Cellular organization in germ tube tips of Gigaspora and its phylogenetic implications

Abstract

Comparative morphology of the fine structure of fungal hyphal tips often is phylogenetically informative. In particular, morphology of the Spitzenkörper varies among higher taxa. To date no one has thoroughly characterized the hyphal tips of members of the phylum Glomeromycota to compare them with other fungi. This is partly due to difficulty growing and manipulating living hyphae of these obligate symbionts. We observed growing germ tubes of Gigaspora gigantea, G. margarita and G. rosea with a combination of light microscopy (LM) and transmission electron microscopy (TEM). For TEM, we used both traditional chemical fixation and cryo-fixation methods. Germ tubes of all species were extremely sensitive to manipulation. Healthy germ tubes often showed rapid bidirectional cytoplasmic streaming, whereas germ tubes that had been disturbed showed reduced or no cytoplasmic movement. Actively growing germ tubes contain a cluster of 10–20 spherical bodies approximately 3–8 μm behind the apex. The bodies, which we hypothesize are lipid bodies, move rapidly in healthy germ tubes. These bodies disappear immediately after any cellular perturbation. Cells prepared with cryo-techniques had superior preservation compared to those that had been processed with traditional chemical protocols. For example, cryo-prepared samples displayed two cell-wall layers, at least three vesicle types near the tip and three distinct cytoplasmic zones were noted. We did not detect a Spitzenkörper with either LM or TEM techniques and the tip organization of Gigaspora germ tubes appeared to be similar to hyphae in zygomycetous fungi. This observation was supported by a phylogenetic analysis of microscopic characters of hyphal tips from members of five fungal phyla. Our work emphasizes the sensitive nature of cellular organization, and the need for as little manipulation as possible to observe germ tube structure accurately.

• character evolution
• cryo-fixation
• electron microscopy
• Glomeromycota
• light microscopy
• Spitzenkörper

Acknowledgments

This work was part of the Assembling the Fungal Tree of Life (AFTOL) project. Financial support was provided by a sabbatical grant to SPB by the UW Oshkosh Faculty Development Program and by a grant from the National Science Foundation ( DEB-0732550) to DJM.

We thank Joseph Morton and William Wheeler at INVAM (West Virginia University) for stock cultures. Teresa Pawlowska (Cornell University) gave valuable suggestions at the early stages of this project. Gail J. Celio (University of Minnesota Imaging Center) provided instruction and performed the thin sectioning. Briana Julius helped scan the photographic negatives. Electron microscopy and scanning laser confocal microscopy were performed at the University of Minnesota College of Biological Sciences Imaging Center. Free usage of the program TNT was sponsored by the Willi Hennig Society.