Evolution of zygomycetous spindle pole bodies: Evidence from Coemansia reversa mitosis.

PREMISE OF THE STUDY: The earliest eukaryotes were likely flagellates with a centriole that nucleates the centrosome, the microtubule-organizing center (MTOC) for nuclear division. The MTOC in higher fungi, which lack flagella, is the spindle pole body (SPB). Can we detect stages in centrosome evolution leading to the diversity of SPB forms observed in terrestrial fungi? Zygomycetous fungi, which consist of saprobes, symbionts, and parasites of animals and plants, are critical in answering the question, but nuclear division has been studied in only two of six clades. METHODS: Ultrastructure of mitosis was studied in Coemansia reversa (Kickxellomycotina) germlings using cryofixation or chemical fixation. Character evolution was assessed by parsimony analysis, using a phylogenetic tree assembled from multigene analyses. KEY RESULTS: At interphase the SPB consisted of two components: a cytoplasmic, electron-dense sphere containing a cylindrical structure with microtubules oriented nearly perpendicular to the nucleus and an intranuclear component appressed to the nuclear envelope. Markham's rotation was used to reinforce the image of the cylindrical structure and determine the probable number of microtubules as nine. The SPB duplicated early in mitosis and separated on the intact nuclear envelope. Nuclear division appears to be intranuclear with spindle and kinetochore microtubules interspersed with condensed chromatin. CONCLUSIONS: This is the sixth type of zygomycetous SPB, and the third type that suggests a modified centriolar component. Coemansia reversa retains SPB character states from an ancestral centriole intermediate between those of fungi with motile cells and other zygomycetous fungi and Dikarya.

Conservation of cytoplasmic organization in the cystidia of Suillus species.

Cystidia of Suillus americanus and S. granulatus (Boletales) were examined cytochemically and ultrastructurally with cells prepared by freeze substitution. We present the first study showing ultrastructural details and cytological functions of the cystidium to be conserved in two closely related species. The results are presented for inclusion in the AFTOL Structural and Biochemical Database to aid in the application of morphological characters to phylogenetic studies. The cystidia of these Suillus species appear to be united by a series of conserved characters, including specialized secretion mechanisms, smooth tubular endoplasmic reticulum and abundant free ribosomes. The conservation of these subcellular traits among members of this genus suggests that ultrastructural details of cystidia may provide a suite of phylogenetically informative characters. Inclusion of such characters in phylogenetic analyses might resolve or provide support for monophyletic groups at the level of family or genus.

Phylogenetic relationships of Auriculoscypha based on ultrastructural and molecular studies.

The phylogeny of Auriculoscypha anacardiicola, an associate of scale insects in India, is investigated using subcellular characters and MP and Bayesian analyses of combined nuLSU-rDNA, nuSSU-rDNA and 5.8S rDNA sequence data. It has simple septa with a pulley-wheel-shaped pore plug, which is diagnostic of phytoparasitic members of the Pucciniomycetes, and hyphal wall break on branching, a phenomenon unique to some simple septate heterobasidiomycetes. The septal ultrastructure of A. anacardiicola is similar to that of the genus Septobasidium. The close relationship to Septobasidium is also confirmed by rDNA sequence analyses. The polyphyletic nature of the order Platygloeales, noted in earlier studies, is evident from the present molecular analysis as well. The placement of Auriculoscypha in the Platygloeales can no longer be justified and both ultrastructural and molecular evidence strongly support the placement of Auriculoscypha in the Septobasidiales.

Septal pore apparatus and nuclear division of Auriscalpium vulgare.

Ultrastructure of the septal pore apparatus and nuclear division of Auriscalpium vulgare (Russulales) was examined with freeze substitution and is presented for inclusion in the AFTOL Structural and Biochemical Database (http://aftol.umn.edu). Previously unreported septal characters for the Russulales (Agaricomycotina) were observed: Septa of the hymenophore had bell-shaped perforated septal pore caps that may extend along the septum and a zone of organelle exclusion surrounded the septal pore apparatus. Metaphase I of meiosis and metaphase of mitosis were similar. Globular spindle pole bodies with electron-opaque inclusions were set within polar fenestrae of the nuclear envelope. The nuclear envelope was mostly intact with occasional gaps. Fragments of endoplasmic reticulum were present near the spindle pole bodies but did not form a polar cap. Structural characters may distinguish one or more clades of the Agaricomycotina and provide additional signal in phylogenetic analyses.

The use of high pressure freezing and freeze substitution to study host-pathogen interactions in fungal diseases of plants.

This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host-pathogen interactions in fungal diseases of plants. The specific host-pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host-pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host-pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.