Cytology and molecular phylogenetics of Monoblepharidomycetes provide evidence for multiple independent origins of the hyphal habit in the Fungi.

The evolution of filamentous hyphae underlies an astounding diversity of fungal form and function. We studied the cellular structure and evolutionary origins of the filamentous form in the Monoblepharidomycetes (Chytridiomycota), an early-diverging fungal lineage that displays an exceptional range of body types, from crescent-shaped single cells to sprawling hyphae. To do so, we combined light and transmission electron microscopic analyses of hyphal cytoplasm with molecular phylogenetic reconstructions. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e. Spitzenkörper), have centrosomes as primary microtubule organizing centers and have stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. To discern the origins of Monoblepharidomycetes hyphae, we inferred a phylogeny of the fungi based on 18S and 28S ribosomal DNA sequence data with maximum likelihood and Bayesian inference methods. We focused sampling on Monoblepharidomycetes to infer intergeneric relationships within the class and determined 78 new sequences. Analyses showed class Monoblepharidomycetes to be monophyletic and nested within Chytridiomycota. Hyphal Monoblepharidomycetes formed a clade sister to the genera without hyphae, Harpochytrium and Oedogoniomyces. A likelihood ancestral state reconstruction indicated that hyphae arose independently within the Monoblepharidomycetes lineage and in at least two other lineages. Cytological differences among monoblepharidalean and other fungal hyphae are consistent with these convergent origins.

A multigene phylogeny of Olpidium and its implications for early fungal evolution.

BACKGROUND: From a common ancestor with animals, the earliest fungi inherited flagellated zoospores for dispersal in water. Terrestrial fungi lost all flagellated stages and reproduce instead with nonmotile spores. Olpidium virulentus (= Olpidium brassicae), a unicellular fungus parasitizing vascular plant root cells, seemed anomalous. Although Olpidium produces zoospores, in previous phylogenetic studies it appeared nested among the terrestrial fungi. Its position was based mainly on ribosomal gene sequences and was not strongly supported. Our goal in this study was to use amino acid sequences from four genes to reconstruct the branching order of the early-diverging fungi with particular emphasis on the position of Olpidium. RESULTS: We concatenated sequences from the Ef-2, RPB1, RPB2 and actin loci for maximum likelihood and Bayesian analyses. In the resulting trees, Olpidium virulentus, O. bornovanus and non-flagellated terrestrial fungi formed a strongly supported clade. Topology tests rejected monophyly of the Olpidium species with any other clades of flagellated fungi. Placing Olpidium at the base of terrestrial fungi was also rejected. Within the terrestrial fungi, Olpidium formed a monophyletic group with the taxa traditionally classified in the phylum Zygomycota. Within Zygomycota, Mucoromycotina was robustly monophyletic. Although without bootstrap support, Monoblepharidomycetes, a small class of zoosporic fungi, diverged from the basal node in Fungi. The zoosporic phylum Blastocladiomycota appeared as the sister group to the terrestrial fungi plus Olpidium. CONCLUSIONS: This study provides strong support for Olpidium as the closest living flagellated relative of the terrestrial fungi. Appearing nested among hyphal fungi, Olpidium's unicellular thallus may have been derived from ancestral hyphae. Early in their evolution, terrestrial hyphal fungi may have reproduced with zoospores.

Actin guides filamentous rhizoid growth and morphogenesis in the zoosporic fungus Chytriomyces hyalinus.

The advantage of filamentous growth to the fungal lifestyle is so great that it arose multiple times. Most zoosporic fungi from phylum Chytridiomycota exhibit a monocentric thallus form consisting of anucleate filamentous rhizoids that anchor reproductive sporangia to substrata and absorb nutrients. Actin function during polarized growth and cytokinesis is well documented across eukaryotes, but its role in sculpting nonhyphal, nonyeast fungal cells is unknown. We sought to provide a basis for comparing actin organization among major fungal lineages and to investigate the effects of actin disruption on morphogenesis in a monocentric thallus. Using fluorescence microscopy, we observed fixed, rhodamine phalloidin-stained actin in chemically fixed Chytriomyces hyalinus, exemplifying monocentric thallus development within the diverse, zoosporic phylum Chytridiomycota. We also compared rhizoid lengths and rhizoid branching of thalli incubated with the actin inhibitor latrunculin B to determine the effects of actin disruption on morphology. Actin was concentrated at the tips of growing rhizoids. Actin cables typically formed cortical, parallel arrays in hyphae, but in mature sporangia they were concentrated in a funnel-shaped array in the central region. Thalli treated with latrunculin B had shorter rhizoids with fewer branches than controls. In both hyphae and monocentric thalli, actin localization coincides with active, polarized growth and cytokinesis. Specific actin localization patterns are largely shared between monocentric species but differ significantly from patterns observed in hyphae. Actin integrity is critical for sustaining filamentous growth in all fungi.