PALYNOLOGY AND SYSTEMATICS OF THE LYTHRACEAE. III. GENERA PHYSOCALYMMA THROUGH WOODFORDIA,ADDENDA, AND CONCLUSIONS

Pollen of the 27 genera presently recognized as comprising the family Lythraceae have been surveyed with light microscopy and scanning and transmission electron microscopy. Results for five genera (Physocalymma, Pleurophora, Rotala, Tetrataxis, Woodfordia), in addition to Duabanga, Sonneratia, and Punica (assigned to the Lythraceae in some classifications), are presented here; the remaining genera were treated previously in the series. The family is revealed as the most diverse palynologically of the order Myrtales. The most simple pollen type and the one common to the largest number of genera is prolate-spheroidal to prolate; tricolporate, without pseudocolpi; psilate, scabrate or finely verrucate; and 16–28 μm or less in length. Specializations include oblate grains, development of pseudocolpi (three or six in number), diversification of exine sculpturing, broadening of the colpal and pseudocolpal areas, and reduction in the conspicuousness of the colpi. Pollen evidence provides qualified support for inclusion of Punica in the Lythraceae, the generalized nature of the pollen tempering the conclusion, and little support for inclusion of Sonneratia and Duabanga in the family. Completion of the survey provides a data base of pollen characters that will be integrated in future studies with other evidence into an overall phenetic and cladistic assessment of the family leading to production of a more natural classification.     

PHYLOGENETIC IMPLICATIONS OF DIVERSE SEED TYPES,CHROMOSOME NUMBERS,AND POLLEN MORPHOLOGY IN HOUSTONIA (RUBIACEAE)

Seed and pollen morphology were studied by light microscopy and scanning electron microscopy in 39 North and Central American species of Houstonia (including Hedyotis, but excluding Oldenlandia). Chromosome counts were obtained for eight taxa, of which five lacked previous chromosome data. A chromosome number of n = 17 for Houstonia gracilis is a new base number for the genus. Seed external morphology in the genus is very diverse, including variation in compression, margins, testa surfaces, and elaboration of ventral cavities or depressions and hilar ridges or their absence. Three types of pollen apertures are recognized: colporate with type A os, colpororate, and colporate with type B os, the last the most advanced type, occurring in H. caerulea and related species. The 39 species are arranged in twelve groups, based on correlation of seed, pollen, and chromosome data. Geographic distribution provides supplementary evidence for the distinctness and integrity of the six principal groups each composed of 2–9 species. Five of the six minor groups each with one species need chromosome data to facilitate future taxonomic decisions. Chromosome numbers of x = 6, 7, 8, 9, 10, 11, 13, and 17 are now known in this genus, and phylogenetic implications of the combined data are discussed.     

First record of resting cysts of the benthic dinoflagellate Prorocentrum leve in a natural reservoir in Gujan‐Mestras,Gironde, France

The resting cysts of the benthic dinoflagellate Prorocentrum leve from a natural reservoir in Gujan‐Mestras (Gironde, France) were described in this study. The incubated urn‐shaped cysts gave rise to cells of P. leve. Morphological observations through light microscopy and scanning electron microscopy, particularly of the periflagellar platelets, combined with large subunit ribosomal DNA sequences obtained through single‐cell analysis confirm their affinity to the species P. leve. The cysts were characterized by a specific shape and the presence of an anterior plug. This is the first conclusive evidence for fossilizable resting stages within the Prorocentrales, one of the major orders within the Dinophyceae. Palynological treatments show that the cysts and endospores withstand hydrochloric and hydrofluoric acids. Micro‐Fourier transform infrared analysis on single specimens suggests that the composition of the endospore is cellulosic and the cyst wall a more robust, noncellulosic β‐glucan. The spectra overall are similar to other published spectra of resting cysts from autotrophic, planktonic dinoflagellates.     

Patterns of cuticular organization in the hybrid Tsuga x jeffryi (Henry) Henry and its putative parents

The structure of the leaf cuticle of Tsuga heterophylla, T. mertensiana and their putative hybrid, T. X jeffryi, is described using light microscopy and scanning electron microscopy. In all three taxa the cuticle, from its response to varying maceration time and from sections stained in Sudan IV, shows clear evidence of two components: an outer one probably corresponding to the cuticle proper and cuticular layer and an inner spongy component separated from the outer by a cutin-deficient layer. Comparison of the three taxa indicates that T. X jeffryi agrees closely in nearly all cuticle features with T. mertensiana indicating that if the hybrid status of T. X jeffryi is correct, cuticle characters have been inherited predominantly from one parent.     

Description of a new species of Nesotriatoma Usinger, 1944 from Cuba and revalidation of synonymy between Nesotriatoma bruneri (Usinger, 1944) and N. flavida (Neiva, 1911) (Hemiptera,Reduviidae, Triatominae)

Nesotriatoma confusa sp. nov. (Hemiptera, Reduviidae, Triatominae) is described based on specimens from Cuba. From one male, one female, and eleven nymphs of a then‐undescribed species of Nesotriatoma collected in Cuba, a colony was formed and its specimens were used to describe N. confusa sp. nov. Characters were observed on the head, thorax, abdomen, female external genitalia, and male genitalia with optical microscopy and scanning electron microscopy. We concluded that N. bruneri (Usinger, 1944 ) was indeed a synonym of N. flavida (Neiva, 1911 ) as previously proposed.     

Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5 min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.     

Notes on gamete production in Lanice conchilega (Annelida,Polychaeta, Terebellidae)

Summary

Male and female gametogenesis in Lanice conchilega are described and illustrated as seen by light microscopy. There is no evidence for size-selectivity in the uptake of male gametes by the hypertrophied reproductive nephromixia, since undissociated motile sperm platelets as well as spermatozoa appear to be taken up by the genital funnels, and such funnels show no sexual dimorphism. Maturation of the coelomic oocytes is marked by a change in shape and the appearance of a surface reticulum of ridges.     

Localization by scanning immunoelectron microscopy of triosephosphate isomerase,the molecules responsible for contact‐mediated killing of Cryptococcus,on the surface of Staphylococcus

In our previous studies, TPI were found to be the molecules responsible for contact‐killing of C. neoformans by S. aureus cells. Since TPI is a glycolytic protein that functions in the cytoplasm, evidence that TPI is present on the surface of S. aureus was required. In the present study, the presence of TPI on the cell surface of S. aureus was demonstrated by agglutination test and scanning immunoelectron microscopy. Furthermore, TPI was found to be present at a lower density than protein A/G molecules on the surface of S. aureus.     

The reproductive system of Osedax (Annelida,Siboglinidae): ovary structure,sperm ultrastructure,and fertilization mode

Osedax is a genus of siboglinid annelids in which the females live on dead vertebrate bones on the seafloor. These females have a posterior end that lies within the bone and contains the ovarian tissue, as well as the “roots” involved with bone degradation and nutrition. The males are microscopic and live as “harems” in the lumen of the gelatinous tube that surrounds the female trunk, well away from the ovary. Females are known to spawn fertilized primary oocytes, suggesting internal fertilization. However, little is known about sperm transfer, sperm storage, or the location of fertilization, and the morphology of the female reproductive system has not been described and compared with the reproductive systems of other siboglinids. A 3D‐reconstruction of the ovisac of Osedax showed ovarian tissue with multiple lobes and mature oocytes stored in a “uterus” before being released through the single oviduct. The oviduct emerges as a gonopore on the trunk and travels along the trunk to finally open to the seawater as a thin cylindrical tube among the crown of palps. Light and transmission electron microscopy of mature Osedax sperm revealed elongate heads consisting of a nucleus with helical grooves occupied by mitochondria. In contrast to other Siboglinidae, Osedax sperm are not packaged into spermatophores or spermatozeugmata, and Osedax females lack a discrete region for sperm storage. Transmission electron microscopy and fluorescence microscopy allowed detection of sperm associated with ovarian tissue of the female ovisac of four different Osedax species. This provides the first evidence for the site of internal fertilization in Osedax. A heart body was found in the circulatory system, as seen in other siboglinids and some other annelids. The possible presence of nephridia in the anterior ovisac region was also documented. These morphological features provide new insights for comparing the regionalization of Osedax females in relation to other siboglinids.     

Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane

The physical characteristics of rubber particles from the four rubber (cis-1,4-polyisoprene) producing species Euphorbia lactiflua Phil., Ficus elastica Roxb., Hevea brasiliensis Müll. Arg., and Parthenium argentatum Gray, were investigated using transmission electron microscopy (TEM) and electron-paramagnetic-resonance (EPR) spin labeling spectroscopy. Transmission electron microscopy showed the rubber particles to be composed of a spherical, homogeneous, core of rubber enclosed by a contiguous, electron-dense, single-track surface layer. The biochemical composition of the surface layer and its single-track TEM suggested that a monolayer biomembrane was the surface structure most compatible with the hydrophobic rubber core. The EPR spectra for a series of positional isomers of doxyl stearic acid, used to label the surface layer of the rubber particles, exhibited flexibility gradients and evidence for lipid-protein interactions for all four rubber particle types that is consistent with a biomembrane-like surface. The EPR spectra confirmed that the surface biomembrane is a monolayer. Thus, rubber particles appear similar to oil bodies in their basic architecture. The EPR spectra also provided information on protein location and degree of biomembrane penetration that correlated with the known properties of the rubber-particle-bound proteins. The monolayer biomembrane serves as an interface between the hydrophobic rubber interior and the aqueous cytosol and prevents aggregation of the particles. An unexpected observation for the probes in pure polyisoprene was evidence of an intrinsic flexibility gradient associated with the stearic acid molecule itself. Received: 22 May 1999 / Accepted: 21 June 1999     

Substructure of spore and pollen grain exines in Lycopodium,Alnus, Betula,Fagus and Rhododendron

We have used atomic force microscopy and scanning tunnelling microscopy to extract new information about the substructure of the Alnus, Betula, Fagus, Lycopodium and Rhododendron pollen grain exine. Our scans of exines using atomic force microscopy and scanning tunnelling microscopy reveal somewhat similar substructures for Lycopodium spores and pollen of Alnus, Betula, Fagus and Rhododendron. They show various levels of alignment and clustering of substructure components. Except for Alnus, which showed polygonal clustering of spheroids and weak alignment, there is pronounced alignment of helical units. In Betula, Fagus, Lycopodium and Rhododendron the subunits appear to be helical or perhaps consisting of elongated spheroids, these spheroids are however arranged in a way that suggest that they are part of a helical structure. The diameter of these helical subunits range from 10–15 nm in Fagus, 20–25 nm in Lycopodium, 35–90 nm in Rhododendron up to 70–120 nm in Betula. Our preparations graded from intact or fractured fresh pollen to pollen that was acetolyzed, chemically fixed and epoxy resin embedded. While our knowledge of the exact radial/lateral orientation of most of our scans is less than perfect there were in all cases substructures or cross connections of exine units. We found results from scanning and transmission electron microscopy to be helpful in understanding images from Atomic Force- and Scanning Tunnelling Microscopy.     

Morphology,Phylogeny, and Diversity of Trichonympha (Parabasalia: Hypermastigida) of the Wood‐Feeding Cockroach Cryptocercus punctulatus

ABSTRACT. Trichonympha is one of the most complex and visually striking of the hypermastigote parabasalids—a group of anaerobic flagellates found exclusively in hindguts of lower termites and the wood‐feeding cockroach Cryptocercus—but it is one of only two genera common to both groups of insects. We investigated Trichonympha of Cryptocercus using light and electron microscopy (scanning and transmission), as well as molecular phylogeny, to gain a better understanding of its morphology, diversity, and evolution. Microscopy reveals numerous new features, such as previously undetected bacterial surface symbionts, adhesion of post‐rostral flagella, and a distinctive frilled operculum. We also sequenced small subunit rRNA gene from manually isolated species, and carried out an environmental polymerase chain reaction (PCR) survey of Trichonympha diversity, all of which strongly supports monophyly of Trichonympha from Cryptocercus to the exclusion of those sampled from termites. Bayesian and distance methods support a relationship between Trichonympha species from termites and Cryptocercus, although likelihood analysis allies the latter with Eucomonymphidae. A monophyletic Trichonympha is of great interest because recent evidence supports a sister relationship between Cryptocercus and termites, suggesting Trichonympha predates the Cryptocercus‐termite divergence. The monophyly of symbiotic bacteria of Trichonympha raises the intriguing possibility of three‐way co‐speciation among bacteria, Trichonympha, and insect hosts.     

A combined morphological,ultrastructural, molecular,and biochemical study of the peculiar family Gomontiellaceae (Oscillatoriales) reveals a new cylindrospermopsin‐producing clade of cyanobacteria

Members of the morphologically unusual cyanobacterial family Gomontiellaceae were studied using a polyphasic approach. Cultured strains of Hormoscilla pringsheimii, Starria zimbabweënsis, Crinalium magnum, and Crinalium epipsammum were thoroughly examined, and the type specimen of the family, Gomontiella subtubulosa, was investigated. The results of morphological observations using both light microscopy and transmission electron microscopy were consistent with previous reports and provided evidence for the unique morphological and ultrastructural traits of this family. Analysis of the 16S rRNA gene confirmed the monophyletic origin of non‐marine repre‐sentatives of genera traditionally classified into this family. The family was phylogenetically placed among other groups of filamentous cyanobacterial taxa. The presence of cellulose in the cell wall was analyzed and confirmed in all cultured Gomontiellaceae members using Fourier transform infrared spectroscopy and fluorescence microscopy. Evaluation of toxins produced by the studied strains revealed the hepatotoxin cylindrospermopsin (CYN) in available strains of the genus Hormoscilla. Production of this compound in both Hormoscilla strains was detected using high‐performance liquid chromatography in tandem with high resolution mass spectrometry and confirmed by positive PCR amplification of the cyrJ gene from the CYN biosynthetic cluster. To our knowledge, this is the first report of CYN production by soil cyanobacteria, establishing a previously unreported CYN‐producing lineage. This study indicates that cyanobacteria of the family Gomontiellaceae form a separate but coherent cluster defined by numerous intriguing morphological, ultrastructural, and biochemical features, and exhibiting a toxic potential worthy of further investigation.     

Cyst‐motile stage relationship,morphology, ultrastructure,and molecular phylogeny of the gymnodinioid dinoflagellate Barrufeta resplendens comb. nov., formerly known as Gyrodinium resplendens,isolated from the Gulf of Mexico

In the present study, we redescribed Gyrodinium resplendens through incubation of process bearing cysts extracted from sediment collected in the northern Gulf of Mexico. The morphology and ultrastructure of the motile stage and cyst stage were examined using light microscopy, scanning electron microscopy, and transmission electron microscopy and this revealed that the species should be transferred to the genus Barrufeta. This genus differs from other gymnodinioid genera in possessing a Smurf‐cap apical structure complex (ASC) and currently encompasses only one species, Barrufeta bravensis. B. resplendens shows a Smurf‐cap ASC that consists of three rows of elongated vesicles with small knobs in the middle one. B. resplendens is very similar to B. bravensis in cell morphology, but can be separated using the ultrastructure such as the shape and location of nucleus and pyrenoids, which highlights the importance of ultrastructure at inter‐specific level in the genus Barrufeta. The unique cysts of B. resplendens are brown and process bearing, and have a tremic archeopyle with a zigzag margin on the dorsal side of the epicyst, and not polar as in cysts of Polykrikos. The cysts do not survive the palynological treatment used here and probably have a wide distribution. Maximum‐likelihood and Bayesian inference were carried out based on partial large subunit ribosomal DNA (LSU rDNA) sequences. Molecular phylogeny supports that the genus Barrufeta is monophyletic, and that the genus Gymnodinium is polyphyletic. Our results suggest that details of the ASC together with ultrastructure are potential features to subdivide the genus Gymnodinium.     

Identification of a new‐to‐science cyanobacterium,Toxifilum mysidocida gen. nov. & sp. nov. (Cyanobacteria,Cyanophyceae)

Cyanobacteria occupy many niches within terrestrial, planktonic, and benthic habitats. The diversity of habitats colonized, similarity of morphology, and phenotypic plasticity all contribute to the difficulty of cyanobacterial identification. An unknown marine filamentous cyanobacterium was isolated from an aquatic animal rearing facility having mysid mortality events. The cyanobacterium originated from Corpus Christi Bay, TX. Filaments are rarely solitary, benthic mat forming, unbranched, and narrowing at the ends. Cells are 2.1 × 3.1 μm (width × length). Thylakoids are peripherally arranged on the outer third of the cell; cyanophycin granules and polyphosphate bodies are present. Molecular phylogenetic analysis in addition to morphology (transmission electron microscopy and scanning electron microscopy) and chemical composition all confirm it as a new genus and species we name Toxifilum mysidocida. At least one identified Leptolyngbya appears (based on genetic evidence and TEM) to belong to this new genus.     

BREEDING SYSTEM OF A HYBRID BETWEEN A SEXUAL AND AN APOMICTIC SPECIES OF AMELANCHIER,SHADBUSH (ROSACEAE,MALOIDEAE)

Amelanchier bartramiana, which has been shown to be sexual and self-incompatible, and A. laevis, in which apospory and self-compatibility occur, grow in a mixed population in western Maine. Also present there is the putative interspecific hybrid, A. × neglecta. Principal components and canonical discriminant analyses of seven morphological characters from 96 parental and putative hybrid individuals substantiate morphological intermediacy of the hybrids. A Nomarski differential interference microscopy study of megasporogenesis and megagametogenesis in five hybrid plants shows a predominantly aposporous mode of reproduction, and controlled pollinations indicate that seed formation between A. bartramiana and A. laevis is possible and that the hybrids are self-compatible. The hybrids, which are tetraploids (n = ca. 34) like both parents in Maine, produce viable pollen and seed, but there is little evidence for backcrossing with either parent.     

STUDIES ON GRACILARIA (GIGARTINALES,RHODOPHYTA): REPRODUCTIVE STRUCTURES1,2,3

The reproductive structures of Gracilaria foliifera (Forsk.) Børg. from England and an unnamed species of Gracilaria from Nova Scotia were studied by light microscopy. These two entities are distinguishable on the basis of the type of gonimoblast tissue in their mature cystocarps. This, together with other evidence, suggests that these are separate taxa.     

Is Diurodrilus an annelid?

Interstitial marine meiofaunal worms of the genus Diurodrilus have always been considered part of Annelida, either as basal or derived, though generally with reference to Dinophilidae. New evidence shows that Diurodrilus has a unique anatomy, and lacks key annelid features, possibly even segmentation. We assessed the systematic position of Diurodrilus among other protostome animals via light microscopy, confocal laser scanning microscopy, and transmission electron microscopy studies of anatomy, focusing on musculature, the nervous system, as well as molecular sequence data. We show that there is little morphological or molecular evidence to support a relationship with Dinophilidae or any other annelids. Diurodrilus has some similarities to Micrognathozoa, though the latter shows complex jaws. On the basis of the configuration of the nervous system and the cuticle we regard Diurodrilus to belong to Spiralia, possibly close to Annelida; however, until further evidence is acquired it should be regarded as incertae sedis in this large animal clade. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

A new genus of athecate interstitial dinoflagellates, Togula gen. nov., previously encompassed within Amphidinium sensu lato: Inferred from light and electron microscopy and phylogenetic analyses of partial large subunit ribosomal DNA sequences

The recent emendation of Amphidinium (Dinophyc‐eae), which now only consists of species with minute left‐deflected epicone, has left more than 100 species without a clear generic affiliation. In the present study, a strain identified as one of the species with a divergent epicone type, Amphidinium britannicum (Herdman) Lebour, and six strains resembling A. britannicum but smaller in size were examined by light, scanning and transmission electron microscopy and by sequence analyses of nuclear‐encoded partial large subunit ribosomal DNA to establish their phylog‐eny. Amphidinium britannicum was not closely related to other genera included in the molecular phylogenetic analyses, but formed a highly supported clade in Bayesian analysis together with the six small‐sized strains. The six strains also formed a highly supported clade, consisting of two closely related, albeit distinct, clades. Light and scanning electron microscopy did not reveal significant differences between the vegetative motile cells; however, cells about to undergo mitosis developed longitudinal grooves on the hypocone in one of the clades but not in the other. Both clades differed substantially from A. britannicum in partial large subunit ribosomal DNA as well as in size and shape. Based on morphological similarity and partial large subunit ribosomal DNA evidence, we erect the new genus, Togula gen. nov. with the emended type species Togula britannica (Herdman) comb. nov. Based on differences in division pattern and partial large subunit ribosomal DNA gene divergence we further describe the species Togula compacta (Herdman) comb. nov. and Togula jolla sp. nov.