much higher A-T content (66.9% in matK vs. 55.8% in rbcL) and a lower transition:transversion ratio (1.23 in matK vs. 2.21 in rbcL). The total number of nucleotide substitutions per site for matK is 2.1 times that of rbcL in Cornales. These findings are similar to recent comparisons of matK and rbcL in other dicots. Variable sites of matK are almost evenly distributed among the three codon positions (1.0:1.0:1.3), whereas variable sites of rbcL are mostly at the third position (1.8:1.0 :7.5). Among- lineages rates of nucleotide substitutions in rbcL are basically homogeneous throughout Cornales, but are more heterogeneous in matK.     

Phylogenetic relationships of the enigmatic angiosperm family Podostemaceae inferred from 18S rDNA and rbcL sequence data

The phylogenetic relationships of some angiosperm families have remained enigmatic despite broad phylogenetic analyses of rbcL sequences. One example is the aquatic family Podostemaceae, the relationships of which have long been controversial because of major morphological modifications associated with their aquatic habit. Podostemaceae have variously been associated with Piperaceae, Nepenthaceae, Polygonaceae, Caryophyllaceae, Scrophulariaceae, Rosaceae, Crassulaceae, and Saxifragaceae. Two recent analyses of rbcL sequences suggest a possible sister-group relationship of Podostemaceae to Crassulaceae (Saxifragales). However, the branch leading to Podostemaceae was long, and use of different outgroups resulted in alternative placements. We explored the phylogenetic relationships of Podostemaceae using 18S rDNA sequences and a combined rbcL + 18S rDNA matrix representing over 250 angiosperms. In analyses based on 18S rDNA data, Podostemaceae are not characterized by a long branch; the family consistently appears as part of a Malpighiales clade that also includes Malpighiaceae, Turneraceae, Passifloraceae, Salicaceae, Euphorbiaceae, Violaceae, Linaceae, Chrysobalanaceae, Trigoniaceae, Humiriaceae, and Ochnaceae. Phylogenetic analyses based on a combined 18S rDNA + rbcL data set (223 ingroup taxa) with basal angiosperms as the outgroup also suggest that Podostemaceae are part of a Malpighiales clade. These searches swapped to completion, and the shortest trees showed enhanced resolution and increased internal support compared to those based on 18S rDNA or rbcL alone. However, when Gnetales are used as the outgroup, Podostemaceae appear with members of the nitrogen fixing clade (e.g., Elaeagnaceae, Ulmaceae, Rhamnaceae, Cannabaceae, Moraceae, and Urticaceae). None of the relationships suggested here for Podostemaceae receives strong bootstrap support. Our analyses indicate that Podostemaceae are not closely allied with Crassulaceae or with other members of the Saxifragales clade; their closest relatives, although still uncertain, appear to lie elsewhere in the rosids.     

Angiosperm phylogeny inferred from sequences of four mitochondrial genes

An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.     

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.     

Phylogeny of the sundews, Drosera (Droseraceae), based on chloroplast rbcL and nuclear 18S ribosomal DNA Sequences

The sundew genus Drosera consists of carnivorous plants with active flypaper traps and includes nearly 150 species distributed mainly in Australia, Africa, and South America, with some Northern Hemisphere species. In addition to confused intrageneric classification of Drosera, the intergeneric relationships among the Drosera and two other genera in the Droseraceae with snap traps, Dionaea and Aldrovanda, are problematic. We conducted phylogenetic analyses of DNA sequences of the chloroplast rbcL gene for 59 species of Drosera, covering all sections except one. These analyses revealed that five of 11 sections, including three monotypic sections, are polyphyletic. Combined rbcL and 18S rDNA sequence data were used to infer phylogenetic relationships among Drosera, Dionaea, and Aldrovanda. This analysis revealed that all Drosera species form a clade sister to a clade including Dionaea and Aldrovanda, suggesting that the snap traps of Aldrovanda and Dionaea are homologous despite their morphological differences. MacClade reconstructions indicated that multiple episodes of aneuploidy occurred in a clade that includes mainly Australian species, while the chromosome numbers in the other clades are not as variable. Drosera regia, which is native to South Africa, and most species native to Australia, were clustered basally, suggesting that Drosera originated in Africa or Australia. The rbcL tree indicates that Australian species expanded their distribution to South America and then to Africa. Expansion of distribution to the Northern Hemisphere from the Southern Hemispere occurred in a few different lineages.     

Secretory structures in plants: Lessons from the Plumbaginaceae on their origin,evolution and roles in stress tolerance

The Plumbaginaceae (non-core Caryophyllales) is a family well known for species adapted to a wide range of arid and saline habitats. Of its salt-tolerant species, at least 45 are in the genus Limonium; two in each of Aegialitis, Limoniastrum and Myriolimon, and one each in Psylliostachys, Armeria, Ceratostigma, Goniolimon and Plumbago. All the halophytic members of the family have salt glands and salt glands are also common in the closely related Tamaricaceae and Frankeniaceae. The halophytic species of the three families can secrete a range of ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl⁻, HCO₃⁻, SO₄^2-) and other elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn). Salt glands are, however, absent in salt-tolerant members of the sister family Polygonaceae. We describe the structure of the salt glands in the three families and consider whether glands might have arisen as a means to avoid the toxicity of Na⁺ and/or Cl⁻ or to regulate Ca²⁺ concentrations with the leaves. We conclude that the establishment of lineages with salt glands took place after the split between the Polygonaceae and its sister group the Plumbaginaceae.     

Generic phylogeny and historical biogeography of Alismataceae, inferred from multiple DNA sequences

Alismataceae is an aquatic or semi-aquatic herb family with a subcosmopolitan distribution. The family is one of the oldest lineages within monocots and plays an important role in the systematics, biogeography and evolutionary processes of flowering plants. However, the generic relationships of the family are still a subject of debate, and its historical biogeography is less studied. In the present study, we carried out a comprehensive phylogenetic analysis based on multiple DNA sequences (nuclear: ITS; chloroplast: psbA, rbcL, matK, rpoB, rpoC1, trnK 5' intron and trnK 3' intron; mitochondria: cob and atp1). The result supports merging Limnocharitaceae into Alismataceae as one family. Two well-supported clades were obtained based on the combined ITS, psbA, rbcL and matK dataset. Clade B consists of Luronium, Damasonium, Baldellia and Alisma; and clade A consists of the remaining genera of Alismataceae as well as Limnocharitaceae. Biogeographic analysis and bayesian molecular dating suggested that Alismataceae originated in West Palearctic or Afrotropical area during the Late Cretaceous, and subsequently split into two clades. Clade A and clade B diversified in Afrotropical area and West Palearctic area, respectively. The intercontinental distribution of this family mainly resulted from dispersals involving migration across land bridges and long-distance dispersal.     

Wood anatomy of Polygonaceae: analysis of a family with exceptional wood diversity

Quantitative and qualitative data are presented for woods of 30 species of woody Polygonaceae. Wood features that ally Polygonaceae with Plumbaginaceae include nonbordered perforation plates, storeying in narrow vessels and axial parenchyma, septate or nucleate fibres, vasicentric parenchyma, pith bundles that undergo secondary growth, silica bodies, and ability to form successive cambia. These features are consistent with pairing of Plumbaginaceae and Polygonaceae as sister families. Wood features that ally Polygonaceae with Rhabdodendraceae include nonbordered perforation plates, presence of vestured pits in vessels, presence of silica bodies and dark-staining compounds in ray cells, and ability to form successive cambia. Of the features listed above, nonbordered perforation plates and ability to form successive cambia may be symplesiomorphies basic to Caryophyllales sensu lato . The other features are more likely to be synapomorphies. Wood data thus support molecular cladograms that show the three families near the base of Caryophyllales s.l. Chambered crystals are common to three genera of the family and may indicate relationship. Ray histology suggests secondary woodiness in Antigonon, Atraphaxis, Bilderdykia, Dedeckera, Eriogonum, Harfordia, Muehlenbeckia, Polygonum , and Rumex . Other genera of the family show little or no evidence of secondary woodiness. Molecular data are needed to confirm this interpretation and to clarify the controversial systematic groupings within the family proposed by various authors. Vessel features of Polygonaceae (lumen diameter, element length, density, degree of grouping) show an extraordinary range from xeromorphy to mesomorphy, indicating that wood has played a key role in ecological and habital shifts within the family; the diversity in ecology and habit are correlated with quantitative wood data.  © 2003 The Linnean Society of London. Botanical Journal of the Linnean Society , 2003, 141 , 25−51.     

Phylogenetic relationships in Saxifragaceae sensu lato: A comparison of topologies based on 18S rDNA and rbcL sequences

Relationships among the morphologically diverse members of Saxifragaceae sensu lato were inferred using 130 18S rDNA sequences. Phylogenetic analyses were conducted using representatives of all 17 subfamilies of Saxifragaceae sensu lato, as well as numerous additional taxa traditionally assigned to subclasses Magnoliidae, Caryophyllidae, Hamamelidae, Dilleniidae, Rosidae, and Asteridae. This analysis indicates that Saxifragaceae should be narrowly defined (Saxifragaceae sensu stricto) to consist of ~30 herbaceous genera. Furthermore, Saxifragaceae s. s. are part of a well-supported clade (referred to herein as Saxifragales) that also comprises lteoideae, Pterostemonoideae, Ribesioideae, Penthoroideae, and Tetracarpaeoideae, all traditional subfamilies of Saxifragaceae sensu lato, as well as Crassulaceae and Haloragaceae (both of subclass Rosidae). Paeoniaceae (Dilleniideae), and Hamamelidaceae, Cercidiphyllaceae, and Daphniphyllaceae (all of Hamamelidae). The remaining subfamilies of Saxifragaceae sensu lato fall outside this clade. Francoa (Francooideae) and Bauera (Baueroideae) are allied, respectively, with the rosid families Greyiaceae and Cunoniaceae. Brexia (Brexioideae), Parnassia (Parnassioideae), and Lepuropetolon (Lepuropetaloideae) appear in a clade with Celastraceae. Representatives of Phyllonomoideae, Eremosynoideae, Hydrangeoideae, Escallonioideae, Montinioideae, and Vahlioideae are related to taxa belonging to an expanded asterid clade (Asteridae sensu lato). The relationships suggested by analysis of 18S rDNA sequences are highly concordant with those suggested by analysis of rbcL sequences. Furthermore, these relationships are also supported in large part by other lines of evidence, including embryology. serology, and iridoid chemistry.     

Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae)

DNA barcoding is a new technology which can identify species rapidly based on short and standardized DNA sequences. Ligularia, a genus of Asteraceae with about 140 species, exhibits high morphological and ecological diversity, which makes the classification and species delimitation difficult, especially in the cases of closely related taxa. In this study, we tested four DNA core barcoding regions (ITS, matK, psbA trnH and rbcL) in 144 samples representing 35 species of Ligularia. The results revealed that the chloroplast regions (matK, psbA trnH and rbcL) have extremely low species identification rate due to low interspecific variation. Conversely, ITS sequence showed higher species identification rate (60%) and could discriminate the species which are difficult to identify. The combination of these four gene fragments did not improve the ability of species discrimination.     

Phylogenetic relationships of Eurasian pines (Pinus, Pinaceae) based on chloroplast rbcL, MATK, RPL20-RPS18 spacer, and TRNV intron sequences

The sequence divergence of chloroplast rbcL, matK, trnV intron, and rpl20-rps18 spacer regions was analyzed among 32 Pinus species and representatives of six other genera in Pinaceae. The total aligned sequence length is 3570 bp. Of the four sequences examined, matK evolved much faster than rbcL in Pinus and in other Pinaceae genera. The two noncoding regions did not show more divergence than the two coding regions, especially within each Pinus subgenus. Phylogenetic analyses based on these four sequences gave consistent results and strongly supported the monophyly hypothesis for the genus Pinus and its two recognized subgenera. Pinus krempfii, the two-flat-needle pine endemic to Vietnam, was placed in subgen. Strobus and showed closer affinity to subsect. Gerardianae. The ancient character of sect. Parrya is further confirmed. However, monophyly of the sect. Parrya is not supported by our data. Among the Eurasian pines of subgen. Pinus, Mediterranean pines formed one clade and the Asian members of subsect. Sylvestres formed another. The Himalayan P. roxburghii showed considerable divergence from all the other hard pines from both regions. Pinus merkusii was distinctly separated from all the Asian members of subsect. Sylvestres. The implications of our results for Pinus classification are discussed.