Molecules and morphology: Congruence of data inColumnea (Gesneriaceae)

The cladistic analysis and comparison of molecular and morphological data has been the source of much recent debate. In this study, independent analyses of molecular and morphological data fromColumnea L. sects.Pentadenia andStygnanthe (Gesneriaceae) are compared. Comparative methods include consensus, visually comparing trees from independent analyses and combined data analysis. Consensus methods provided little resolution. Comparison of trees obtained from the independent analyses revealed some differences although the trees are highly similar. However, a combined analysis found that the level of incongruence between the two data sets was low. The tree resulting from the combined data has aspects of both the morphological and molecular trees despite the larger number of molecular characters. In addition, the combined data tree has greater resolution than either of the two data sets singly, indicating that the two types of data are congruent, and complementary to each other.     

Comparative seed morphology of <Emphasis Type="Italic">Leandra</Emphasis> (Miconieae,Melastomataceae)

The seed morphology of 79 species of neotropical Miconieae (Melastomataceae) is presented. These species have been chosen, in majority, from the polyphyletic genus Leandra. A few other species from the polyphyletic genera Miconia, Ossaea, and Clidemia were also sampled, because of potential similarities. Sixteen morphological seed types are defined after analysis through light microscopy and scanning electron microscopy. The seed morphology appears to possess a great deal of variability on the level of the overall shape as well as the structure and the surface of the testa. The different types defined here do not match with genera or sections, but rather are composed of species coming from different genera. In comparison with a preliminary molecular phylogeny study done on Leandra, some types of seeds are related to well supported clades. In some cases seed morphology corresponds with natural groups of species, thus being of high phylogenetic importance.     

Estimating genetic conformity between related ryegrass (Lolium) varieties. 1. Morphology and biochemical characterisation

In this study the morphological and protein diversity of twelve diploid perennial ryegrass accessions (Lolium perenne L.) was examined. These accessions comprised five closely related groups, each containing an `initial variety' (IV) and one or more declared `essentially derived varieties' (EDV), with differing degrees of relatedness. `Essential derivation' is a legal concept relating to intellectual property in plant varieties and is additional to Plant Breeders Rights (PBR). An EDV is defined as clearly distinct from, but conforming in its expression of the essential characteristics of an IV, from which it is found to have been predominantly derived. Where an essential derivation has been confirmed, the breeder of the IV may be entitled to some royalty sharing of the EDV. Clearly, therefore, in any successful EDV claim, evidence of a high degree of conformity in either the phenotype or genotype would be required. Examination of plant morphology indicated that all the EDVs were morphologically distinct from their corresponding IV in one or more morphological characteristic. using a principal co-ordinates analysis to give an overall measure of morphological difference, all twelve accessions were correctly clustered into their related groups and the magnitude of the differences within groups reflected their known breeding histories. Examining protein diversity by methods that targeted single- and multiple-locus genes also clustered the accessions into their correct groups, in most cases. However, only by examining diversity in seed storage proteins were within-group relationships accurately represented. The methods used provided no consistent representation of between-group relationships. It was concluded that the morphological method provided a creditable measure of genetic conformity, but to avoid incurring excessive time, work and cost, results from existing national PBR trials would need to be openly available. Within the limits of the genetic material examined, seed storage protein diversity appeared to provide a suitable combination of accuracy and efficiency on which to base a routine test. However, given more complex breeding relationships than those in this study, methods such as AFLP¹ markers that sample more genetic diversity, may be necessary to maintain this level of accuracy.     

From axenic spore germination to molecular farming

The first bryophyte tissue culture techniques were established almost a century ago. All of the techniques that have been developed for tissue culture of seed plants have also been adapted for bryophytes, and these range from mere axenic culture to molecular farming. However, specific characteristics of bryophyte biology—for example, a unique regeneration capacity—have also resulted in the development of methodologies and techniques different than those used for seed plants. In this review we provide an overview of the application of in vitro techniques to bryophytes, emphasising the differences as well as the similarities between bryophytes and seed plants. These are discussed within the framework of physiological and developmental processes as well as with respect to potential applications in plant biotechnology.     

Comparative seed morphology and character evolution in the genus Lysimachia (Myrsinaceae) and related taxa

Summary We investigated seed morphology in 34 species of the genus Lysimachia and in 14 species and two subspecies of six additional genera (Anagallis, Ardisiandra, Asterolinon, Glaux, Pelletiera, Trientalis), which have been shown to be closely related to, or are placed within Lysimachia in previous molecular studies. We studied seed shape, seed coat structure, and seed coat surface patterns. Three major types of seed shape were identified: (1) sectoroid, (2) polyhedral, and (3) coarsely rugose with concave hilar area. In addition, seeds may be keeled or winged. The outer layer of the seed coat is either sponge-like and adhering only loosely to the inner seed coat or it is thin and tightly adhering to the underlying tissue. Seed surface patterns can be divided into six main types: (1) reticulate, (2) tuberculate, (3) vesiculose, (4) colliculate, (5) undulate, or (6) poroid-alveolate. Seed surface patterns are mostly congruent with molecular phylogenetic relationships. A reticulate surface pattern is diagnostic of, e.g. Lysimachia subgenera Palladia and Hawaiian Lysimachiopsis. Mapping seed characters onto a recent phylogenetic tree, reveals that they provide potentially synapomorphic character states for various subclades of Lysimachia. Salient examples include a rugose seed shape, which turns out to be synapomorphic for the clade comprising the genus Pelletiera plus Asterolinon linum-stellatum and a sponge-like outer seed coat layer, which characterizes a clade with Lysimachia vulgaris, L. thyrsiflora, and L. terrestris, with an analogue that apparently evolved in parallel in Trientalis europaea. We also discuss possible habitat factors that may have favored the independent evolution of particular seed types such as winged seeds in various lineages.     

Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

Re-thinking the classification of corticioid fungi

Corticioid fungi are basidiomycetes with effused basidiomata, a smooth, merulioid or hydnoid hymenophore, and holobasidia. These fungi used to be classified as a single family, Corticiaceae, but molecular phylogenetic analyses have shown that corticioid fungi are distributed among all major clades within Agaricomycetes. There is a relative consensus concerning the higher order classification of basidiomycetes down to order. This paper presents a phylogenetic classification for corticioid fungi at the family level. Fifty putative families were identified from published phylogenies and preliminary analyses of unpublished sequence data. A dataset with 178 terminal taxa was compiled and subjected to phylogenetic analyses using MP and Bayesian inference. From the analyses, 41 strongly supported and three unsupported clades were identified. These clades are treated as families in a Linnean hierarchical classification and each family is briefly described. Three additional families not covered by the phylogenetic analyses are also included in the classification. All accepted corticioid genera are either referred to one of the families or listed as incertae sedis.     

Validation of manometric microrespirometers for measuring oxygen consumption in small arthropods

Scientists have used numerous techniques to measure organismal metabolic rate, including assays of oxygen (O(2)) consumption and carbon dioxide (CO(2)) production. Relatively few studies have directly compared estimates of metabolic rate on the same groups of animals as determined by different assay methods. This study directly compared measures of the metabolic rate of three lines of Drosophila simulans as determined either from direct measures of CO(2) production using infrared gas analysis (IRGA), or from estimates of O(2) consumption based on manometeric techniques. Determinations of metabolic rate of the same cohorts of flies using these two methods produced results that often differed widely. Typically metabolic rate as determined by the manometric method was significantly greater than that determined by CO(2) output. These differences are difficult to explain by simple biotic or abiotic factor(s). Because of the idiosyncratic nature of these differences it is not possible to use a simple factor to convert from metabolic rate measurements done using manometric techniques to those expected from direct measures of CO(2) output or O(2) consumption. Although manometric devices are simple to construct and use, measurements of metabolic rate made with this method can vary significantly from measurements made by directly assaying CO(2) production or O(2) consumption.     

Utility of psbA and nSSU for phylogenetic reconstruction in the Corallinales based on New Zealand taxa

A number of molecular studies of the Corallinales, a calcified order of the red algae, have used the conservative nSSU gene to investigate relationships within the order. However interspecific variation at this locus is low for closely related species, limiting resolution of recently diverged groups. In this study, we obtained psbA sequence data from specimens of the order from New Zealand that had been identified according to current taxonomic criteria. We compared phylogenetic analyses based on psbA with those based on nSSU for the same dataset, and also analysed nSSU sequences of the New Zealand material with nSSU sequences of Corallinales taxa from other parts of the world. Our study shows that psbA has considerable potential as a marker for this group, being easily amplified and considerably more variable than nSSU. Combined analyses using both markers provide significant support for relationships at both distal and terminal nodes of the analysis. Our analysis supports the monophyly of all three families currently defined in Corallinales: the Sporolithaceae, Hapalidiaceae and Corallinaceae, and indicates cryptic speciation in Mesophyllum and Spongites.     

Molecular characterization of ochratoxin A producing strains of the genus Penicillium

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.     

A taxometric analysis of seed proteins in the genusBriza s. l. (Poaceae)

The species ofBriza L. s. l. are disjunctly distributed between Eurasia and South America. A taxometric analysis has been carried out, using data from electrophoresis of seed polypeptides and published morphological characters. Evidence from a cluster analysis of seed polypeptide data strongly supports the recognition of two different natural groups, a S. American group and a Eurasian group, and is consistent with karyotype data, but does not support the generic groupings of South American taxa derived from the morphological data.     

Problems caused by new approaches in fungal taxonomy

An array of new techniques including biochemical, physiological and molecular biological methods are being introduced to modify or improve the systematics of fungi of biotechnological, medical and industrial importance. In some genera e.g. Penicillium and Aspergillus such a multidisciplinary approach has shown to be useful and has resolved confusing species concepts. However, in other cases the significance of new techniques is still unclear and results drawn from such studies may even increase nomenclatural instability. Taxonomists should be careful and users aware and whenever possible a pragmatic approach should be welcomed.     

Extraction of RNA from tissues containing high levels of procyanidins that bind RNA

Commonly used methods for extraction of RNA from plants are not effective for isolation of high quality RNA from the pigmented seed coats of soybeans that produce procyanidins (tannins) during seed coat development. We demonstrate a significant modification of the phenol-LiCl method that yields high quality RNA from a black seed coat variety. In this method, seed coat material was ground in a buffer containing a high concentration of bovine serum albumin (100 mg BSA/50 mg of lyophilized seed coats) to competitively inhibit proanthocyanidin binding. The presence of hydrated insoluble polyvinylpoly-pyrrolidone (PVPP) was also necessary to bind proanthocyanidins and remove them from solution. Proteinase K was added to digest the remaining BSA, and phenol extraction was used to remove both the proteins and small molecular weight complexes formed by BSA and proanthocyanidins. After LiCl and ethanol precipitations, the RNA quality was examined by UV absorbance spectra, gel electrophoresis, and hybridization. Using this method, good quality RNA can be extracted from pigmented seed coats of soybean varieties that are homozygous for the recessivei allele and also contain the dominantT gene that results in production of procyanidins in the seed coat. The method is also effective for tissues from other plant species that contain abundant polyphenolic compounds.     

2种块菌的物种分子鉴定与系统地位分析

以采自内蒙古贺兰山地区的2种块菌为实验材料,分别运用形态学和分子生物学方法进行物种鉴定,并通过与GenBank数据库信息比对,利用Mega5.1软件构建系统进化树,分析其进化关系并确定2种块菌的进化地位。结果表明:两菌株子囊果都为地下生;其中,菌株order-a为灰黄色,近球形到不规则形,较硬,表面被覆微绒毛;菌株tuber-b呈黄褐色,质地均匀,表面有疣状物,顶端呈凹陷状,全株带有干果般特殊的香气。经分子生物学方法分析认为菌株order-a为块菌属的一种,tuber-b为冬块菌(Tuber brumale),并初步确定为内蒙古新记录种。     

The chloroplast generps 4 as a tool for the study ofPoaceae phylogeny

Phylogenetic analyses of 28Poaceae species based on the chloroplastrps 4 gene are presented using parsimony and distance methods. Two monocots from other families were used as outgroups. The chloroplast generps 4 was amplified, cloned, and sequenced for each species. The inferred phylogenetic trees were compared to recent classifications and are shown to fit their general features. There is a dichotomy in our tree between the pooid group and the other grasses. This is in contradiction with other molecular phylogenies, where the bamboos appear first within the family. This result led us to discuss some hypotheses about the relationships of the bambusoids with the other groups of grasses, and also about the relative position of rice and bamboo, which are found close to each other in our trees.     

Systematics of Chaetognatha under the light of molecular data, using duplicated ribosomal 18S DNA sequences

While the phylogenetic position of Chaetognatha has became central to the question of early bilaterian evolution, the internal systematics of the phylum are still not clear. The phylogenetic relationships of the chaetognaths were investigated using newly obtained small subunit ribosomal RNA nuclear 18S (SSU rRNA) sequences from 16 species together with 3 sequences available in GenBank. As previously shown with the large subunit ribosomal RNA 28S gene, two classes of Chaetognatha SSU rRNA gene can be identified, suggesting a duplication of the whole ribosomal cluster; allowing the rooting of one class of genes by another in phylogenetic analyses. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the molecular data, and statistical tests showed (1) that there are three main monophyletic groups: Sagittidae/Krohnittidae, Spadellidae/Pterosagittidae, and Eukrohniidae/Heterokrohniidae, (2) that the group of Aphragmophora without Pterosagittidae (Sagittidae/Krohnittidae) is monophyletic, (3) the Spadellidae/Pterosagittidae and Eukrohniidae/Heterokrohniidae families are very likely clustered, (4) the Krohnittidae and Pterosagittidae groups should no longer be considered as families as they are included in other groups designated as families, (5) suborder Ctenodontina is not monophyletic and the Flabellodontina should no longer be considered as a suborder, and (6) the Syngonata/Chorismogonata and the Monophragmophora/Biphragmophora hypotheses are rejected. Such conclusions are considered in the light of morphological characters, several of which are shown to be prone to homoplasy.     

Survival of a bacterial/archael consortium on building materials as revealed by molecular methods

The analysis of microbial communities using molecular techniques has become a common method to describe their components. In this study some building materials were inoculated with a microbial consortium of 14 microorganisms, subjected to alternate wetting/drying cycles for six months, and left to rest for eight years. At the end of the resting period, most members of the consortium were detected by molecular methods, which indicate a remarkable preservation of the DNA, although only one bacterium was able to grow in a culture medium. The experimental approach we carried out indicates that the list of microorganisms obtained from DNA analyses reflects not only the actual composition of the microbial communities, but is also a reflection of the microorganisms that were once active on the building materials.     

Molecular phylogenetic analysis of Violaceae (Malpighiales) based on plastid and nuclear DNA sequences

A phylogenetic analysis of Violaceae is presented using sequences from rbcL, atpB, matK and 18S rDNA from 39 species and 19 genera. The combined analysis of four molecular markers resulted in only one most parsimonious tree, and 33 of all 38 nodes within Violaceae are supported by a bootstrap proportion of more than 50%. Fusispermum is in a basal-most position and Rinorea, Decorsella, Rinoreocarpus and the other Violaceae are successively diverged. The monogeneric subfamily Fusispermoideae is supported, and it shares a number of plesiomorphies with Passifloraceae (a convolute petal aestivation, actinomorphic flowers and connate filaments). The other monogeneric subfamily Leonioideae is sunken within the subfamily Violoideae and is sister to Gloeospermum, sharing some seed morphological characteristics. The present molecular phylogenetic analysis suggests that the convolute, apotact and quincuncial petal aestivation is successively derived within the family. The evolutionary trends of the other morphological characteristics, such as a filament connation, the number of carpels and floral symmetry, are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Summary Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.     

The structure of the USP/RXR of <Emphasis Type="Italic">Xenos pecki</Emphasis> indicates that Strepsiptera are not closely related to Diptera

The receptor for the insect molting hormone, ecdysone, is a heterodimer consisting of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The ligand binding domain sequences of arthropod USPs divide into two distinct groups. One group consists of sequences from members of the holometabolous Lepidoptera and Diptera, while the other arthropod sequences group with vertebrate retinoid-X-receptors (RXRs). We therefore wondered whether USP/RXR structure could be used to clarify the contentious phylogenetic position of the order Strepsiptera, which has proposed affinities with either Diptera or Coleoptera. We have cloned and sequenced the USP/RXR from the strepsipteran Xenos pecki. Phylogenetic analyses are not consistent with a close affinity between Strepsiptera and Diptera.Electronic Supplementary Material Supplementary material is available for this article at Edited by D. Tautz