Phylogenetic relationships of ferns deduced from rbcL gene sequence

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene (rbcL) was sequenced from three fern species: Adiantum capillus-veneris, Botrypus strictus, and Osmunda cinnamomea var. fokiensis. This region included 1,333 base pairs, about 90% of the gene. Maximum likelihood analysis of the deduced amino acid sequences indicated that (1) Botrypus (Ophioglossaceae) clustered monophyletically with other ferns (Adiantum, Angiopteris, Osmunda); the closest relative to Botrypus among the three species was Osmunda, which did not support the hypothesis that the Ophioglossaceae are linked to the progymnosperm-seed plant lineage. (2) Eusporangiate ferns containing Botrypus (Ophioglossaceae) and Angiopteris (Marattiaceae) were a paraphyletic group. (3) Seed plants and the four fern species examined formed a monophyletic group, but ferns and bryophytes (liverwort) did not. Variations in rates of substitution for synonymous and nonsynonymous codons were found in fern lineages.Correspondence to: M. Hasebe     

Relations between submarginal and marginal sori in ferns IV. The sori of Osmunda and selected Schizaeaceae and their morphological relations

Developmental types of sori in Osmunda and Schizaeaceae are comparatively studied and discussed in a systematic context. (1) The ``simultaneous marginal sorus' is characterized by a minute knob-like receptacle representing the thickened tip of a short costa, simultaneous initiation of massive sporangia, and absence of indusia. (2) The ``acropetal marginal sorus' is characterized as an accessory outgrowth at the end of a costa or at the margin of a sorophore, comprising a minute or elongated bifacial receptacle, acropetal initiation of massive marginal sporangia, and minute or long-extended or hood-shaped upper indusia. The state of superficial singly-arranged massive sporangia such as in Todea may give rise to the Osmunda type of the simultaneous marginal sorus and to the Schizaeaceae types of the acropetal marginal sorus. Phase differentiation in leaf development combined with complete laminar reduction leads to the Osmunda type, while phase differentiation in leaf lamina development and additional processes may give rise to the Mohria, Anemia, and Lygodium types. These morphological results support recent phylogenetic studies by other authors in disclaiming close relationships between Osmundaceae and Gleicheniaceae, and between Marattiaceae and Ophioglossaceae (``Eusporangiatae'). They also potentially support relationships between Osmunda- ceae and Schizaeaceae or between Ophioglossaceae and Schizaeaceae. The high complexity of the sori in Marattiaceae and Ophioglossaceae is surprising if the two families are viewed as basal groups.     

OSMUNDA WEHRII,A NEW SPECIES BASED ON PETRIFIED RHIZOMES FROM THE MIOCENE OF WASHINGTON

Silicified rhizomes from Miocene strata near Yakima, Washington represent a new species of Osmunda. The stems are 8–13 mm in diameter and are surrounded by a thick sheath of adherent leaf bases, each of which shows stipular expansions typical of the Osmundaceae. The new species has an ectophloic siphonostele in which the xylem cylinder is dissected by leaf gaps with 12–14 strands being visible in a given stem cross section. Such sections also show 12–16 leaf traces in the cortex. The xylem of each leaf trace diverges from the xylem cylinder of the stem as an adaxially concave strand with its protoxylem organized into a single medial adaxial cluster. Initial bifurcation of the leaf-trace protexylem occurs as the leaf trace passes through the outer cortex of the stem. In the basal part of the stipular region of the petiole base, thick-walled fibers form an arch on the abaxial side of the sclerenchyma ring around the petiolar bundle. This arch persists throughout most of the length of the stipular region, with the thick-walled fibers becoming reorganized into two lateral masses in the distal part of the stipular region. Similar thick-walled fibers form an elongate strip of tissue in each wing of the stipule along with several small clusters scattered near the sclerenchyma ring. The new species belongs to the subgenus Osmunda and shows that during the Neogene, the latter existed as a group of closely related species much as it does today. Furthermore, Osmunda wehrii combines features of the modern O. regalis, O. japonica, and O. lancea with those of O. claytoniana and thus supports the inclusion of the latter species in the subgenus Osmunda.     

Die Beeinflussung der Farnsporen-Keimung [Osmunda cinnamomea (L.) undO. claytoniana (L.)] über das Phytochromsystem und die Photosynthese

Summary The control by light of the spore germination ofOsmunda cinnamomea L. andO. claytoniana L. has been investigated.This light control is complex, and in order to investigate this control quantitatively the process of germination had to be divided into at least two stages. The first stage of germination leads to the rupture of the exospore, the second step is characterized by the outgrowth of a rhizoid. In a very few percent of our spore population the first stage can occur in total darkness, the second step however does not ever occur in darkness.In the present paper it has been demonstrated, usingO. cinnamomea spores, that the first step of germination is phytochrome controlled, whereas the second step is under photosynthetic control. This was observed withO. claytoniana by a rough action spectrum and in experiments with a CO₂-less atmosphere.The germinating spores of these two species ofOsmunda show a very similar behaviour as the spores of the mossFunaria hygrometrica (L.) Sibth. (Bauer andMohr 1959). They behave very differently compared with spores of other ferns, e.g.Dryopteris filix-mas (L.) Schott, where the whole process of germination is exclusively phytochrome controlled (Mohr 1956).

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Studies on DNA sequences in the Osmundaceae

Summary Phylogenetic relationships ofOsmunda cinnamomea, O. claytoniana, andO. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences ofO. claytoniana are nearly equally diverged from those ofO. cinnamomea andO. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the threeOsmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hybridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.     

Characterization of Stelar Initiation in Shoot Apices of Ferns

Procambium is commonly recognized as a vascular meristem inshoot apices of vascular plants. Prestelar tissue comprisingprovascular tissue (PVT) and pith mother cells (PMCs) immediatelysubjacent to the single cell layer of promeristem has been consideredto represent the initial stage of stelar differentiation precedingprocambium and rib meristem in ferns. In addition to characterizationof PVT and PMCs on the basis of cell morphology, cytologicalfeatures and developmental continuity with procambium and ribmeristem, four lines of evidence from studies of shoot apicesof Matteuccia struthiopteris and Osmunda cinnamomea supportthis interpretation of initial differentiation. (1) Differentialstaining by safranin-fast green and crystal violet-erythrosinshows that PVT and PMCs differ in colour reactions from promeristemand resemble procambium and pith meristem, respectively. (2)Comparative ultrastructural study reveals qualitative differencesin the cell membrane system, nuclei, cytoplasm, vacuoles andplastids between promeristem and PVT but similarity of PVT toprocambium. (3) Large droplets of tannins occur in promeristembut not in PVT, PMCs and procambium. (4) Cytochemical studyof the shoot apex of Osmunda shows that carboxylesterase activityis strongly demonstrated in PVT and procambial cells but notin promeristem cells and PMCs. These observations further substantiatethe interpretation that PVT represents initial vascular differentiationand PMCs reflect a commitment to pith development.Copyright1995, 1999 Academic Press Initial vascular differentiation, provascular tissue, differential staining, ultrastructure, tannins, carboxylesterase, shoot apex, Matteuccia struthiopteris, Osmunda cinnamomea     

Reticulate evolution of the hybrid produced artificially by crosses between Osmunda banksiifolia and Osmunda lancea

Although ferns have been developed by hybridization and chromosome doubling, no natural polyploidy has yet been recorded in Osmundaceae. So, we produced hybrids artificially by crosses between Osmunda banksiifolia (2n = 2x = 44) and Osmunda lancea (2n = 2x = 44), and investigated their sporogenesis. From the O. banksiifolia × O. lancea hybrid with 44 univalent chromosomes, allotetraploids with 44 bivalent chromosomes were produced by chromosome doubling, and allotriploids with 22 univalent chromosomes and 22 bivalent chromosomes were then produced by back crosses. The results show when and how chromosome doubling occurs in hybrids. The success of artificial hybridization between O. banksiifolia and O. lancea, did not, however, reflect any product of natural hybridization between the two species.     

A specialized new species of Ashicaulis (Osmundaceae,Filicales) from the Jurassic of Liaoning,NE China

A new species of structurally preserved fern rhizome, Ashicaulis plumites (Osmundaceae, Filicales), is described from the Middle Jurassic Tiaojishan Formation in western Liaoning Province, NE China. The new species is characterized by a peculiar sclerenchyma mass in the petiolar vascular bundle concavity. This sclerenchyma mass varies from a linear-shape to a mushroom-like shape with a remarkable outward protuberance, which distinguishes the present new species from other Ashicaulis species. Such a protuberance is very rare among osmundaceous ferns, and should represent a unique type for sclerenchymatous tissue in the osmundaceous vascular bundle concavity. Recognition of the peculiar structure of this new fossil species enriches anatomical diversity of permineralized osmundaceous ferns, indicating that the family Osmundaceae might have experienced a remarkable diversification during the Middle Jurassic in NE China. The new species show anatomical similarities to Osmunda pluma Miller from the Palaeocene of North America. The occurrence of A. plumites in the Middle Jurassic of China provides a new clue for understanding the evolution of some members of the living subgenus Osmunda.     

A tourist element in the 5'-flanking region of the catalase gene CatA reveals evolutionary relationships among Oryza species with various genome types

Tourist-OsaCatA, a transposable element, was found in the 5′-flanking region of the rice gene CatA. The characteristics of this element are similar to those of the other Tourist elements so far found in Oryza sativa. PCR and sequence analyses of 37 accessions of 18 species revealed that all the Oryza species examined, except for one accession, have either a full-length or a partial Tourist element at this locus. Unlike the Tourist elements previously reported, this Tourist element is found in all four Oryza species complexes in the Oryzeae tribe. All AA genome Oryza species, except O. longistaminata, contain the full-length Tourist element. O. longistaminata and the species of the O. officinalis, O. meyeriana and O. ridleyi complexes contain the partial element. A phylogenetic tree of Oryza species based on the nucleotide sequences of these Tourist elements was constructed. The O. longistaminata accessions were placed near the neighboring cluster of the officinalis complex. We propose that the ancestor of O. longistaminata and that of other species with the AA genome diverged, and the ancestor(s) of the O. officinalis, O. ridleyi and O. meyeriana complexes then diverged from the ancestor of O. longistaminata in the course of the evolution of the Oryza species. The Tourist elements associated with CatA and its orthologs thus provide useful tools for examining evolutionary relationships among Oryza species. Received: 12 March 1999 / Accepted: 7 July 1999     

Phylogeny and Evolution in Cariceae (Cyperaceae): Current Knowledge and Future Directions

The goal of this study was to review the impact of DNA sequence analyses on our understanding of Cariceae phylogeny, classification and evolution. To explore character evolution, 105 taxa from four different studies were included in an nrDNA ITS + ETS 1f analysis of all recognized genera (Carex, Cymophyllus, Kobresia, Schoenoxiphium, Uncinia) and Carex subgenera (Carex, Psyllophora, Vignea, Vigneastra). As in previous analyses, four major Cariceae clades were recovered: (1) a “Core Carex Clade” (subg. Carex, Vigneastra, Psyllophora p.p); (2) A “Vignea Clade” (subg. Vignea, Psyllophora p.p.); (3) a “Schoenoxiphium Clade” (Schoenoxiphium, subg. Psyllophora p.p.), and (4) a “Core Unispicate Clade” (Uncinia, Kobresia, subg. Psyllophora p.p.). All studies provide strong support (86–100% BS) for the Core Carex and Vignea Clades, but only weak to moderate support (<50%–78% BS) for the Core Unispicate and Schoenoxiphium Clades. The relationships of these groups are unresolved. Studies suggest that Carex is either paraphyletic with respect to all Cariceae genera or to all genera except Schoenoxiphium. Kobresia is a grade, but Uncinia and possibly Schoenoxiphium are monophyletic. The monotypic Cymophyllus is indistinct from Carex subg. Psyllophora species. Character analyses indicate that inflorescence proliferation and reduction have occurred in all major clades, and that the Cariceae’s unisexual flowers have evolved from perfect flowers. The ancestor to Cariceae possessed a multispicate inflorescence with cladoprophylls and female spikelets with tristigmatic gynoecia and closed utricles. This morphology is most similar to extant Carex subg. Carex species, which contradicts the nearly unanimous assumption that the highly compound inflorescences of Schoenoxiphium are primitive. Since taxonomic sampling and statistical support for phylogenies have generally been poor, we advocate the temporary maintenance of the four traditional Carex subgenera with androgynous unispicate species placed within subg. Psyllophora and dioecious and gynaecandrous unispicate species distributed amongst subgenera Carex and Vignea. A collective effort focused on developing new nuclear markers, on increasing taxonomic and geographic sampling, and on studying development within the context of phylogeny, is needed to develop a phylogenetic classification of Cariceae.     

A Phylogeny of Euphorbieae Subtribe Euphorbiinae (Euphorbiaceae) Based on Molecular Data

Phylogenetic relationships within Euphorbiinae were inferred from our analysis of the 3′; end of the chloroplast gene ndhF. A sampling of that subtribe covered 88 species; 3 closely related species from the subtribes Anthosteminae and Neoguillauminiinae and the tiribe Hippomaneae were included as outgroups. A phylogenetic assessment was carried out using the parsimony approach. The relationships revealed via these ndhF data supported the monophyly of subg.Esula, subg.Chamaesyce, subg.Euphorbia, and subg.Lacanthis. However, the polyphyly of subg.Agaloma, subg.Lyciopsis, and subg.Eremophyton also was strongly suggested. The African succulent Euphorbiinae can be divided into primarily two independent groups: 1) spiny succulents, which form a strongly supported clade with three subclades (subg.Euphorbia, subg.Lacanthis, andMonadenium+Synadenium); and 2) non-spiny succulents, which consist of sect.Meleuphorbia, sect.Medusae, sect.Anthacantha, sect.Trichadenia, sect.Pseudeuphorbium, sect.Treisia, and sect.Pseudacalypha. In the ndhF tree, the subg.Esula clade is placed as a sister to the rest of the Euphorbiinae. Thus, the origin of theEuphorbia s.I. should be sought within the herbaceous species of subg.Esula. The core North American endemicEuphorbia groups --Agaloma, Chamaesyce, andPoinsettia — are monophyletic and independent of the South American subg.Agaloma. Instead, they are derived from the AfricanEuphorbia subg.Lyciopsis andEremophyton. The Eurasian subg.Esula clade forms two subclades, which are concordant to sect.Esula and sect.Tithymalus.     

Chloroplast Genome Evolution in Early Diverged Leptosporangiate Ferns

In this study, the chloroplast (cp) genome sequences from three early diverged leptosporangiate ferns were completed and analyzed in order to understand the evolution of the genome of the fern lineages. The complete cp genome sequence of Osmunda cinnamomea (Osmundales) was 142,812 base pairs (bp). The cp genome structure was similar to that of eusporangiate ferns. The gene/intron losses that frequently occurred in the cp genome of leptosporangiate ferns were not found in the cp genome of O. cinnamomea. In addition, putative RNA editing sites in the cp genome were rare in O. cinnamomea, even though the sites were frequently predicted to be present in leptosporangiate ferns. The complete cp genome sequence of Diplopterygium glaucum (Gleicheniales) was 151,007 bp and has a 9.7 kb inversion between the trnL-CAA and trnV-GCA genes when compared to O. cinnamomea. Several repeated sequences were detected around the inversion break points. The complete cp genome sequence of Lygodium japonicum (Schizaeales) was 157,142 bp and a deletion of the rpoC1 intron was detected. This intron loss was shared by all of the studied species of the genus Lygodium. The GC contents and the effective numbers of co-dons (ENCs) in ferns varied significantly when compared to seed plants. The ENC values of the early diverged leptosporangiate ferns showed intermediate levels between eusporangiate and core leptosporangiate ferns. However, our phylogenetic tree based on all of the cp gene sequences clearly indicated that the cp genome similarity between O. cinnamomea (Osmundales) and eusporangiate ferns are symplesiomorphies, rather than synapomorphies. Therefore, our data is in agreement with the view that Osmundales is a distinct early diverged lineage in the leptosporangiate ferns.     

Determination of the complete mitochondrial DNA sequence of <Emphasis Type="Italic">Octopus minor</Emphasis>

In this study, we have determined the complete nucleotide sequence of the mitochondrial genome of Octopus minor. It is 15,974 nucleotide pairs and encodes 13 proteins, two ribosomal RNAs and 22 tRNAs of the mitochondrion’s own protein synthesizing system. Seven of thirteen proteins are encoded by the H-strand, while the other six proteins, as well as the two ribosomal RNAs are encoded by the L-strand. The nucleotide composition of the proteins showed a nucleotide bias against G encoded by the H-strand, while they showed a nucleotide bias against A and C encoded by the L-strand. Two of the 13 protein coding genes of O. minor began with the unorthodox translation initiation codon ATA and all others use the standard ATG. In addition, six of thirteen mt proteins of O. minor have unambiguous termination codons. There are four cases where tRNA genes appear to overlap. The long noncoding region (LNCR) of O. minor was 930 nucleotides and no repeated sequences were found in this LNCR. The gene arrangements of O. minor showed remarkable similarity to that of O. ocellatus and O. vulgaris. Phylogenetic analysis demonstrated that O. minor appears as sister taxan to the monophyletic group combined by O. ocellatus and O. vulgaris, suggesting a relative distant genetic relationship between O. minor and the other two octopus species.     

ORIENTATION AND LOCUS OF TROPIC PHOTORECEPTOR MOLECULES IN SPORES OF BOTRYTIS AND OSMUNDA

Study of the tropic responses of Botrytis cinerea and Osmunda cinnamomea spores to blue light shows the photoreceptor molecules to be highly dichroic and oriented: in Botrytis their axes of maximum absorption lie perpendicular to the nearby cell surface; in Osmunda, parallel. The chief evidence lies in a comparison of their responses to plane polarized light—both germinate parallel to the vibration planes (defined by the axis of vibration of the electric vector and the axis of light propagation)—with those to partial illumination with unpolarized light: Botrytis grows from its brighter part; Osmunda, from its darker. The degree of orientation produced by polarized light corresponds, at high intensities, to that produced by the imposition of such large (about 100 per cent) intensity differences across a cell as to preclude all alternatives to oriented dichroic receptors. The photoreceptors of the Botrytis spore lie within the cell wall's inner half. The chief evidence lies in the component of its tropic responses to polarized light within the vibration plane: germination peaks about 10° off the vibration axis. This deviation arises from focusing which is effective only in the wall's inner half. At high intensities, anomalies appear in Botrytis which are interpreted as "centering," i.e., a tendency toward growth from the center of two or more equally illuminated points of a cell rather than from one of them.     

AUREALCAULIS CROSSII GEN. ET SP. NOV., AN ARBORESCENT,OSMUNDACEOUS TRUNK FROM THE FORT UNION FORMATION (PALEOCENE), WYOMING

Aurealcaulis crossii gen. et sp. nov., is based on permineralized trunks of an osmundaceous tree fern from the Paleocene Fort Union Formation from near Bitter Creek Station of southwestern Wyoming. This new species is characterized by centripetal (exarch) development of its xylem strands which form part of the leaf traces. Most of the leaf traces depart the stele as two segments that fuse into a single C-shaped petiole vascular strand outside of the outer cortex. Stipular expansions of the petiole bases of this species lack sclerenchyma, and roots arise from the lateral edges of leaf traces in the inner cortex. The family Osmundaceae and subfamily Osmundoideae are slightly emended to accept genera assignable to this family and subfamily with exarch protoxylem in their steles. Foliage similar to Osmunda greenlandica (Heer) Brown, which is possibly the leaf form of A. crossii, occurred next to an axis of this species which was in growth position. This axis was anchored in a lignite suggesting that this species grew under swampy conditions. Aurealcaulis crossii is the first arborescent member of the Osmundaceae of Tertiary age and the second arborescent form in this family reported from the Northern Hemisphere.     

SYSTEMATIC SIGNIFICANCE OF SEED-SURFACE FEATURES IN ORTHOCARPUS (SCROPHULARIACEAE—SUBTRIBE CASTILLEJINAE)

Scanning electron microscope and light microscope examination of seed-coat features of 26 species of Orthocarpus have allowed recognition of many species-level differences (summarized in a key) and of three seed-coat types that parallel taxonomic subgroups but support realignments at generic and infrageneric levels. Type 1 seeds (subg. Orthocarpus, sect. Orthocarpus) have a lateral hilum, sculptured inner tangential seed-coat walls, and a tightly fitting outer seed coat. They are very similar to seeds of Cordylanthus. Seeds of Types 2 and 3 have a terminal hilum and membranous inner tangential cell walls. Type 2 seeds (subg. Orthocarpus, sects. Castillejoides and Cordylanthoides, with one exception) have a net-like, loosely fitting outer seed coat that shows close relationship to seeds of Castilleja. Inner tangential walls of Type 2 seeds normally rupture. Type 3 seeds (subg. Triphysaria, with two exceptions) have a tightly fitting outer seed coat and inner tangential walls are always retained. Seed features support evidence from floral morphology and chromosome numbers that Orthocarpus as currently recognized is not a monophyletic lineage.     

Relationships among Old and New World Alliums according to ITS DNA sequence analysis

 Phylogenetic analysis of ITS DNA sequences of various Old and New World Alliums suggests the reinclusion of Nectaroscordum and affirms the reestablishment of Caloscordum as subgenera of genus Allium. The results sanction the elimination of the Old World Allium species from subg. Amerallium Traub and endorse Wendelbo’s definition of subg. Molium (excluding sect. Porphyrason). Received: 29 July 1998 / Accepted: 13 August 1998     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.