Masatoshi Saikawa

Molecular phylogeny of Zygomycota based on EF-1α and RPB1 sequences: limitations and utility of alternative markers to rDNA

Earlier molecular phylogenetic analyses based on nuclear small subunit ribosomal DNA (nSSU rDNA) suggest that the Zygomycota are polyphyletic within the Chytridiomycota. However, these analyses failed to resolve almost all interordinal relationships among basal fungi (Chytridiomycota and Zygomycota), due to lack of sufficient characters within the nSSU rDNA. To further elucidate the higher-level phylogeny of Zygomycota, we have sequenced partial RPB1 (DNA dependent RNA polymerase II largest subunit) and EF-1alpha (translation elongation factor 1 alpha) genes from 10 and 3 zygomycete fungi, respectively. Independent molecular phylogenetic analyses were performed based on each sequence by distance and maximum likelihood methods. Although deep phylogenetic relationships among basal fungi still remain poorly resolved using either gene, the RPB1-based phylogeny identified a novel monophyletic clade consisting of the Dimargaritales, Harpellales, and Kickxellales. This result suggests that regularly formed septa (cross walls that divide hyphae into segments) with a lenticular cavity are plesiomorphic for this clade, and indicates the importance of septal pore ultrastructure in zygomycete phylogeny. In addition, a peculiar mucoralean genus Mortierella, which was considered to be distantly related to the other Mucorales based on previous nSSU rDNA analyses, was resolved as the basal most divergence within the Mucorales, consistent with traditional phenotypic-based taxonomy. Although the taxa included in our analysis are restricted, the monophyly of each order suggested by nSSU rDNA phylogeny is supported by the present RPB1-based analysis. These results support the potential use of RPB1 as an alternative marker for fungal phylogenetic studies. Conversely, the overall fungal phylogeny based on EF-1alpha sequence is poorly resolved. A comparison of numbers of observed substitutions versus inferred substitutions within EF-1alpha indicates that this gene is much more saturated than RPB1. This result suggests that the EF-1alpha gene is unsuitable for resolving higher-level phylogenetic relationships within the Fungi.

Fungal predators of rotifers-a comparative study of Zoophagus, Lecophagus and Cephaliophora

Ultrathin sections of two rotifer-capturing species of Cephaliophora, C. muscicola and C. longispora , were compared with those of Zoophagus insidians . Accumulation of electron-dense vehicles in the adhesive pegs, and mitochondria with lamellar cristae, are common to all three species. The outer layer of the apical portion of the adhesive peg in Z. insidians has distinctive ridge-like markings. In the Cephaliophora species, the cell wall at the apex of the peg is somewhat irregular in outline but lacks ridge-like markings. In Z. insidians , the living, vegetative hyphae are nonseptate. Adventitious septa are formed in senescing hyphae to delimit the living portion from the dead, evacuated portion. In the two species of Cephaliophora , septa occur regularly and frequently in hyphae and conidia. Such septa have a central pore which is similar in its ultrastructural detail to those reported for the Ascomycota/Deuteromycota. Anastomoses are common in all isolates of the rotifer-trapping Cephaliophora species examined. Anastomoses were not observed in the isolates of Z. insidians . We conclude that C. musciola and C. longispora are properly placed in the Hyphomycetes.

Capture of rotifers by Acaulopage pectospora, and further evidence of its similarity to Zoophagus insidians

Acaulopage pectospora is a nematode-trapping zoopagaceous fungus in which the hyphae bear a number of short lateral branches (ca 10-25 tm long, 2.5-3.0 Am wide) as trapping organs. Each branch terminates in a small, globose knob from which an adhesive is squeezed out to capture nematodes (Drechsler, 1962). This species is unique in the genus Acaulopage in that all other known species capture amoebae using the entire surface of their undifferentiated hyphae. The extremely large elongated conidia (ca 180-240 x 7-14 ,um) are also exceptional for the genus. Recently, Saikawa and Morikawa (1985) reported several ultrastructural aspects of the fungus as follows: (1) the traps cytoplasm was occupied by large electron-dense vesicles (0.4-0.8 ,um) before secretion of the adhesive; (2) the secreted adhesive derived from the large vesicles had a high electron density and contained a number of bubbles; (3) the cell walls of the traps became doubled after the secretion of the adhesive and an aliquot of the adhesive remained in the intervening spaces between the outer and newly-formed inner cell walls; (4) the outer wall of the terminal knob exhibited a rimose appearance. These ultrastructural characteristics in A. pectospora are similar to those of Z. insidians (Whisler and Travland, 1974) except that the rimose appearance in the terminal knob is replaced in the latter by the fine ridges in the apical, rounded end of the short branches. These similarities led Saikawa and Morikawa (1985) previously to suggest a close relationship between the two species. In the present study, A. pectospora was tested for its ability to capture rotifers instead of nematodes in an aquatic environment, and Z. insidians was tested for its ability to trap nematodes. MATERIALS AND METHODS

Ultrastructure of Stylopage rhabdospora, an amoeba-capturing zoopagaceous fungus

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Electron microscopy on Sommerstorffia spinosa, a watermold parasitic on rotifers

Sommerstorffia spinosa Amaudow (Saprolegniales, Oomycetes) infects loricate rotifers by both endoparasitic and predacious means, using sporelings and pegs, respectively. The sporeling is a lecythiformshaped structure derived from the encysted, secondary zoospore, whereas the peg is a narrow terminal protuberance of a short, hyphal branch. In electron microscopy of thin sections, however, infective organs are very similar to each other, being packed with many, large (approx. 1.0 Atm diam), electrondense vesicles in their apical portion. When that portion is engulfed by rotifers, both infective organs secrete an amorphous, electron-dense, adhesive mass containing a number of bubbles. Sommerstorffia spinosa Arnaudow, a watermold parasitic on small loricate rotifers, has been reported from Bulgaria (Arnaudow, 1923), several places in the U.S.A. (Karling, 1952; Sparrow, 1929) and England (Prowse, 1953). In the present study, we obtained the fungus species in Japan and used it as material for electron microscopic observations. It is of interest that the fungus has two different manners of infection, endoparasitic and predacious, by different infective organs respectively. In the former case, the fungus infects the animals with sporelings. The sporelings are lecythiformor flask-shaped structures with narrow necks which are always terminated with globose knobs. Infection of rotifers is initiated by the animals engulfing the globose knob of sporelings. After penetration from the knob, an infection thallus soon develops inside the body of the rotifer and it eventually consumes the body content. Soon after the endozoic thallus forms, the fungus also develops two (or rarely only one) short, hyphal branches from the basal part of the sporeling external to the infected rotifer. Each of the two hyphal branches terminates with narrow protuberances called pegs with which other, healthy, animals are captured by the predacious manner of infection. The fungus then further develops its hyphal system by consumption of the captured rotifers. In this paper, we show transmission electron micrographs from ultrathin sections of these two different kinds of parasitic structures.

Electron microscopy of amoeba-capturing Dactylella tylopaga, showing the morphology of a basidiomycete

Dactylella tylopaga, a hyphomycete capturing terrestrial amoebae with adhesive knobs, showed the ultrastructural morphology of the Basidiomycota in that the cross walls in the hyphae were dolipore septa with parenthesomes. The parenthesomes were flat? tened and imperforate. The conidium (40 x 2-3 um) of the fungus was composed of two cells with the upper cell bearing an apical, tubular extension. On one oc- casion, one of the conidia on an agar plate produced teardrop-shaped microconidia (2.5-3 x 1.5-1.8 um) on sterigmatic outgrowths of each cell of the two- celled conidium.

ULTRASTRUCTURE OF COCHLONEMA ODONTOSPERMA, AN ENDOPARASITE IN AMOEBAE

Cochlonema odontosperma, a zoopagalean fungus endoparasitic in terricolous amoebae, was examined using ultrathin sections. Conspicuous cellular components in the young thallus were many nuclei (ca 1 lim in diam), electron-dense vesicles (ca 0.5 ,m) and small vacuoles (0.2-1.0 um). In older thalli there was usually one or more large vacuoles and a number of small vacuoles. Both the large and small vacuoles were often connected by a number of electron-dense vesicles. With vacuole expansion, the protoplasm migrated into the conidiogenous and zygosporangial hyphae outside the thallus. The conidia contained one or more nuclei and several vesicles (ca 0.3 Aum). The latter tended to

Germination and morphology of zygospores in two Cochlonema and one Acaulopage species

Germination of zygospores in three amoeba-destroying zoopagalean fungi was observed for the first time in Cochlonema cerasphorum and C. megalosomum in the Cochlonemataceae and Acaulopage lophospora in the Zoopagaceae. Zygospores were maintained on agar plates in their resting state 2–6 mo before germination. Active protoplasmic movement of minute granules, 0.3–0.5 μm diam, was observed within 24 h of germination and was clearly visible due to the disappearance of the thick cell wall of the zygospore proper. In the two species of Cochlonema the germ tube formed an aerial conidiogenous hypha with a few branches, each of which modified to form a chain of conidia. In A. lophospora the germ tube became a vegetative hypha that captured amoebae by adhesion. Zygospores of C. cerasphorum were found for the first time in the present study.

Nonconstricting-ring formation in two species of nematode-capturing hyphomycetes

Abstract Dactylella leptospora and Dactylaria candida, and Arthrobotrys dactyloides (Hyphomycetes), capture nematodes by nonconstricting- and constricting-ring traps, respectively. In the formation of the constricting-ring trap of the latter fungus, the basal portion of a curved hyphal branch put forth a bud to fuse with its advancing tip to make a ring. However, in nonconstricting-ring formation in the former two fungi, the portion behind the tip of the curved branch did not develop such a bud before fusion with the tip.

Electron microscopy of infection of nematodes by Dactylaria haptotyla

Infection of nematodes byDactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membrane-bound vesicles, 0.2–0.5 µm in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. When the adhesive knob came into contact with the nematodes cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0.7 µm in diam, was found in serial thin sections of the knobs cytoplasm. At the site facing the vesicle, the peripheral part of the nematodes cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.