SYSTEMATIC IMPLICATIONS OF CHLOROPLAST DNA VARIATION IN THE SUBTRIBE MICROSERIDINAE (ASTERACEAE: LACTUCEAE)

Phylogenetic relationships among six of the seven currently recognized genera of the subtribe Microseridinae were examined using comparative restriction site mapping of the chloroplast genome. Complete cleavage maps of 17 enzymes were constructed for 35 of the 46 species in the subtribe and six representative outgroup species from four other subtribes of the Lactuceae. A total of 373 restriction site mutations was detected, 180 of which were phylogenetically informative. The chloroplast DNA mutations were used to generate phylogenetic trees using Dollo, Wagner, and weighted parsimony, and the resulting monophyletic groups were evaluated by the bootstrap method. The results indicate that the Microseridinae is paraphyletic because Stephanomeria is nested within the six core genera of the subtribe. Five of the genera of the Microseridinae are monophyletic, whereas Microseris is paraphyletic because one of its species, M. lindleyi, is more closely related to Agoseris and Nothocalais. The distant relationship of M. lindleyi to the rest of the species of Microseris is enigmatic, especially in view of its known involvement in the origin of two allotetraploid species, M. decipiens and M. heterocarpa. The taxonomic implications of these results lead to a revised classification of Microseris in which the genus Uropappus is revived to accommodate M. lindleyi, and a new genus, Stebbinsoseris, is described to include the allotetraploids. The cpDNA phylogeny is used to evaluate the patterns of character evolution within the Microseridinae, as well as to test previous hypotheses that differences in generation time result in unequal rates of chloroplast DNA evolution.     

Mitotic cycle time and DNA content in annual and perennialMicroseridinae (Compositae,Cichoriaceae)

Within eight annual and perennialMicroseridinae species studied, the duration of the mitotic cycle is positively correlated with the nuclear DNA content, cycle time (hrs) = 7.3 + 0.32 × pg DNA/nucleus. Within the generaAgoseris andMicroseris, the annuals have lower DNA contents and more rapid mitotic cycle times than do the perennials. This relationship is predicted by the nucleotypic theory ofBennett. Annual species ofPyrrhopappus have relatively high DNA contents and a proportionately longer mitotic cycle time, but contrary to that expected by the nucleotypic theory as originally proposed have the fastest growth rate and shortest generation time observed in theMicroseridinae. This rapid developmental rate is discussed, nucleotypically, however, by analyzing relationships between DNA content, mitotic cycle time, and cell size.     

DROUGHT STRESS RESPONSES OF MICROSERIS SPECIES DIFFERING IN NUCLEAR DNA CONTENT

Anatomical and physiological responses to drought stress were compared in two Microseris species differing in DNA content and originating from contrasting habitats relative to water availability (M. bigelovii, DNA = 2.6 pg nucleus^–1, more xeric; M. laciniata, DNA = 6.8 pg nucleus^–1, mesic). Leaf mesophyll cell volume was positively correlated with DNA content and negatively correlated with tissue elasticity, i.e., low ϵ̄ and thin cell walls. Drought stress increased leaf tissue elasticity (lower ϵ̄, thinner cell walls). Cell volume, cell wall thickness, cell number, and leaf area were decreased most by drought stress in M. laciniata. Osmotic adjustment with a 20% increase in total solutes (mostly amino acids) after stress was observed in both species, but their estimated contribution to the change in osmotic potential was larger in M. bigelovii. These findings indicate that the Microseris species studied respond to low water availability by maintaining turgor with 1) small cell volumes, 2) elastic tissues (low ϵ̄, thin cell walls), and 3) osmotic adjustment. Both enhanced tissue elasticity and small cell volume appear to be inherent characteristics in M. bigelovii and drought-induced responses in M. laciniata. These data are compatible with the hypothesis that natural selection may influence DNA content through differential sensitivity of cell growth to environmental stress.     

Molecular phylogenetics of subtribe Aeridinae (Orchidaceae): insights from plastid matK and nuclear ribosomal ITS sequences

We conducted phylogenetic analyses using two DNA sequence data sets derived from matK, the maturase-coding gene located in an intron of the plastid gene trnK, and the internal transcribed spacer region of 18S–26S nuclear ribosomal DNA to examine relationships in subtribe Aeridinae (Orchidaceae). Specifically, we investigated (1) phylogenetic relationships among genera in the subtribe, (2) the congruence between previous classifications of the subtribe and the phylogenetic relationships inferred from the molecular data, and (3) evolutionary trends of taxonomically important characters of the subtribe, such as pollinia, a spurred lip, and a column foot. In all, 75 species representing 62 genera in subtribe Aeridinae were examined. Our analyses provided the following insights: (1) monophyly of subtribe Aeridinae was tentatively supported in which 14 subclades reflecting phylogenetic relationships can be recognized, (2) results are inconsistent with previous classifications of the subtribe, and (3) repeated evolution of previously emphasized characters such as pollinia number and apertures, length of spur, and column foot was confirmed. It was found that the inconsistencies are mainly caused by homoplasy of these characters. At the genus level, Phalaenopsis, Cleisostoma, and Sarcochilus are shown to be non-monophyletic.     

Condensed chromatin in diploid and allopolyploid microseris species with different genome size: a quantitative electron microscopic study

Summary The species-specific proportion of chromatin in the condensed state was estimated by quantitative electron microscopic morphometry of nuclear sections in 9 diploid and 5 allopolyploid species of Microseris (Asteraceae). A positive correlation between the genome size (haploid DNA content, or C value) and the percentage of chromatin in the condensed state (as visible in ultrathin sections) was found in diploids (r=0.89). Nuclei of allopolyploid (tetraploid) species exhibit condensed chromatin in a percentage which corresponds to the average of the values found in the parents. This suggests that each parental genome controls chromatin condensation at interphase independently within the nucleus, and that the degree of condensation is not directly determined by the nuclear DNA content per se. Genome size differences among Microseris species may depend preferentially, but not entirely, on DNA fractions located in, and perhaps being the cause of, condensed chromatin.Dedicated to Professor F. Mechelke in honour of his 60^th birthday.     

Phylogenetic relationships of the enigmatic species Serratula chinensis and Serratula forrestii (Asteraceae - Cardueae)

The phylogenetic relationships of the Chinese species Serratula forrestii and S. chinensis within the subtribe Centaureinae (Asteraceae – Cardueae) were investigated based on nuclear ribosomal DNA external and internal transcribed spacer sequences. Results indicated that Serratula s. l. is clearly polyphyletic, none of the two species showing close affinities to either Serratula s. str. or Klasea, a genus traditionally included in Serratula. Serratula forrestii has a basal position within the subtribe and has retained most plesiomorphic character states. Serratula chinensis is related to the genera of the Rhaponticum group. Its chromosome number was determined as 2n = 26, and the relationship between this species and the Rhapon-ticum group is discussed from a morphological and karyological point of view.     

EVIDENCE FOR A CLOSE RELATIONSHIP BETWEEN IOSTEPHANE AND VIGUIERA (ASTERACEAE: HELIANTHEAE)

The phylogenetic relationship of Iostephane is assessed using data from morphology, flavonoid chemistry, and chloroplast DNA and nuclear ribosomal DNA restriction fragment analysis. Morphological evidence supports placement of Iostephane in subtribe Helianthinae, but fails to clarify the placement of the genus within this assemblage. Further evidence for the placement of Iostephane in subtribe Helianthinae is provided by the presence in all species of the genus of floral flavonoids of the chalcone/aurone type, which provides a distinctive trait for the subtribe within the tribe Heliantheae. Analysis of chloroplast DNA from two species of Iostephane, I. heterophylla and I. madrensis, in comparison to Viguiera and related genera indicates that the restriction site patterns with 16 enzymes for the Iostephane species are virtually identical to one another as well as to those of Viguiera sect. Maculatae. Data from restriction fragment patterns of nuclear rDNA are concordant with the results from chloroplast DNA in suggesting a direct relationship between the two groups. The close phylogenetic relationship between Iostephane and Viguiera sect. Maculatae suggested by the DNA restriction fragment data was not suggested by any other set of data.     

Evolution of microsporangium numbers in Microseris (Asteraceae: Lactuceae)

The genus Microseris contains species with disporangiate stamens and species with tetrasporangiate stamens. We determined the number of microsporangia per stamen in serial sections of heads from all 13 species of Microseris, its close relative Uropappus lindleyi, and the two allopolyploid species of Slebbinsoseris. Four Microseris species, three diploid and one tetraploid, have two microsporangia per stamen; all other species investigated have four. The most parsimonious assumption is that the disporangiate condition is derived and arose once in the evolution of Microseris. The inheritance of the number of microsporangia per stamen in crosses between M. bigelovii (disporangiate) and M. douglasii (tetrasporangiate) was determined. Segregation of microsporangium number per stamen in F2s derived from these crosses is quantitative rather than Mendelian. The average number of microsporangia per stamen in the F2 plants ranges from 2.0 to 4.0. There is a predominance of tetrasporangiate stamens in the F1 and in most F2 plants. The observed pattern of inheritance suggests a major gene with dominance and quantitative modifiers.     

Incipient adaptive radiation of New Zealand and Australian Microseris (Asteraceae): an amplified fragment length polymorphism (AFLP) study

The disjunct allotetraploid lineage of the North American genus Microseris in New Zealand and Australia originated from one or a few diaspores after a single introduction via long‐distance dispersal. The plants have evolved into four morphologically distinct ecotypes: ‘fine‐pappus’, ‘coastal’, ‘murnong’, and ‘alpine’, from which the first two are grouped as Microseris scapigera, mainly from New Zealand and Tasmania, and the latter two as M. lanceolata, endemic to the Australian mainland. Three chloroplast (cp) DNA types were distinguished in each of the species, but their distribution, especially in M. lanceolata, showed discrepancies with ecotype differentiation. Here, we analyse the genetic structure of the nuclear (n) DNA among two plants of each of 55 New Zealand, Tasmanian, and Australian Microseris populations for amplified fragment length polymorphisms (AFLPs). The nuclear genetic structure is compared to geographical, ecotype, and cpDNA distribution, in order to resolve and illustrate the early process of adaptive radiation. The strongest signal in the AFLP pattern was related to geographical separation, especially between New Zealand and Australian accessions, and suggested an initial range expansion after establishment. The ecotypic differentiation was less‐well reflected in the AFLP pattern, and evidence was found for the occurrence of hybridization among plants at the same geographical region, or after dispersal, irrespective of the cpDNA‐ and ecotypes. This indicated that the ecotype characteristics were maintained or re‐established by selection. It also showed that genetic differentiation is not an irreversible and progressive process in the early stage of adaptive radiation. Our results illustrate the precarious balance between geographical isolation and selection as factors that favour differentiation, and hybridization as factor that reduces differentiation.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Summary Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1–7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G₂ phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G₂ phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.This work was supported by the Polish Academy of Sciences as a part of project no 09.7.1.4.5     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

Nuclear DNA Content of 26 Orchid (Orchidaceae) Genera with Emphasis onDendrobium

2C DNA content values for 70 orchid species from 26 genera,including 37Dendrobiumspecies from eight taxonomic sections,were analysed using flow cytometry. The resulting nuclear DNAcontent values for species other thanDendrobiumranged from 1.91pg 2C^-1to 15.19 pg 2C^-1nuclei forCadetia tayloriandVanilla phaeantha,respectively.Dendrobiumnuclear DNA content values ranged from1.53 pg 2C^-1to 4.23 pg 2C^-1nuclei forD. cruentumandD. spectabile,respectively. DNA content measurements varied greatly withinDendrobiumsectionsLatouria and Spatulata. Nuclear DNA content values for the sixspecies analysed within Latouria ranged from 1.88 pg 2C^-1nucleiforD. macrophyllumto 4.23 pg 2C^-1nuclei forD. spectabile. NuclearDNA content values for the 16 species analysed within Spatulataranged from 1.69 pg 2C^-1nuclei forD. discolorto 4.05 pg 2C^-1nucleiforD. samoense. The least variation in DNA content was foundwithin the section Phalaenanthe, with nuclear DNA content valuesof 1.79 pg  2C^-1, 1.86 pg 2C^-1and 1.98 pg 2C^-1forD. bigibbum,D.affineandD. phalaenopsis, respectively.Copyright 1998 Annalsof Botany Company Orchidaceae,Dendrobium, flow cytometry, propidium iodide, nuclear DNA, genome size, 2C values.     

A revised classification of subtribe Helianthinae (Asteraceae: Heliantheae) II. Derived lineages

Sequence data for internal transcribed spacer (ITS) and partial external transcribed spacer (ETS) regions were combined in a phylogenetic analysis with previously obtained plastid DNA restriction site data to provide a comprehensive molecular phylogenetic hypothesis for derived members of subtribe Helianthinae. Analyses of the two molecular datasets provided conflicting evidence on relationships among some groups, supporting the hypothesis that hybridization has played a significant role in the divergence of the subtribe. A revised generic‐level classification is presented that divides the approximately 350 species of the subtribe among 21 genera. The paraphyletic Viguiera is narrowed to embrace only the type species, V. dentata. Four newly described genera, Dendroviguiera, Gonzalezia, Heiseria and Sidneya, are composed of species formerly included in Viguiera. Aldama is expanded to include 118 species extending from southwestern North America and Mexico to South America. This requires 116 new combinations, including 58 that were recently transferred into Rhysolepis, which is a synonym of Aldama, based on molecular phylogenetic results. One species of Viguiera is transferred to Tithonia, and two combinations in Hymenostephium are validated. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 311–331.     

Phylogenetic reticulation in subtribe Helianthinae

Incongruence between phylogenetic estimates based on nuclear and chloroplast DNA (cpDNA) markers was used to infer that there have been at least two instances of chloroplast transfer, presumably through wide hybridization, in subtribe Helianthinae. One instance involved Simsia dombeyana, which exhibited a cpDNA restriction site phenotype that was markedly divergent from all of the other species of the genus that were surveyed but that matched the restriction site pattern previously reported for South American species of Viguiera. In contrast, analysis of sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region showed Simsia to be entirely monophyletic and placed samples of S. dombeyana as the sister group to the relatively derived S. foetida, a result concordant with morphological information. A sample of a South American species of Viguiera was placed by ITS sequence data as the sister group to a member of V. subg. Amphilepis, which was consistent with cpDNA restriction site data. Samples of Tithonia formed a single monophyletic clade based on ITS sequence data, whereas they were split between two divergent clades based on cpDNA restriction site analysis. The results suggested that cpDNA transfer has occurred between taxa diverged to the level of morphologically distinct genera, and highlight the need for careful and complete assessment of molecular data as a source of phylogenetic information.     

POLLEN MORPHOLOGY AND DETAILED STRUCTURE OF FAMILY COMPOSITAE,TRIBE CICHORIEAE. II. SUBTRIBE MICROSERIDINAE

A survey of pollen morphology of 40 species representing eight genera of the primarily North American subtribe Microseridinae reveals seven of the eight genera to have caveate, echinolophate, tricolporate grains, Picrosia being the only taxon with echinate pollen. Sectioned grains reveal the exine to consist of an ektexine and endexine. The ektexine, composed of spines, columellae, and foot layer appears to be of two basic types, one with six or seven levels of horizontally anastomosing columellae which are reduced to a single columellar layer under the paraporal lacunae and the second, a bistratified ektexine not reduced to a single layer below the paraporal lacunae. Sectioned exines of Pyrrhopappus are unusual, having very large columellae fused to the foot layer below ridges and highly reduced columellae under lacunae. Endexine organization is similar in most of the genera. Exceptions to this are Pyrrhopappus and some species of Agoseris, which have an “endexine 2” layer. Subtribe Microseridinae is essentially stenopalynous. The pollen data support most of the relationships suggested by Stebbins in his classification. The genera Agoseris, Microseris, Nothocalais, and Phalacroseris seem to form a natural group while Krigia and Pyrrhopappus form another cohesive series. The position of Picrosia, as an advanced offshoot of Pyrrhopappus, is not supported by the pollen data.     

Relationship between ploidy level and genome size in strawberries

This study investigated the pattern of variation in nuclear DNA content at different ploidy levels in Fragaria (Strawberry, Rosaceae) using flow cytometry based on mean fluorescent intensity (MFI) reflected by propidium-iodide-stained nuclei. On average, MFI values were 237 for diploids F. vesca, F. viridis, and F. nubicola, 416.5 for tetraploid F. orientalis, 621.5 for hexaploid F. moschata, and 798 for octoploids F. × ananassa, F. virginiana, and F. chiloensis. Within diploids MFI ranged from 225.9 in F. vesca ssp. vesca to 255.4 in F. nubicola, and within octoploids varied from 766 in F. × ananassa to 808 in F. virginiana. The nuclear DNA variation was significant among diploid species (N = 21, P < 0.008), but not across octoploid species (N = 17, P>0.386). MFI values were also variable among different genotypes of a given species though not significant. The values of mean basic genome DNA (MFI divided by ploidy level) were 118.5, 104, 103.5, and 99.8, respectively, for diploids, tetraploid, hexaploid, and octoploid species. This indicates that relative genomic size decreases by increasing ploidy level, and that there is no direct proportional relationship between DNA content and ploidy levels in Fragaria, supporting the idea of genome downsizing during polyploidization in plants.     

Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines

Eight species of Gracilariaceae from the Philippines, representing the generaGracilaria, Gracilariopsis andHydropuntia, were investigated to quantify and characterize their nuclear genomes. DNA reassociation kinetics were used to determine nuclear genome organization and complexity in six of these species. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–74% and unique DNA ranged from 26–84%. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA contents. Comparisons of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.38–0.43 pg/2 C genomes for seven of the species investigated. Preliminary analyses of agar content and quality confirm the economic potential ofGracilaria firma, Gracilaria sp. 2 from Sorsogon andGracilariopsis bailinae. Nuclear genome profiles developed from data for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity.Author for correspondence     

Phylogenetic relationships and generic realignments in the early diverging subtribe Pentziinae (Asteraceae,Anthemideae)

Pentziinae are a subtribe of tribe Anthemideae (Asteraceae), comprising seven almost exclusively southern African endemic genera and c. 60 species. Generic delimitations and relationships in the subtribe are explored using parsimony and Bayesian analyses of nuclear (internal transcribed spacer, ITS) and plastid (ndhF, psbA‐trnH, trnK‐rps16 and rpl32‐trnL) DNA regions, with morphological and fruit anatomical studies. Phylogenetic analyses for 72 accessions (43 species) representing 72% of the subtribe indicate that several of the genera are not monophyletic as currently circumscribed. Pentzia tortuosa differs from its congeners in its inconspicuously three‐ribbed fruits and in several other distinctive morphological characters. In the phylogenetic trees based on the nuclear dataset, P. tortuosa is recovered in the perennial clade with its congeners, but in the plastid data set, it is strongly placed among the early diverging lineages comprising annual taxa with three‐ribbed fruits. Even with the exclusion of P. tortuosa, Pentzia remains paraphyletic by the inclusion of at least Rennera and possibly also Cymbopappus and Marasmodes. A possible sister relationship between Marasmodes and Pentzia, however, cannot be excluded. The four Rennera specise are therefore here transferred to Pentzia, whereas Marasmodes and Cymbopappus are retained pending further investigation. Among the annual lineages, Foveolina is also found to be polyphyletic, with F. dichotoma (the type species) and F. tenella clearly allied with Oncosiphon, and the anomolous species, F. burchellii, recovered with Myxopappus in the nuclear trees. Both Myxopappus species share the disciform capitula and heteromorphic fruits with Foveolina burchellii (characters previously overlooked in Myxopappus and reported here for the first time). © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 633–647.