BALLISTICS IN CERTAIN ASCOMYCETES

BALLISTICS IN CERTAIN ASCOMYCETES BY C. T. INGOLD Birkbeck College, University of London (Received 5 January 1961) (With 3 figures in the te.xt) SUMMARY Horizontal spore discharge has been studied in the genera Daldinia, Sordaria, Pleurage and Ascobolus. The average distance of discharge for the full spore-complement of the ascus increases with the size of the projectile. In Pleurage taenioides a consideration of the form of the ascus jet, as caught on a rapidly rotating disk, suggests that as the four spores are shot from the ascus the vigour of discharge tends to fall with the escape of successive spores. INTRODUCTION In an earlier paper (Ingold and Hadland, 1959) an account was given of ballistics in Sordaria with special reference to the horizontal distance of discharge and to the behaviour of the spores in the ascus jet. The present paper is concerned with a comparative consideration of six members of the Ascomycetes, covering a considerable range of spore size (Fig. ib), namely Daldinia concentrica Ces. and de Not., Pleurage minuta (Fuckel) Kuntze, P. taenioides Griffiths, Ascobolus leveillei Boud. and three isolates of Sordaria. The genus Sordaria, as delimited by Moreau (1953), contains a small number of species in which the black ascospores, each with a mucilaginous sheath, are without elongated appendages and in which the ascus dehisces by a pore clearly visible in the intact ascus. Within the genus there is an assemblage of very similar homothallic species namely S. fimicola (Rob.) Ces and de Not., S. destruens (Shear) Hawker, S. humana (Fuckel) Winter and S. macrospora Auerswald. These have no accessory spores and have the same type of rapid mycelial growth. Perithecium and ascus structure are essentially the same in all. These species appear to have been separated from one another largely on the basis of spore size and, to a lesser extent, spore shape. It may well be that these species should be combined in a single rather variable species. In spite of the considerable differences in spore size, I consider, that the three isolates (A, B and C) of Sordaria used in the present investigation might all be referred to 5. fimicola. DISTANCE OF HORIZONTAL DISCHARGE A Study was made of the horizontal discharge of the seven fungi. The method used has already been described (Ingold and Hadland, 1959). The only modification was that the elongated black box employed in the earlier work was modified to accommodate greater distances of throw than are encountered in S. fimicola. 143

144 C. T.INGOLD With the exception of Daldinia the fungi were grown on a suitable nutrient agar poured brimfull into 2.5x2.5 cm glass specimen tubes. In each experiment the discharge of spores from a culture, with its fruiting surface in the vertical plane, was followed. Phototropism of the perithecial necks in Pleurage spp. and in the Sordaria isolates, and of the individual protruding asci m Ascobohis leveillei made possible the horizontal alignment of the discharging asci under the influence of a horizontal beam of light. As in the work on Sordaria previously reported, a horizontal graduated glass slide divided into transverse sections each 0.9 cm wide was used to catch the spore deposit. However, when using Pleurage taenioides a much longer slide had to be used and on this the graduations were 1.0 cm apart. Imm Fig. I. A, ascus jet of Pleurage taenioides discharged upwards and caught on the under surface near the margin of a rapidly rotating perspex disk. To fit the jet into the figure it has been bent through a right angle at the points X and Y. B, single ascospores: a, Pleurage taenioides; b, Ascobolus leveillei; c, Pleurage minuta; d, Sordaria fimicola (isolate B); e, Daldinia concentrica. In the stomatal pyrenomycete Daldinia, however, a different procedure was necessary. A rectangular block was cut from a large discharging stroma and arranged so that a flat perithecium-studded surface formed a perfectly vertical face. The elongated perithecia may be considered as fairly accurately orientated at right angles to this face. No phototropic orientation of the perithecial necks is here possible as they are completely embedded in black, rigid stromatal tissue. The block was arranged a few millimetres above, and with its discharging surface coinciding witb, the end of a horizontal graduated slide. The spores caught, after an appropriate period, on a 3 mm -wide strip of this leading at right angles from the stromatal surface were considered, the strip being divided into transverse bands each 2 mm in width. In the previous work on Sordaria fimicola the distances of discbarge of projectiles of all the eight possible sizes were investigated, but in tbe present comparative study attention was concentrated on the distances to which the largest projectiles, consisting of

Ascomycete ballistics 145 the full complement of ascospores, were shot. In Pleurage taenioidcs there are four spores per ascus but in all the other species used the number is eight. All experiments were performed in the temperature range 20-23 C, The results of experiments on the distances of horizontal discharge are illustrated in Fig, 2 where, to avoid confusion, the curves for the three Sordaria isolates are separated from the rest. The mean distances of discharge are given in Table i together with spore dimensions determined from a random sample (25 or 50) of freshly discharged spores. In these dimensions, and in the calculation of spore volumes, mucilage associated with the spores has been ignored, as also has the minute cellular appendage connected to the spore of Pleurage miiiuta. Table i. Projectile size and distance of spore discharge bpecies Average dimensions of ascospore (M) Calculated total volume (V) of spore complement in ascus (u'^) Average distance (cm) of discharge of full ascus complement Daldinia concentrica 14.2 6,8 2,626 190 0.9 Sordaria fimicola (A) 17.3 II.O 8,325 411 3.2 S. fimicola (B) Pleurage minuta Ascobolus leveillei Sordaria fimicola (C) 21.5 21,0 28.8 32,1 / 13,1 14.8 15.6 16,3 14.465 18.413 28,016 34.432 594 697 922 1058 6.4 10,6 11-5 12,0 Pleurage taenioides 46.0 24.0 52.992 1411 32,3 It may be seen that when these fungi are arranged in order of increasing volume of the spore complement of the ascus, they are also in order of increasing mean distance of discharge. Now the distance 'd' to which a minute spherical projectile of radius 'r' issuing with a particular initial velocity is shot is given by J = Kr-, where 'A'' is a constant. Thus if the asci in all the species explode with equal violence (i.e. impart the same initial velocity to the escaping spore mass), then the average distance of horizontal discharge might be expected to be proportional to the square of the cube root of the spore mass in the ascus (F-f in Table i), assuming that, in flight, the ascospores are compacted into a more or less spherical projectile. In Fig. 3 mean distance is plotted against 'I'- '. It will be seen that, with one outstanding exception, the points lie on, or close to, a straight line drawn through the origin and through the points for Sordaria A and B, suggesting that it is spore mass rather than force of discharge, that is the really significant factor in determining differences in distance of throw. However, the point for Pleurage taenioides is completely out of line and, amongst those which approximate to the line, P. minuta departs most significantly from it. In both species of Pleurage the average distance of discharge exceeds expectation. This may well be related to the remarkable structure of the ascus in Pleurage. In Sordaria, Ascobolus and Daldinia the ascus is essentially cylindrical, but in Pleurage it becomes enormously inflated in its middle regions when fully grown. Because of the inflated ascus it is probable that the spore projectiles in this genus, as compared with the other genera, are increased in size by a relatively great volume of accompanying ascus sap.

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Ascomycete ballistics TENDENCY OF SPORES TO STICK TOGETHER Although this study is concerned with those spore projectiles to which the whole ascospore complement of the ascus contributes, it is of interest to note that, as in Sordaria (Ingold and Hadland, 1959) projectiles of all the possible sizes are formed in all species. However, the tendency of the spores to separate on discharge varies considerably from species to species. This tendency can be expressed by the 'd value' (Ingold and Hadland, 1959). If the spores from an ascus never separate the value is o, if they never stick together it is 1,0. d values for the fungi used in this work are given in Table 2. H7 35 30 Pleurage taenioides 25 i 20.5 15 Pleurage Ascobolus leveitlei^^^ minuta -^ ^^^ Sordaria c m :linia 4OO 8OO 1200 I60O V Fig, 3, Mean distance of horizontal discharge for the full ascus complement plotted again V^. V = the total volume of the ascospore complement excluding mucilage and sap. It will be seen that in Daldinia there is a considerable tendency for spores to separate from one another at discharge and, indeed, projectiles consisting of groups of eight spores are in the minority. However, in Pleurage taenioides only rarely do the spores from a single ascus separate from one another so the d value is extremely low. 1 BEHAVIOUR OF THE ASCUS JET IN Pleurage taenioides Because of the large spores and because there are only four in the ascus, P. taenioides is exceptionally suitable for studying the behaviour of ascus jets. An experimental study of ascus jets in Sordaria has already been reported (Ingold and Hadland, 1959). The procedure was to catch the contents from asci discharging upwards on the underside of a perspex disk rotating rapidly in a horizontal plane very close to the

148 C. T.INGOLD bursting asci. In this way the constituents of each ascus become spread out horizontally and, although the distances between the various portions of the jet are determined by the speed of rotation of the disk, their relative positions are clearly indicated. Table 2. Tendency of spores in ascus to separate at discharge {6 value) Species Daldinia concentrica Sordariafimicola (B) Ascobolus leveillei Pleurage minuta P. taenioides 0 determined at approx. 20'C 0.40 0.23 0.29 0.04 0.07 0.03-0.09 O.OI Using a disk of 12 cm diameter rotating at 1450 rev/min the ascus jet in Pleurage taenioides, when caught near the edge of the disk, is often spread out as a streak which may be 1-2 mm long. It consists of the four spores usually in a single line and only rarely with one or more spores hunched together. Although normally spread out and separated by 0.5 mm or more, the spores almost invariably remain roped together by the long mucilaginous appendages of whieh there is one at each pole of the spore. Very frequently the spores are extended in a straight line followed by a tail, in the same line, of separate drops of ascus sap (Fig. i A). When, however, asci are allowed to discharge their contents horizontally and these are then free to fall, under gravity, onto a horizontal glass slide, they are always in circular masses each consisting of the four spores, the entangled mucilaginous appendages and accompanying ascus sap. It, therefore, appears that, although at discharge the spores, escaping one at a time through an apical pore in the ascus, form an elongated streak held together only by the elastic appendages, this streak rounds off in the air, no doubt as a result of surface tension, finally forming a spherical drop. All this, of course, happens in a minute fraction of a second. Table 3. Distance in microns between centre of spore and that of its neighbour in ascus jets caught on a rotating disk 1st and 2nd spore 2nd and 3rd spore 3rd and 4th spore Mean of so jets 497 v- 563 w 668 n Range 120-1070 v 150-1795 u 200-1660 ^ Since it would appear that normally the spores escape from the ascus one at a time, and since they are so well spaced when caught on the rotating disk, it was felt that there was here an opportunity of testing the idea that as spores are liberated the pressure in the ascus falls so that each successive one tends to be discharged with less velocity. This should be reflected in the spread-out of the spores on the disk. It might be expected that, on the whole, the distance between the second and third spores would exceed that hetween the first and second, and the distance between the third and fourth spores exceed that between the second and third. The first fifty spore-jets in which the spores were spread out in a straight line and in whieh all the spores were separated from one another were considered, the distances between the centre of each spore and that of its neighbour being measured. The results are summarized in Table 3. It will be seen (Table 3) that the means do in fact accord with theory. However, the

Ascomycete ballistics 149 variability between individual ascus jets is very considerable and it is not, therefore, surprising that the significance of the difference, as determined by Student's method, between the first mean (that for the distance between the first and second spore) and the second is not established (P = 0.2-0.3) nor is that between the second and third means (P = 0.05-0.1). However, the difference between the first and third means is highly significant (P = <o.oi). My thanks are due to Susan Hadland who carried out most of the experimental work upon which this paper is based. REFERENCES INGOLD, C. T. & HADLAND, S. A. (1959). The ballistics of Sordaria. New PhytoL, 58, 46-57. MOREAU, C. (1953). Les genres Sordaria et Pleurage. Paris.