INHERITANCE BY TETRAD SIBS IN SPHÆROCARPOS.

BY CHARLES E. ALLEN.

(Read April 25, 1924.)

In Sphærocarpos Donnellii Aust., as in some other members of the same genus, the four spores formed by the division of a single spore mother cell are (certain strains excepted) regularly held together at maturity. If the spores of such a tetrad germinate, four haploid plants (gametophytes), and ultimately four clones, are produced whose genotypic constitutions result from the distribution of chromosomes in the course of the reduction divisions that formed the spore nuclei. Sphærocarpos thus offers a special opportunity for observing what is virtually the immediate result of the segregation of chromosomes in the reduction divisions.

To realize the advantage here presented, it is necessary to secure germination of a large proportion of the spores sown, and to grow the resultant clones to a size sufficient for the satisfactory determination of their characters. The genetic work thus far done with Sphærocarpos has encountered serious difficulties in the germination of spores and the growing of sporelings. Two sets of germinations, as reported in a previous paper (Allen, 1919), were sufficiently successful to give some information as to the inheritance of sex and of characters closely associated with sex. But in the study of the inheritance of other characters, the most extensive results, reports on some of which are now in press, have until very recently been obtained by sowing the spores or spore tetrads broadcast upon soil, and by later isolating the sporelings. By this method it may be learned what genotypes occur, and in approximately what proportions, among the offspring of a given mating-results comparable with those obtained in the genetic study of other organisms-but not what is the distribution of the different genotypes within a single tetrad.

During the last two years, however, the use of improved methods has resulted in a larger proportion of germination on the part of the

spores of isolated tetrads. While the results that can be reported at present are still comparatively meager, and while some statements as to the character of individual plants must still, as will appear, be made in tentative form, yet the facts at hand are sufficient to indicate the possibilities inherent in this method of analysis of inheritance. The sowings of spore tetrads whose results are here recorded were made in the autumn of 1922; the sporelings were separated during the early months of 1923 and transplanted to individual pots, where they developed into clones.

In these studies I have had the valued assistance of Mr. James A. Lounsbury, and the work has been carried on with the aid of an appropriation from the research fund of the University of Wisconsin.

POLYCLADY AND Sex.

Among the divergent races of Sphærocarpos Donnellii whose inheritance has been studied is one to which the term "polycladous" is applied. Polycladous plants of either sex are characterized by profuse and irregular branching, by marked variation in the form of the lateral lobes of the thallus, and by the occurrence of dorsal lobes and cilia. Antheridia are fewer than on typical male plants, sometimes lacking altogether on many, or even on all, of the plants of a clone. The antheridial involucres are, with rare exceptions, reduced in various ways, leaving the antheridia in consequence partly or entirely exposed. In vegetative structure, apart from their greater average size, polycladous female plants resemble the males. Only in very few instances have these female plants been observed to bear archegonia, and even when present archegonia have proved functionally sterile. The rare archegonia are enclosed in involucres more or less closely approximating those of typical form; elsewhere the polycladous females bear, in varying abundance, solid columnar upgrowths which seem to correspond to involucres-containing, however, no structures that suggest archegonia.

If a typical female is mated with a polycladous male, the gametophytic offspring (in the f, generation) consist of four classes: typical females, polycladous females, typical males, and polycladous males. The four classes occur, as previous studies have shown, in

at least approximately equal numbers. The sterility of polycladous females, of course, makes the reciprocal cross impossible.

Table I. gives the results of the germination of spores of 29 spore tetrads borne by two sporophytes (14 and 15 tetrads respectively) produced by the mating of typical females with polycladous males. The distribution of characters within the two families was so similar that, for economy of space, both are tabulated together. Only those clones are included in this classification, as well as in that of Table II., which grew to a large enough size to permit of a satisfactory determination of their character.

The facts summarized in Table I. make possible the formulation of certain general rules.

First, the facts harmonize with the expectation that, of the spores of a tetrad, two will carry the female, and two the male, potentiality. The results in this respect agree with those previously reported (Allen, 1919), as well as with the observation that in spore-formation two of the spores of a tetrad receive the X-chromosome character

istic of females and two receive the Y-chromosome characteristic of males.

Second, the results indicate that two of the spores of each tetrad carry the potentiality for the production of the typical (non-polycladous), and two that for the production of the polycladous, character. In terms of current genetic conceptions, this implies that the appearance or non-appearance of the polycladous character is determined by the distribution of physical bases borne by the chromosomes of a single pair. In the course of the reduction divisions, a chromosome bearing the physical basis necessary to the appearance of polyclady passes to two of the four spores of each tetrad; the other two spores receive a different chromosome belonging to the same pair, and consequently the physical basis corresponding to non-polyclady.

Third, in all except the one instance noted as an "exception," the results are in harmony with a supposition that the four spores of a tetrad give rise either to two typical females and two polycladous males (classes a-e, Table I.), or to two polycladous females and two typical males (classes f−j), each tetrad in either case being composed of two and only two types of spores. However, classes d, h, and j, in each of which a clone was secured from only one spore of a tetrad, and classes c and g, in each of which one clone of each sex appeared, are not conclusive upon this point. Omitting these classes, it appears that there were 7 tetrads (classes a, b, and e) composed of two typical and two polycladous male spores (or, more strictly, of spores bearing the physical bases for these respective characters); and 3 tetrads (classes f and i) composed of two polycladous female and two typical male spores. These numbers, though small, suggest that there is probably no linkage between polyclady and sex-that is, that the physical bases determining the appearance or non-appearance of polyclady are borne on a pair of chromosomes other than the X-Y pair. This conclusion had been previously reached on the basis of more extensive studies of plants resulting from the broadcast sowing of spore tetrads.

Fourth, there was one clear case (class k, Table I.) in which the character-distribution was such that the four spores of the tetrad represented as many different genotypes, and therefore all the combinations possible when two pairs of characters (in this case, female

male and polycladous-typical) are concerned. In classes c, d, g, h, and j, the possibility of a similar distribution of factors is not excluded. But, as already seen, it is reasonably certain in 10 cases. that the spores of each tetrad fell into only two classes, as compared with one case in which the spores were certainly of four types. It seems safe, therefore, to conclude that, so far as the physical bases of the characters here studied are concerned, in the majority (and) probably the large majority) of cases the reduction divisions proceed in such a manner as to result in the production of two and only two differently constituted types of spore nuclei; but that in some (probably few) cases, these divisions bring about the formation of four qualitatively different spore nuclei. The bearing of this conclusion upon the problem of the method of segregation of the chromosomes in the course of the reduction divisions will be considered after the distribution of a second pair of characters has been described.

TUFTEDNESS AND SEX.

Another character distinguishing certain strains is designated as "tufted." Tufted strains, both male and female, are characterized especially by aberrancies of varied nature and degree in involucral form. Some branches of a tufted clone bear extremely atypical involucres; other branches of the same clone display a mixture of typically and atypically formed involucres; while still other branches depart in no noticeable way from a strictly typical appearance. There is strong reason for thinking that different tufted clones differ in the magnitude of their inherent tendency to produce aberrant involucres; although, in view of the variations from typical to extremely aberrant forms within a single clone, it is difficult to determine to what extent differences of this nature between clones are genotypic and how far merely phenotypic.

Matings of tufted females by typical males, as previous work has shown, result in f1 offspring of four classes: typical females, tufted females, typical males, and tufted males. The reciprocal cross (Table II.) gives the same four classes among the f, gametophytes. The tufted offspring of such crosses show marked differences in degree of tuftedness, ranging from clones of the total number of whose branches produced during a period of two or three years only a frac