THE study of development among the Ascobolacese began in

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1 156 E. J. Welsford. FERTILIZATION IN ASCOBOLUS FURFURACEUS, PERS., BY E. J. WELSFORD, F.L.S., Botanical Assistant, I^oyal Holloway College. [WITH PLATB IV.] THE study of development among the Ascobolacese began in 1866 when Woronin (13) gave a brief description of Ascophanus pulcherrimus Cr. (Ascobolus pulcherrimus Cr.) He describes the archicarp as a row of swollen cells which he terins a scolecite. It becomes enclosed in a sheath of mycelial branches, some of which Woronin regards as probably antheridial. In 1871, Janczewski (12) published a somewhat similar account of Ascobolus furfuraceus, Pers.; here also the scolecite arises as a curved multicellular branch, and becomes surrounded, as development proceeds, by a dense sheath. As in Ascophanus pulcherriiims, the earliest branches of the envelope are supposed to be antheridial. One of the cells near the apex of the scolecite increases in size and gives rise to branches upon which asci are eventually borne. The spores were also germinated, but only after they had b^n passed through the alimentary canal of an animal. In 1896, Harper (8) published a fuller account of this species. He described the young scolecite as consisting of a row of uninuclear cells connected by large pores, and surrounded by a sheath. The cells of the scolecite become multinuclear, one of them increases in size, and into it nuclei from the other cells migrate through the pores. From the large cell branches arise as described by Janczewski, and into them the nuclei pass. Dangeard (6) however in 1904 stated that the'scolecite, even in the young stages before the sheath is formed, is multinucleate. The pores described by Harper he regards as evidence of protoplasmic connections similar to those found in the vegetative hyphae. He asserts that the nuclei do not pass through them, but degenerate in situ, except in the case of that cell which eventually gives rise to the ascogenous hyphse.

2 THE NEW PHYTOLOGIST..^Ascobolus. ifm

3 Fertilisation in Ascobolus furfuraceus Pers. 157 In 1905, Claussen (5) investigated a species of Boudiera in whicb he found that the young arcliicarp consists of a stalk, fertile cell and trichogyne, and that after fertilization the ascogonium becomes multicellular, and gives rise to ascogenous byphae. Previous to" this investigation no cytological evidence of a sexual process had been obtained in tbe members of this group. In 1906, Blackman and Fraser (2) described a process of reduced fertilization in Hnniaria grannlata; in this form an antheridiuni is not found and the female nuclei fuse in pairs on the ascogonium. The authors suggest that a similar parthenogenetic fusion probably exists in such other forms as Ascoboltis where an archicarp but no antheridium had been reported. A further investigation of Ascobolus fnrfttraceus, Pers, seemed therefore desirable, and was undertaken in the autumn of 1905, abundant material being obtained on cow dung. Two well-marued colour varieties were found, but they graded into each other, and were therefore used indiscriminately. No definite relation between the colour of the ascocarp and the nature of the substratum could be made out. Methods. All stages except the ydungest were readily obtained on the natural substratum and such material was therefore employed throughout tbe researcb. Attempts were also made to obtain cultures on agar, but these proved unsuccessful and were ultimately abandoned. Material was collected at various times of tbe day, but was found to be most satisfactory when fixed about noon. Various fixing agents were used, but the best results were obtained witb Flemming's weaker fiuid. Material was embedded through either chloroform or cedar oil. Sections were usually cut 5/1,. in thickness, and were stained either with Flemming's triple stain or with Heidenbain's iron haematoxylin and a solution of eosin in clove oil (Fraser (7). ACTUAL OBSERVATIONS. Mycelium and Chlamydospores. The cells of the vegetative mycelium are usually miiltiiiucleate, rich in proto'plasm and as Harper (8) has observed characterised by the presence of granules on their transverse walls. Chlamydospores closely resembling those figured hy Woronin (13) for Ascophanus piilcherrimus were frequently observed.

4 Germination of Ascospores. E.J.Welsford. The ripe ascospores are ejected in large numbers at about mid-day, and may be readily collected on a cover slip arranged above the ascocarp. After exposure for about twenty-four hours, in watery extract of cow dung, to a temperature of 38 c. that of the body of a cow, the spores germinated. The dung extract was either used alone, or was preceded by treatment with the various digestive fiuids. It was also found possible to obtain germination in a centinormal solution of sodium carbonate, but in this case the germ tubes were badly developed, probably owing to the lack of food material. The methods used are fully described by Fraser (7) for Lachnea stercorea in which similar results were obtained. The Development of the Ascocarp. The ascocarp, in its earliest stages, shows a scolecite of from six to ten usually similar cells arising from a dense tangle of mycelial hyphae. As a rule the scolecite soon becomes very much curved over, and a differentiation of the cells takes place, those towards the middle of the branch becoming considerably larger than the rest, and the whole structure at the same time rapidly covered by a sheath of branched hyphse. In section the cells of the young scolecite are seen to be rich in protoplasm, and to contain each a single large nuctetis. This stage was rarely found, and probably lasts only a short time, the cells soon becoming multinucleate ; figure 3 shows a young scolecite in which the nuclei have already begun to divide, and in figure 4 each cell contains several nuclei. As the cells become multinucleate they increase in size and the ascocarp is seen to consist of a many layered sheath, surrounding the archicarp, of which one cell, generally the fourth from the apex, is larger than the rest and shows small outgrowths which eventually develop as the ascogenous hyphae. This is therefore the ascogenous cell and corresponds morphologically to the ascogonium of such forms as Hutnaria grannlata. The transverse walls of the scolecite are perforated medianly by large circular pores; these are shown in figures 5 and 6, that in figure 6 measuring 5i* in diameter. Pores could not be identified in the earliest stages of development and it seems possible they may be of secondary formation. The nuclei with the cytoplasm of the several cells pass through the pores till they reach the ascogenous cell, where they fuse in pairs (figures 7, 8, 9). The fusion nuclei pass into the ascogenous hyphae (figure 10) and the ascogonium becomes

5 Fertilisation in Ascobolus furfuraceus Pers. 159 emptied of its contents, though a little vacuolate cytoplasm with scattered nuclei remains for a time. The empty scolecite eventually collapses. The ascogenous hyphje develop rapidly, their apices bend over, a terminal and a penultimate cell are cut off and the two nuclei in the latter fuse to fgrm the definitive nucleus of the ascus from which the spore nuclei are derived. The development of the ascus was not studied in detail, as it has already been fully described by Harper (8). GENERAL CONCLUSIONS. Among Ascomycetes the union of the pronuclei in fertilization was first observed by Harper (10) in 1896 for Sphaerothecd Iiniiiuli. The sexual organs are here uninucleate and the fertilized oogonium gives rise to a row of cells. These with the exception of the penultimate binucleate cell, are uninucleate ; they are regarded by Harper as an " ascogonium," but by Blackman and Fraser (3) as a single ascogenous hypba. A similar fertilization occurs in Erysiphe commnnis. Harper (8) Sind in Pliyllactinia Corylea, Harper (11). The oogonium in these species gives rise to a row of cells, but bere several ascogenous byphae grow out from the penultimate cell and, in Pliyllactinia at least, may arise from the other cells also. In Pyronema conjluens (9) the male and female cells are coenocytic and fusion between a very large number of nuclei takes place in tbe ascogonium which without further development branches freely to form tbe ascogenous byphae. Among parthenogenetic forms, a reduced fertilization has been.observed in Hnmaria grannlata, and Laclmea stercorea. In Lachnea stercorea, Fraser (7) a trichogyne and antheridium are present but not functional; fusion takes place between the nuclei of the ascogonium. Hnmaria granulata, Blackman and Fraser (2), probably shovis a further stage of reduction since a trichogyne and antheridium are not developed. In view of the above facts it seems obvious that the nuclear fusions occurring in the ascogenous cell of Ascobolns fnyftiracens constitute a reduced sexual process. Here, as in Hnmaria granulata, an antheridium is not present. Though no definite knowledge could be obtained of the relationship of the fusing nuclei or indeetl as to whether they necessarily came from the different cells of the scolecite, yet it seems probable that they are more distantly

6 i6o E.J. Welsford. related than are those which fuse in the one-celled ascogonium of Humaria granulata. The nuclei which fuse in the ascogenous cell are partly derived from the neighbouring cells of the scolecite. If these cells be regarded as female, the fusions in Ascobolus /ur/uraceus are quite comparable to those seen Humaria granulata and Lachnea stercorea or to the association of female nuclei in pairs flrst observed by Christman (4) in Phragmidium speciosum. If however the cells of the scolecite be vegetative, the fusions in Ascobolus fur/uraceus are to be related, rather, to the fertilization of a female by a vegetative nucleus which occurs in Phragmidium violaceum, Blackman (1). I have to thank Miss H. C. I. Fraser, at whose suggestion this work was undertaken, for valuable help and criticism throughout its progress. REFERENCES. 1. Blackman, V. H. "On the Fertilization, Alternation of Generations and General Cytology of the Uredinex." Ann. Bot., XVIII., 1904, p Blackman, V. H., and Fraser H. C. I. " Sexuality and Development of the Ascocarp in Humaria granulala, Quel." Proc. Roy. Soc. Ser. B. LXXVII., 1906 p Blackman, V. H., and Fraser, H. C. I. " Fertilization in Sphaerotheta." Ann. Bot. XIX., 1905, p Christman, A. H. " Sexual Reproduction in the Ru^s." Bot. Gaz. XIX., 1905, p Claussen, P., ** Zur Entwickelungsgeschichte der Ascomyceten Boudiera." Bot. Zeit. LXIII., 1905, pi Dangeard, P. A. " Recherches sur le O^veloppement du Perithece chez les Ascomycltes." Le Botaniste. 9me. ser., Fraser, H. C. I. 8. Harper, R. A. " On the Sexuality and Development of the Ascocarp in Lachneastercorea Pers." Ann. Bot. July ' Ueber das Verhalten der Kerne bei der Fruchtentwickelung einiger Ascomyceten." Jahr. f. wies. Bot. XXIX., Harper. R. A. " Sexual Reproduction in Prronema coiifiucns," etc. Ann. Bot. XIV., 1900, p. 321.

7 Fertilisation in Ascobolus furfuraceus Pers. i6i 10. Harper, R. A. Die Entwicklung des Peritheciums bei Sphaerotheeaeeulagnei." Ber. d. Deutsch. Bot. Gesell. XIII., 1895, p Harper, R. A. " Sexual Reproduction and the Organization o{ the Nucleus in Certain Mildews," Carnegie Institution of Washington. 12. Janczewski, E. " HlorpholonicdeaAseobolus/urfuraceiis." Bot. Zeit. XXIX., 1871, p Woronin, M. "Zur Entwicklungsgeschichte des.ascobolus pulcherrimut Cr., u.s.w." Beitr. rur Morphol. u. Physiolog. : der Pike, Zweite Reihe, DESCRIPTION OF FIGURES ON PLATE IV. ILLUSTRATING MISS E. J. WELSFORD'S PAPER ON "FERTILISATION IN ASCOBOLUS FURPURACEJJS. Fig. 1. Young Ascocarp. From fresh material. (xi500). Fig. 2. Mycelial hyphae showing numerous nuclei. (x 1250). Fig. 3. Transverse section of a young ascocarp in which the cells of the scolecite are binucleate. (x 625). Fig. 4. Transverse section ot a slightly older asocarp. visible in the cells of the scolecite. (x &is) NunMrous nuclei Fig. 5. Longitudinal section of a scolecite showing passage of cytoplasm through the pores, (x 625). Fig. 6. Oblique section of scolecite showing pore. ( x 625.) Fig. 7. Scolecite showing migration of nuclei and cytoplasm. (x 1250). Fig. 8. Longitudinal section of scolecite showing nuclear fusion in ascogenous cell. Nuclei shown in which fusion is complete and others i«which the two nucleoli are still separate. ( x 1250)..Fig. 9. A single fusion nucleus and another in which the nucleoli have not fused, (x 1250). Fig. 10. Longitudinal section of scolecite, fusion nucleus in-an ascogenous hyphae. (x 625). Fig. II. A chlamydospore. From fresh material. (x625).

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