ALTHOUGH Piloboliis has been extensively studied both by myco-

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1 [58] THE SPORANGIOPHORE OF PILOBOLUS BY C. T. INGOLD Department of Botany, University of Reading (With 2 figures in the text) ALTHOUGH Piloboliis has been extensively studied both by myco- ^ logists and by physiologists, there does not appear to be any clear account of the exact manner in which the sporangium is discharged. In this short note it is proposed to give an illustrated description of the development and dehiscence of the sporangiophore in Piloholns Kleinii based on original observations, Pilobolits appears almost invariably on the dung of most herbivorous animals. If fresh horse-dung baus are kept on moist filter paper in a dish and covered with a beu-jar, sporangiophores of P. Kleinii develop after a few days and then crops of mature sporangiophores are produced daily for a week or two. DEVELOPMENT OF THE SPORANGIOPHORE The non-septate mycelium of the fimgus develops in the dung bau, germination in the first instance being induced by the relatively high temperature of the horse's intestine. The sporangiophore primordium is first visible to the naked eye as a minute spherical orange bulb protruding shghtly above the general level of the substratum. Microscopic examination shows that this bulb is cut off from the greatly enlarged end of a hypha, and that the spherical primordium is packed with minute globules of yeuow ou (Fig. i, a). This is the condition of the young sporangiophores in the forenoon. During the course of the afternoon an intensely yeuow branch grows up from the primary bulb (Fig. i, b). If a horse-dung culture bearing Pi/o&o/Ms is examined between 3 and 5 p.m.^ these httle yellow processes (about 2 mm. high) are very conspicuous on account of their brilliant colour. From this stage onwards the tip of the sporangiophore is markedly heliotropic (Jolivette, 1914; Parr, 1918). Towards evening (6-8 p.m.) the tip sweus up into a minute spherical bulb destined to become the sporangium (Fig. i, c), and at 1 The times referred to in this paper are in Summer time.

2 Fig. I. Pilobolus Kleinii. a. spherical primordium stage found in forenoon; b. bright yellow process developed from the primordium (3-5 p.m.); c. swelling of the tip of the process to form the sporangium (6-8 p.m.); d. massing of the contents in the terminal swelling. Subsporangial swelling just beginning. Stalk becoming colourless (8-10 p.m.); e. black cap completely formed, stalk almost colourless, subsporangial region considerably swollen (about midnight);/, mature sporaagiophore (8-11 a.m.); g. sporangiophore a few seconds after dehiscence exuding a drop of liquid; h. the deflated sporangiophore some hours after dehiscence showing the circular line of dehiscence. All the figures are drawn to approximately the same scale.

3 6o C. T. INGOLD this stage the oil drops giving the yellow colour to the sporangiophore are evenly distributed throughout. As night approaches (8-10 p.m.) the oil tends to accumulate in the upper spherical sweuing and the stalk region becomes more or less colourless. At the same time the wall of the upper part of the terminal swelling begins to blacken and the subsporangial region starts to swell. This condition is shown in Fig. i, d. Towards midnight the subsporangial swelling develops considerably and the black cap of the sporangium becomes fully formed, but the spores are still undifferentiated (Fig. i, e). In the early morning (6-8 a.m.) the sporangiophores are practically mature (Fig. i,/). At this stage the subsporangial swelling is swollen to its full size, the stalk region is completely elongated and the spores are fully formed. Throughout the development of the aerial portion of the sporangiophore minute drops of water are to be seen covering the surface, presumably exuded on account of the great hydrostatic pressure within the cell. These drops become particularly conspicuous and abundant on the mature sporangiophore. Immediately before explosion the sporangiophore consists of (i) a lower bulb immersed in the substratum, (2) an erect stalk, and (3) an enlarged subsporangial bulb ending in a dome-shaped columeua which protrudes into the sporangium. The stalk and the upper bulb of the sporangiophore contain a clear watery liquid except at the point where the stalk joins the bulb; here a conspicuous zone of oil is invariably found. This oil appears to the naked eye as a minute orange spot at the base of the subsporangial swelling and has been interpreted by BuUer (1921) as an eye spot, the bulb itself being regarded as a lens focussing the light on the sensitive spot. The sporangium wall is differentiated into a hard upper blackened region and a very thin non-resistant lower region. Before discharge this thin wall partially breaks down. Within the sporangium, and occupying the upper part of it, is a mass of yellow-orange spores completely hidden by the black cap. The remainder of the sporangium is occupied by clear mucilage. The structure of the mature sporangium is illustrated diagrammatically in Fig. 2, II. Sporangium discharge occurs in the morning between 9 and 12 o'clock. The formation of the sporangiophore, from the spherical primordium until discharge occurs, therefore occupies just one day. At any time all the sporangiophores are at approximately the same stage in development. It is probable that the production of daily

4 The Sporangiophore of Pilobolus crops of sporangiophores is connected with the periodic changes in hght intensity associated with the alternation of day and night. 6i m Fig. 2. Pilobolus Kteinii. I, diagram showing the supposed form of the projectile. Below is the contracted subsporangial bulb, above is the projectile consisting of a drop of liquid carrying an inverted sporangium. If, diagram of the sporangium. B, the subsporangial bulb; L, the line of dehiscence; P, the columella; IF, the thin part of the sporangium wall; C, the black cap; S, the mass of spores; M, the clear mucilage; III, the columella dissected out from a discharged sporangium, IV, spores, V, surface view of the sporangium caught on a glass slide. In the centre is the black cap, fringing this is what remains of the thin region of the wall. The remainder is clear mucilage. No spores can be seen. VI, sporangium caught on a glass slide and viewed through the slide. In the centre is the hollow cone of the columella. around this can be seen a mass of spores and outside this mass the edga of the black cap. Outside this again is the clear mucilage. DISCHARGE OF THE SPORANGIUM The pressure inside the sporangiophore finauy becomes so great that an explosion results, rupture occurring along a very definite Hne of weakness where the columeua joins the subsporangial bulb. The elastic wall of the sporangiophore contracts almost instan-

5 62 C. T. INGOLD tancously with a very audible click. The explosion takes place so quickly that it is impossible to follow the e.xact details at this stage. It is, however, the opinion of the writer that a small drop of liquid is ejected from the sporangiophore and on escaping tears off and carries away the sporangium. If sporangia are caught on a glass slide held several centimetres above a culture of exploding Piloboli it can readily be seen that the sporangium is accompanied by a considerable amount of clear watery liquid presumably derived from the subsporangial bulb. The amount of this water appears to be about twice the volume of the sporangium itself. The sporangia caught thus on a slide are always the same way up unth the black cap looking downwards, the mucilage and the rim of the columella being stuck to the surface of the glass (Fig. 2, V and VI). It is clear that the sporangium on leaving the sporangiophore, and before it hits an object held above it, must in some way be turned upside down. The manner in which this inversion takes place may be visualised in the following way. The columella probably begins to tear away from the sporangiophore at a point on the circumference of the line of dehiscence and the tear rapidly spreads. Through the aperture thus produced water exudes forming a drop which, as it grows, increases the tear. This drop is moving with great velocity and as it rounds off, in separating from the sporangiophore, tears away the sporangium completely, so that in the projectile the sporangium is at the bottom of the drop with the black cap undermost forming an unwetted base to the drop (Fig. 2, I). The time occupied from the initial break in the dehiscence line to the separation of the projectile occupies only a very small fraction of a second. Immediately on the discharge of the sporangium the stalk region of the sporangiophore loses its rigidity and the bulb, which has contracted to about half its original volume, is brought down to the level of the substratum. The bulb is, however, still full of liquid, part of which is soon squeezed out, as a small drop, by further slow contraction of the wall (Fig. i, ^). After a short time the old sporangiophore becomes completely deflated (Fig. i, /;). Microscopic examination of the sporangiophore immediately after explosion shows that the line of dehiscence is a very perfect circle. The discharged sporangia exhibit certain interesting features; these are illustrated in Fig. 2, III, V and VI. The mucilage of the sporangium serves to stick it to whatever object it happens to strike. The black cap of the sporangium completely covers the spores and possibly protects them from the injurious action of light. The remains

6 The Sporangiophore of Pilobolus 63 of the unthickened region of the sporangium wall can still be identified in the discharged mass and the columella is a cap-like cone which may with care be dissected out. The number of spores contained within a single sporangium is very great. Buller has found using Pilobolus Kleinii and P. longipes that the sporangia are shot to a maximum height of six feet and a horizontal distance of eight feet. With P. Kleinii the writer has found that sporangia may be shot to a height of 118 cm. The ease with which this fungus may be obtained in quantity and the interesting features which it exhibits render it specially suitable for class work. An intensive study of the development of the sporangiophore and the discharge of the sporangium is both instructive and interesting for advanced students. For microscopic examination the sporangiophore must be removed from the substratum, care being taken not to rupture the lower bulb which is partially immersed in the substratum. The sporangiophore must be mounted in water since glycerine causes immediate deflation. REFERENCES (1) BULLER, A. H. R. Upon the ocellus function of the subsporangial swelling of Piloholus. Trans. Brit. Mycol. Soc. 7, 6i (2) JoLivETTE, H, D. M. Studies in the reactions of Pilobotus to light stimuli. Bot. Gaz. 57, (3) PARR, R. The response of Pilobolus to light. Ann. Bot. 32,

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