WHOLE SPORES

COLOR: Hyaline/white in most recently formed spores to yellow-brown (0-5-40-0)
in older spores (especially those from field soils).

SHAPE: Globose, subglobose, often elliptical or strongly oblong.

SIZE DISTRIBUTION: 220-380 µm (mean = 302 µm, n = 100)

SUBCELLULAR STRUCTURE OF SPORES

SPORE WALL: Four layers (L1, L2, L3 and L4), with L1 sometimes separating from L2 and L3 (which usually are adherent), and L4 separating readily and often grouped with germinal walls when detectable.

Spore wall layers	Papillate surface	L4 of spore wall

L1: A single hyaline layer less than 1 µm thick with a papillate surface. Papillae are rarely more than 1 µm high and 1-1.5 µm wide and often are closely crowded. This layer forms with L2 and L3 in juvenile spores and separates sometimes only in mature spores. L1 and L2 together are homologous with the L1 layer (which may or may not be ornamented) in other Scutellospora species. It is treated as a separate layer because it is capable of separation as a discrete component and in deference to the authors (Spain and Miranda, 1996).

L2: A single hyaline layer that is distinguishable from the laminate layer (L2) only in Melzer's reagent, where it produces only a pale pinkish-red (0-40-40-0) reaction in Melzer's reagent compared to the much darker reaction of L2 (see below); 1.5-3.0 µm thick.

L3: A layer consisting of very fine adherent hyaline to pale yellow (0-0-20-0) sublayers (or laminae), 2.5-8 µm thick (mean of 6.2 µm) in mature spores; staining dark red-purple (40-80-40-0) to reddish-black (60-80-50-10) in Melzer's reagent.

L4: A thin hyaline layer, less than 1 µm thick, which separates readily from the remainder of the spore wall except at its attachment to the spore wall at the subtending hypha. This layer, when it is not traced back to its connection to the spore wall, can easily be mistaken for a separate flexible inner wall. It is analagous to the separable flexible layer associated with some Glomus species and also species in Acaulospora and Entrophospora.

GERMINAL WALLS: Two bi-layered flexible inner walls (gw1 and gw2) are present that readily separate from each other and from the spore wall. Both walls form in sequence and begin synthesis only after the spore wall has completed differentiation.

In PVLG
In PVLG and Melzer's reagent (1:1 v/v)

GW1: Two layers (L1 and L2) that usually are adherent, but sometimes L1 separates slightly and produces numerous fine folds. L1 is less than 0.5 µm thick and L2 is 1.0-2.8 µm thick. Neither layer reacts in Melzer's reagent.

GW2: Two layers (L1 and L2) that rarely separate. L1 is 2.0-4.8 µm thick and can produce a weak pink reaction (0-10-20-0) in Melzer's reagent that is detected only when it separates from L2. L2 is hyaline and plastic enough that it has been termed "amorphous". It is 4.0-18.0 µm thick (measured in PVLG), depending on amount of pressure applied to it while breaking the spore; staining red-purple (20-80-20-0) to dark red-purple (40-80-60-0) in Melzer's reagent.

SUBTENDING HYPHA

WIDTH OF SPOROGENOUS CELL: 32-45 µm (mean = 37.6 µm).

SPOROGENOUS CELL WALL: Two layers (L1 and L2) probably are present (continuous with the two layers of the spore wall), but only L2 is readily discernible at the level of the compound microscope.

L2: Pale yellow (0-0-20-0) to brownish yellow (0-20-80-0), 2.5-3.2 µm thick near the spore and then thinning to 0.8-1.2 µm beyond the sporogenous cell.

OCCLUSION: Closure by a plug concolorous with the wall of the sporogenous cell.

GERMINATION SHIELD

COLOR: Pale yellow-brown (0-10-80-0) to dark yellow-brown (0-30-100-0)

SHAPE: Almost spherical, with length and width near equal. Margins of shields generally are smooth, with few folds (each with paired germ holes). The shield is sufficiently robust to separate intact from the inner flexible walls when spores are broken. Position of the shield is on gw2.

SIZE: Not measured

AUXILIARY CELLS

Aggregate (1-12) cells borne on coiled light brown (0-40-80-0) hyphae, thin-walled (< 1 µm thick), pale brown (0-40-80-0) to dark brown (0-60-100-10) in transmitted light, each cell with shallow swellings 0.5-2 µm m high and 7-10 µm wide.

MYCORRHIZAE

Intraradical arbuscules and hyphae consistently stain darkly in roots treated with trypan blue. Arbuscules with many fine tips from a swollen trunk. Hyphae often with knobs or projections, usually densely coiled near entry points. Auxiliary cells and external hyphae often amass around the root, with the former most abundant in pot cultures just prior to spore formation and declining thereafter.

Arbuscules in corn roots		Other mycorrhizal structures in corn

NOTES

Small spores of S. cerradensis can be mistaken for S. pellucida or S. gilmorei under a dissecting microscope, but inner wall structure clearly distinguishes this species. Spain and Miranda (1997) were the first to detect a third layer of the spore wall, probably because it is so obvious in this species (see photos of sporogenous cell region above). It has since been detected in spores of S. pellucida and S. heterogama and thus probably is a feature of most, if not all, Scutellospora species.

Both germinal walls (gw1 and gw2) do not separate easily, even in vigorously crushed spores and thus resembles one very thick hyaline wall, much like that found in spores of S. erythropa. However, the germination shield clearly establishes the separation of two inner walls (forming on iw2). The layers of each wall are hard to distinguish except with differential interference contrast optics.

Spain and Miranda (1997) describe gw2 as having three layers, but in our judgement, they confuse numerous folds of the highly plastic L2 as being a separate layer. If evolutionary pattern is any indication, such a bi-layered organization is a constraining feature of all germinal walls in both Gigasporaceae and Acaulosporaceae.

REFERENCES