LECTURE XVI

FERTILIZATION IN PLANTS AND UNICELLULARORGANISMS, AND ITS IMMEDIATESIGNIFICANCE

Fertilization in a lichen, Basidiobolus—In Phanerogams—Here too there is reduction of the number of chromosomes by a half—'Polar cells' in lower and higher plants—Conjugation among unicellular organisms—Noctiluca—The maternal and paternal chromosomes remain apart—Actinophrys—Infusoria—Sexual differentiation of the two conjugates in Vorticella—Importance of the process of Amphimixis—Not a 'life-awakening' process—May occur independently of multiplication—The Rejuvenescence hypothesis—Pure parthenogenesis—The cycle idea—Does Amphimixis prevent natural death?—Maupas' experiments with Infusorians—Bütschli's view—Potential immortality of unicellular organisms—The immortality of unicellular organisms and of the germ-cells depends on the fact that there is no time-limit to the multiplication of the smallest living particles—Parthenogenesis is not self-fertilization—Petrunkewitsch's observations on the ova of bees—Is the chromatin really the 'hereditary substance'?—Nägeli's conclusion from the difference in size between ovum and spermatozoon—Artificial division of Infusorians—Boveri's experiments with the fertilization of pieces of ova not containing a nucleus—Fertilization gives an impulse to development even to non-nucleated pieces of ova—Merogony—The female and male nuclear substances are essentially alike—Summary.

I nowturn to the consideration of the process of fertilization in plants and unicellular organisms.

With regard to plants, it can now be definitely asserted that in them, too, fertilization is essentially a conjugation of nuclei; it depends on the union of the nuclei of the two 'sex-cells.' These sex-cells are usually very small among lower plants, indeed up to the phanerogams; this is especially true of the zoosperm-like male germ-cells, but it usually holds also true of the ovum, which is but seldom burdened with an abundant supply of yolk. In spite of the many difficulties which this smallness of size puts in the way of observation, the untiring exertions of a host of excellent investigators have succeeded in following the process of fertilization in all the larger groups of plants—in algæ, fungi, mosses, ferns, and horse-tails among cryptogams, and in phanerogams.

I shall first give an example from among the lower plants (Fig. 81). In one of the lichens,Basidiobolus ranarum, each of two adjacent cells in the fungus-thread gives off a bill-like process, and thetwo processes become closely apposed (Fig. 81,a). The nucleus of each cell moves into the bill-shaped process, is there transformed into a nuclear spindle (B,ksp) and divides, so that one daughter-nucleus comes to lie in the apex point of the bill, the other at the base. The cell-body also divides, though very unequally, and the final outcome of the process is two cells in each, of which one is small and occupies the apex of the bill, while the other is large and fills all the rest of the cell-space. The former do not play any further part of importance, but break up, the latter are the sex-cells, the cytoplasm of which now coalesces through a gap in the cell-walls, while their nuclei become closely apposed and ultimately unite (C, ♂ and ♀k). From this union arises the fertilized spore, the so-called 'zygote' (D). The two small abortive cells so greatly resemble in their origin the polar cells of the animal ovum that it is difficult to resist the supposition that they bring about a reduction in the number of chromosomes. But the number of the chromosomes has not yet been determined either in them or in the sex-nuclei.

Fig. 81.Formation of polar bodies in a lichen,Basidiobolus ranarum.A, the two conjugating cells with the bill-like processes in which the nuclei lie.B, the nuclei dividing.ksp, the nuclear spindle.C, after the division into a polar body (rk) and a sex-nucleus (♂kand ♀k).D, after the union of the nuclei to form a conjugation nucleus (copk); the fertilized ovum is surrounded by envelopes and modified into a lasting spore. After Fairchild.

We have come to know the processes of fertilization among phanerogams chiefly through Strasburger, Guignard, and more recently through the Japanese botanist Hirase. The agreement with the animal process is surprisingly great, notwithstanding the notable differences in the external conditions of fertilization.

As is well known, the male cells in the highest flowering plants are not zoosperms but roundish cells, each of which, enclosed, together with a sister-cell—the so-called 'vegetative' cell—in a thick cellulose capsule constitutes a pollen-grain. The pollen-grains reach the stigma, under which, buried deep within the 'ovule,' the female sex-cell rests, enclosed in a long, sac-like structure called the 'embryo-sac' (Fig. 82,A). Beside it (eiz) there lie several other cells, usually seven in number, two of which, the so-called 'synergidæ' (sy), have their place at one end of the embryo-sac, just in front of the ovum (eiz). Probably these give off a secretion which exercises an attractive (chemotactic) influence on the male fertilizing body ('the pollen-tube'), and thus, so to speak, show it the way to the ovum.

Fig. 82.Fertilization in the Lily,Lilium martagon, after Guignard.A, the embryo-sac before fertilization;sy, synergidæ;eiz, ovum;opandup, upper and lower 'polar nuclei';ap, antipodal cells.B, the upper part of the embryo-sac, into which the pollen-tube (pschl) has penetrated with the male sex-nucleus (♂k) and its centrosphere; below that is the ovum with its (also doubled) centrosphere (csph).C, remains of the pollen-tube (pschl); the two sex-nuclei are closely apposed. Highly magnified.

When a pollen-grain has reached the stigma it sends out a tube, usually after a few hours, which penetrates into the soft tissue of the style, and grows deep down into the interior of the ovule, ultimately penetrating as far as the embryo-sac through a special little opening in the covering of the ovule, the so-called 'micropyle' (Fig. 82B,pschl). Its blunt end is now closely apposed to this, so that the true sperm-nucleus (B, ♂k), surrounded by some protoplasm, can leave the pollen-tube and wander in among the cells of the embryo-sac. Later on we shall see that two generative nuclei migrate from the pollen-tube, but in the meantime we shall devote our attention only to oneof them, the fertilizing nucleus, which immediately moves towards the ovum-nucleus and apposes itself closely to it. Then follows the fusion or conjugation of the two nuclei, which are alike in size and appearance, just as in the fertilization of the animal ovum (C, ♂kand ♀k). Whether in this case, too, the sperm-nucleus brings with it a central corpuscle, or whether, as Guignard believed he observed, the ovum retains its central corpuscle (C,csph), or finally, whether both modes occur, is not yet known with certainty. The fact that, as a rule, seeds capable of reproduction only form in an ovule when the stigma has been previously dusted with pollen, leads us to suppose that, in this case, as among animals, the ovum lacks something that is necessary to induce embryonic development, only retaining this power in very exceptional cases, namely, when adapted for parthenogenesis. And this something may very well be the dividing apparatus of the cell, the centrosome with the centrosphere. But whether this supposition prove correct or not, a nuclear spindle always forms simultaneously with the fusion of the two sex-nuclei into a segmentation nucleus, and this spindle is the starting-point of the young plant, thus exactly corresponding to the first segmentation of the animal ovum. It agrees with it also in the important respect that it again contains the full number of chromosomes—twenty-four in the lily—while the two nuclei, male and female, only exhibit half the number each, that is, twelve.

Thus a reduction in the number of chromosomes to half takes place in plants also, but it is not yet known with certainty whether this is brought about in the same way as among animals, namely, by reducing divisions. Without entering more fully into this still unsolved and very complex problem, I should like to state that I consider this very probable; indeed, I agree with the view of V. Häcker[13], that the reducing divisions of plants are only more difficult to recognize as such, and, furthermore, are often disguised by the fact that they often occur alongside of, or between divisions which are not reducing. If it were possible to reduce the number of chromosomes in a cell to half without the aid of cell-division, if, for instance, only half were to integrate again from the chromatin-network, this must have been quite as possible in the case of animal cells, and then, moreover, the single chromosome would not have had the significance of an individuality, and no special form of nuclear division would have been introduced to reduce their number. That it has been introduced seems to me to prove that it was necessary, and since itwas so among animals, it could not have been dispensed with among plants either.

[13]See V. Häcker,Praxis und Theorie der Zellen- und Befruchtungslehre, Jena, 1899, pp. 144-5.

Moreover, throughout the vegetable kingdom divisions often occur in connexion with the origin of the sex-cells which can be compared, in occurrence and result, with the maturation divisions of animal germ-cells. In the lichen,Basidiobolus, we have already seen that an abortive cell separates itself off from the sex-cell before the latter becomes capable of reproduction (Fig. 81,C). Similar cell-divisions occur in many if not in all groups of plants. In the marine algæ of the genusFucusit has even been proved that the division of the first primordial cell of the ovary into the so-called 'stalk-cell' and the primitive egg-cell is a reducing division, and brings down the number of chromosomes from thirty-two to sixteen. In vascular plants the reduction is not postponed until the formation of the sex-cells, but occurs earlier in the formation of the spores, as Calkins has demonstrated for ferns; in the Conifers and other Gymnosperms several so-called 'preparatory' divisions precede the formation of the germ-cells, and we know by comparison with the alternation of generations in vascular plants that these are related to the gradual waning of the strictly sexual generation. As the 'polar bodies' or 'directive corpuscles' of the animal ovum are rudimentary egg-cells, so the cells which, in the pollen-grains, separate themselves from the sex-cells proper are rudimentary Prothallium-cells, and, like the animal cells, they perish without playing any further physiological rôle. I will not assert that it is precisely in these divisions that the reducing divisions are concealed, for the analogy with the spore-formation of ferns leads us rather to suppose that it may lie further back; but in any case there is no lack of opportunity in the ontogeny of phanerogamic plants for the interpolation of a reducing division, and as long as it remains unproved that a reduction of the chromosomes can take place directly, that is, without the help of nuclear division, we shall continue to expect with confidence that the reducing divisions of phanerogams will be discovered in the future. Processes of a similar kind are known among unicellular organisms, and there, too, they are associated with nuclear divisions.

In passing to the so-called 'sexual reproduction' of unicellular organisms, I should like first to call attention to the fact that the expression 'reproduction' is not very suitable in this case, for the process in question does not always effect an increase in the number of individuals as reproduction ought to do, but leads, in fact, in many cases, even to a decrease, when two individuals unite to form one. Even if the phenomena of sexual 'reproduction' among higherorganisms, which we have already studied, had not made it clear to us that there are two associated processes, quite different in nature, the conjugation of unicellular organisms would have led us to that conclusion. It has long been known that two unicellular plants or animals occasionally become closely apposed and fuse; and this process of 'conjugation' was many years ago regarded as an analogue to 'fertilization,' although it is only through the laborious investigations of the last two or three decades that this supposition has been proved to be correct. We now know that a process quite analogous to that which we have learnt to know as 'fertilization' takes place among unicellulars, only in this case it is not directly connected with reproduction and multiplication, but occurs independently of them, and, in its most primitive form, it results, not in an increase but—for a short time at least—in a diminution of the number of individuals. This occurrence of the process independently of reproduction appears to me of inestimable value theoretically, for it frees us completely from the old deep-rooted preconceptions in the interpretation of fertilization.

Fig. 83.Conjugation of Noctiluca, after Ischikawa.A, two Noctilucas beginning to coalesce;pr, the protoplasmdrawn out into processes which traverse thegelatinous substance of the cell;k, the nucleus.B, the cells and their gelatinous substance have fused;the nuclei, in which the chromosomes are visible, areclosely apposed;CK, centrospheres.C, the two nucleiare united in one nuclear spindle; beginning ofdivision.D, completion of the division. Highlymagnified.

First let us briefly sketch the process itself in the main forms of its occurrence.

The most primitive form of conjugation is undoubtedly the complete fusion of two unicellular organisms of the same species, as we see it to-day in unicellular plants, and also among the lowest unicellular animals, such as the flagellate Infusorians, Gregarines, and Rhizopods. It is well seen, for instance, in the Noctilucæ, those unicellular flagellate organisms which cause the familiar marine phosphorescence extending uniformly over wide surfaces of water (Fig. 83). In these forms Prof. Ischikawa of Tokio was able to trace the whole process of conjugation. To begin with, two Noctilucas range themselves side by side (Fig. 83) and coalesce at the surfaces in contact, both as to the spherical gelatinous envelope (A,G) and the protoplasm (pr) itself, which branches in amœboid fashion into the jelly. The union becomes gradually complete, and the two animals form a single sphere (B) with one cell-body. But the two nuclei (K) also place themselves side by side (B), and though they do not actually fuse, they form together, under the guidance of two centrospheres (C), a single nuclear division-figure, which is obviously analogous to the segmentation spindle of the fertilized egg. Then follows a division, by means of which the chromatin substance of the nuclei of both animals is divided between the two daughter-nuclei, and after this has been accomplished the united individual again separates into two independent Noctilucas (D). Although I have spoken here—that is, in referring to the Protozoa—of chromosomes, I must immediately add that these have not yet been seen with full clearness in Noctiluca itself; nothing more has been recognized than deeply staining thickenings of the spindle fibrils, which move from the equator of the nuclear spindle towards the pole. Since, however, in other Protozoa, as, for instance, in the beautiful freshwater Rhizopod (Euglypha alveolata), these thickenings of the nuclear spindle fibrils have been clearly recognized as chromosomes, doubt on this point is hardly justifiable. Apart from this, the assumption that each of the two daughter-nuclei receives half the chromosomes of each of the conjugated nuclei rests on a secure basis, not only because otherwise the whole process would have no meaning, but because the position of the mitotic figure conditions this. Even the fact that the two conjugation-nuclei lying side by side remain apart during nuclear division is not without parallel; Häcker and Rückert observed it also in the segmentation-nucleus of much higher animals, the Copepods, and it has no effect in altering the process of division, but only proves that the chromosomes of maternal and those of paternal origin in the combination-nucleus remain independent—a fact the significance of which I shall discuss later on.

The process of conjugation occurs, in the same manner as inNoctiluca, in a freshwater Rhizopod, the well-known Sun-animalcule,Actinophrys sol(Fig. 84), but in this case complete fusion of the two nuclei takes place (Fig. 84,V) before the formation of the division-spindle (VI,sp), which, with the simultaneous division of the cell-body, gives rise to two new individuals. The process in this case is especially interesting, because Schaudinn has succeeded in observing a maturation division (III,Rsp, directive spindle) as well as in demonstrating polar bodies (IV,Rk). Thus the analogy with theprocess of fertilization in the Metazoa and the Metaphyta is almost complete.

But that the conjugation of unicellular organisms, like the fertilization of multicellular organisms, is essentially a matter of nuclear conjugation is shown more distinctly still by the ciliated Infusorians, the most highly organized of the Protozoa.

Fig. 84.Conjugation and polar body formation in the Sun-animalcule,Actinophrys sol, after Schaudinn.I, two free-swimming conjugated individuals, which inIIhave become surrounded by a transparent gelatinous cyst.III, formation of the directive spindles (RSp).IV, the polar bodies are formed (RK);K, the two sex-nuclei.V, these are fused to form the conjugation-nucleus (K).VI, the conjugation-nucleus is transformed into the division-spindle; the polar bodies (RK) have penetrated the internal cyst-wall, and are in process of degeneration.

Here there is usually no complete union of the cell-bodies of the two animals, but only an adhering of the apposed surfaces. In the relatively largeParamœcium caudatumthe process of conjugation is very exactly known through the beautiful investigations of Maupas and R. Hertwig. In this case the mouth-surfaces of the two animals come together and unite over a short area, and then the two animals swim about together in this conjugated state. During this time very remarkable changes take place in their nuclei.

It is well known that these Infusorians have a double nucleus, a large one, the macronucleus (Fig. 85,ma), and one which is usually very small, the micronucleus (mi). We may ascribe to the former of these the guidance and regulation of the everyday processes of life, that is, briefly, of metabolism, and the preservation of the integrity of the whole animal. The small nucleus has often been designated the 'reproductive nucleus,' but as it plays no other part in reproduction, as far as can be recognized, than that of dividing into two daughter-nuclei, I cannot regard this designation as suitable; it obviously originated in the mistaken interpretation, prevalent till very lately, of conjugation as a 'kind of reproduction,' and this in its turn depends on the conception, transferred from multicellular organisms, of fertilization as a 'sexual reproduction.' We shall immediately see that the micronucleus plays the main part in conjugation, and from this we may suppose that it otherwise fills no rôle in the life of the animal, and therefore it may best be designated the 'supplementary' or reserve nucleus. In every conjugation the macronucleus, which has hitherto been active, breaks up and becomes completely absorbed, very much like a ball of food. This of course takes place slowly; the large nucleus elongates, becomes indented, falls into several pieces, and these are so gradually absorbed that, even after the act of conjugation has been accomplished, irregular fragments of the macronucleus often lie about in the animal (Fig. 85, 9).

But while the macronucleus falls to pieces the previously minute micronucleus grows enormously and forms a distinct longitudinally striated spindle (1,mi). About the same time these divide in both animals, and each of the daughter-nuclei immediately divides again, so that after these two divisions four spindle-shaped descendants of the micronucleus are to be seen in each animal (Fig. 85, 4). We have previously noted that the apparatus for nuclear division in unicellular organisms was similar to that in multicellular organisms, and yet was different from it. In these ciliated infusorians we see an essential difference, for the striated spindle, after the division into daughter-chromosomes has taken place, lengthens out enormously, and becomes so thin in the middle of its length (2) that the two daughter-nuclei at the ends of this long stalk suggest the appearance of a very long and thin dumb-bell, or of a long silk purse. Of asters (centrospheres) there is nothing to be seen, and the mechanism of division is still very obscure; it almost seems as if a rapidly growing substance forced the two groups of chromosomes apart.

Hardly have these four descendants of the micronucleus arisen when three of them begin to break up and very shortly disappear; only the fourth is of any further importance, and it divides once more (5), and so gives rise to the two nuclei which play the chief part in the process of conjugation—the copulation-nuclei, exactly analogous to the male and female pronuclei in the fertilized ovum (5,mi4). But in this case each of the two animals functions doubly, that is, both as male and female, for each sends one of the two copulation-nucleiacross the bridge formed by the union of the apposed surfaces into the other animal (6,mi♂), so that it may form, by union with the nucleus which has remained there, a double nucleus (7), a structure which corresponds to the segmentation nucleus of the ovum (copk). From it there then arises by division a new macronucleus and a new micronucleus, not usually directly, however, that is, not by a single division, but through several successive nuclear divisions, into the meaning of which I cannot here enter. Immediately after the union of the two sex-nuclei the two animals sever their connexion with each other; each begins again to feed, and is subject to multiplication by division just as it was before conjugation took place (8 and 9).

Fig. 85.Diagram of the conjugation of an Infusorian,Paramœcium, after R. Hertwig and Maupas. 1, two animals with the mouth-openings apposed;ma, the macronucleus beginning to degenerate;mi1, the micronucleus has already increased considerably in size and is beginning to divide. 2. each micronucleus has divided into two daughter-nuclei (mi2), which are connected only by the division-strand (ts). 3, to the left each of the daughter-micronuclei (mi2) is beginning to divide; to the right this division is already completed and the grand-daughter-nuclei of the original micronucleus hang together by their division-strands (ts). 4, in each of the animals there are now four grand-daughter-micronuclei (mi3). 5, three of these are in process of dissolution, the fourth is dividing into two great-grand-daughter-nuclei (mi4), which are the two sex-nuclei. 6, one (the male) sex-nucleus (mi♂) migrates into the other animal, and there unites with the remaining (female) sex-nucleus. 7, the conjugation-nucleus (copk) being formed. 8, the animals have separated; the conjugation-nucleus divides into (9) the new macronucleus (n ma) and the new micronucleus (n mi).

Although the course of this remarkable process exhibits all manner of differences in detail in different species, it is everywhere the same in its essential feature, and this essential feature is undoubtedly the union of an equal quantity of the nuclear substance of two animals to form a new nucleus. It is thus essentially the same process which we have already recognized among higher animals as 'fertilization.' The differences are of minor importance, and they arise partly from the fact that the sex-cells of multicellular animals are not independent self-supporting units, and partly from their differentiation into 'male' and 'female' cells. The minuteness of the sperm-cell, for instance, conditions its penetration of the ovum, which is always much larger and passive, and also the thorough fusion of its cell-body with the cell-body of the ovum. That this difference has very little deep significance is best seen from the fact that, even among Infusorians, there are forms in which the two conjugating individuals are quite different, especially in size, and in which the much smaller 'male' animal fuses completely with the much larger 'female,' and indeed bores its way into it after the manner of a sperm-cell. This is the case among the bell-animalcules (Vorticellinæ) (Fig. 86), the conjugating pairs of which had been observed long before our present insight into these processes had been attained. Indeed, the facts had been interpreted as a kind of 'budding process,' the minute and differently shaped 'male' animal (mi), which at the time of conjugation is attached to the larger 'female' (ma), was regarded as its bud. This supposed bud, however, does not grow out from the animal, but into it!

Thus we see here again that a differentiation of individuals as males and females may occur among unicellular organisms, just as in the sex-cells of higher animals and plants, and this proves to us once more that all these differences of sex, whether in reproductive cells of multicellular organisms, or in the entire multicellular animal or plant,or finally, in unicellular organisms, are not of essential, but only of secondary significance, however important they may be for securing fertilization or conjugation in each special case. They are always only adaptations to the special conditions, and only occur where they are necessary to ensure the union, and always in such a manner that the union of the two cells is facilitated. In most Infusorians such a differentiation into male and female animals was not necessary, because these organisms are very motile, and are thus readily able to meet and unite; it was therefore sufficient for them to remain hermaphrodite. The bell-animalcules, however, are sedentary, and for them it was obviously an advantage that, at the time of conjugation, smaller, free-swimming, and also more simply organized individuals should arise, which were able to seek out the larger sedentary forms. Here, then, as in many other unicellular animals, these little male individuals only occur when they are necessary, that is, at the time of conjugation. Similarly, in the green alga,Volvox, male and female cells arise only at the time of conjugation, reproduction being at other times effected by means of parthenogonidia, that is, by elements which require no fertilization.

Fig. 86.Conjugation of an Infusorian.Vorticella nebulifera, showing sexual differentiation of the whole organism. After Greef.I, the 'microgonidium' or male individual (mi) attaches itself to the 'macrogonidium' or female individual (ma);cv, contractile vacuole;st, contractile stalk.II, the ciliated circle on the male individual has disappeared. The male has become firmly embedded in the female by means of a sucker-like retraction of its lower end.III, the fusion of the two individuals has been completed; the bristly residue of the male (ct) is about to be thrown off; the stalk (st) is contracted into a spiral. Magnified about 300 times.

As these differences are only adaptations to the necessity that the animals or cells shall find each other and unite, so also are all the other differences of a sexual kind, the thousand-fold differences between the sperm-cell and the egg-cell, and the not less numerous differences between male and female animals, both in 'primary' and especially in the diverse 'secondary' sexual characters which we havepreviously discussed; all these are only means for bringing about the process of the union of two germ-cells to form a fertilized 'ovum' which is capable of development. The essential part of this so-called 'sexual reproduction' does not, however, depend on these differences, neither on the sexual differences of the germ-cells nor on those of the whole organism; it lies solely in the actual union of the two germ-cells. Let us remember the idea we have already emphasized, that theessential partof the so-called 'sexual reproduction' does not depend on these differences, and let us hold fast to the idea already indicated, that the chromosomes of the nucleus are the real bearers of the hereditary tendencies; then we see that the mingling, or, better, the union of the hereditary substances of two different individuals, whether single-celled or many-celled, is the result of the process which we have hitherto called fertilization or conjugation, but which we shall henceforward designate by the more general term 'Amphimixis' which means the mingling of substances contributed from two distinct sources.

Having made ourselves acquainted with the phenomena of amphimixis in animals, plants, and unicellular organisms, we have to face the problem of the significance of this remarkable and complicated process. What is it that happens, and what meaning can we attach to it?

The first thing to be done is to show that the old and long-prevailing conception of fertilization asa life-awakening processmust be entirely abandoned. That a new individual can arise even among highly organized animals, quite independently of fertilization, is proved by the parthenogenetic eggs of insects and crustaceans; fertilization is not the spark 'which falls into the powder-cask' and causes the explosion; it is only an indispensable condition of development. As we have seen, there are germ-cells which are not sexually differentiated, such as the spores of the lower plants, which are all capable of development without amphimixis; and parthenogenetic ova prove that even differentiated female germ-cells, that is, germ-cells originally adapted for amphimixis, may in certain circumstances develop without it; amphimixis is thus not the fundamental cause of development, but is only, for many germ-cells, one of the conditions which must be fulfilled before development can set in. It is a condition which, under certain circumstances, may be dispensed with.

If, then, the multiplication of individuals by single-celled germs can take place independently of amphimixis, we may conclude that the establishment of amphimixis has nothing to do with the capacityfor multiplication, that it is not a life-awakening process, but is a process of a unique kind, which means something quite different. The whole conception of the awakening of life in the germ is antiquated and out of harmony with the present state of our knowledge.Life never begins anew, as far as we can see, and apart from the possibility that, unknown to us, a spontaneous generation (Urzeugung) of the lowest forms of life is still taking place, life is continuous and consists of an infinite series of living forms between which there is no real interruption. Life, in fact, is like a continuous stream, the larger and smaller waves of which are particular species and individuals. Only a few decennia ago a morphologist, who was rightly held in high esteem, could champion the idea that the mature ovum of animals was lifeless material, which had to be quickened in order to develop, but now such a theory is untenable, since we have become aware of the phenomena of maturation in the ovum, and know that most important vital processes, the reducing divisions, take place at the time of maturation, quite independently of fertilization.

Thus we do not even require to take into account the conjugation of unicellular organisms to make it clear that amphimixis is not the cause of the origin of new individuals, but a process,sui generis, which may indeed be associated with the beginning of embryonic development, but which may also occur independently of it, as we see in the case of unicellular organisms. If, on the one hand, we see development taking place in spores and parthenogenetic ova independently of amphimixis, and on the other hand amphimixis occurring without reproduction in unicellular organisms, we must regard the two phenomena, amphimixis and reproduction, as processes of a distinct kind, which may, however, occur in association with and interdependence upon each other.

It was by chance that human observation brought the latter fact to light first, and therefore led us for so long to accept the idea thatfertilization, that is, amphimixis, anddevelopment, that is, reproduction, are one and the same; and thus it happens that even now there are many naturalists who cannot rid themselves of the idea that amphimixis, if not a life-awakening, is at least alife-renewingprocess, a so-called 'process of rejuvenescence.'

More than ten years ago[14]I disputed this view, and since then the facts which make it untenable have become more and more clear. Notwithstanding this I see that it is still adhered to, at least in amodified form, by many esteemed naturalists, and so it does not seem superfluous to discuss it in more detail.

[14]Die Bedeutung der sexuellen Fortpflanzung für die Selektionstheorie, Jena, 1886.

I have already noted that we see in conjugation an amphimixis without reproduction, and in spores and parthenogenetic ova reproduction without amphimixis, and I do not doubt that every unprejudiced critic will admit this; many among us, however, are not unprejudiced, but are under the spell of earlier ideas, so that they cannot forget that it was long believed that fertilization was an indispensable condition of development; they therefore regard the divisions which recommence sooner or later after conjugation, and which may be repeated hundreds of times,as conditioned by the conjugation which preceded them, and compare them to the series of cells which, in the Metazoa, lead from the fertilized ovum to the fully-formed animal. They regard both series of cell-generations as a developmental cycle, which leads from fertilization to fertilization again, from conjugation to conjugation, and which would be impossible without either fertilization or conjugation.

This play with the idea of a 'cycle' reminds me vividly of similar fantastic play from the time of the much-despised 'Naturphilosophie' of a hundred years ago. As men sought to find the analogues of 'solar' and 'planetary' systems in animal and plant, and believed they had stated something when they compared the motile animals to planets and the sedentary plants to the sun (!), so it is now imagined that a deeper insight has been gained by the recognition of cycles of development. By all means let us regard the development of a multicellular organism as cyclic; it returns again to its starting-point, but this no more explains the forces which produce the cycle, and thus the meaning of fertilization, than a comparison with the circling planets explains the causes of locomotion in animals. With quite as much reason the cycle of development might be made to start from the parthenogenetic ovum, and then the whole conclusion of the fanciful cycle idea in regard to the meaning of fertilization falls to the ground, for in this case the cycle begins without fertilization. Attempts are made to get over this difficulty by showing that in many cases parthenogenesis alternates regularly or irregularly with sexual reproduction, as in the water-fleas (Daphnids), the Aphides, and so on. The mysterious rejuvenating power of amphimixis is supposed to suffice for several generations, a purely gratuitous assumption, which is also in open contradiction to the facts. For there are species which now reproduce exclusively by parthenogenesis, among plants for instance, a number of fungi, among animals a few species of Crustaceans. Of the latter it can be demonstrated that ages ago theyreproduced sexually, for they still possess the sac which serves for receiving spermatozoa, but this sac remains empty, for there are now no males, at least in any habitat of the species known to us. To this set belongs an inhabitant of stagnant water,Limnadia hermanni, a species of Crustacean which was found thirty years ago in hundreds, all of the female sex, near Strassburg, and also many of the little Ostracods (Cypris) which inhabit especially the muddy bottom of our pools and marshes. I bred one of these (Cypris reptans) in numerous aquaria for sixteen years, during which there were about eighty generations, and throughout this time no male ever appeared, nor did the sperm-sac of the female ever contain spermatozoa. The after-effects of the 'rejuvenating' power of an amphimixis supposed to have taken place earlier must in this case have been enduring indeed!

For these reasons it seems to me useless to make comparisons between the developmental cycle of unicellular organisms and the ontogeny of multicellular organisms. Both processes have indeed many points of resemblance—long series of cells, then interruption of the divisions and the occurrence of amphimixis—so that we may quite well speak of cyclic development in the physiological sense, in as far as certain internal conditions periodically recur and compel the organism to conjugation, but we must not suppose that there is more in this than, for instance, in the 'cyclic development' of Man, which consists in the fact that he finds himself periodically impelled to take food. The feeling of hunger which forces him to do so is the signal which warns the organism that it is time to supply fresh combustible material to the metabolism. In the same way, after a long series of generations of Infusorians the necessity for conjugation arises; the whole colony suffers an 'epidemic of conjugation,' and the animals unite in pairs; in the meantime we know not why, and must content ourselves with formulating what is observable, thatthe nuclear substances of two individuals are thereby mingled in each conjugate.

Obviously the impulse to conjugation is a signal in the same sense as the feeling of hunger is, and we know well from the higher animals what a mighty influence it exerts, an influence hardly less potent than that of hunger. In Schiller's words, 'Durch Hunger und durch Liebe, erhält sich dies Weltgetriebe.'

We can see clearly enough why Nature should have given animals the feeling of hunger, but the reason for the need of conjugation is not so plain; we can only say in the meantime that it must be of some value in maintaining the forms of life, for only that which fulfils a purpose can be permanently established.

I shall return later to the problem of the meaning of 'sexualreproduction,' and try to probe more deeply into the meaning of its establishment; in the meantime I must restrict myself to having shown its significance in the union of the hereditary substances of two individuals, and at the same time to controverting the theory of the 'rejuvenating power' of amphimixis. I use this expression in its original sense, which indicates that every life is gradually wearing itself away and would become extinct were it not fanned to flame again by amphimixis—by an artifice of Nature, we may say. This conception rests on the fact that the cells of the multicellular body possess for the most part only a limited length of life, for they are used up by the processes of life, and they break up and die, some sooner, some later. As it is observed that all true somatic cells, among higher animals at least, are subject to this law of mortality, but that the germ-cells are not, and that, furthermore, the germ-cells only develop when they are fertilized, the cause of the potential immortality of the germ-cells is believed to lie in amphimixis, and a 'rejuvenating' power in fertilization, or, more generally, in amphimixis, is inferred. Mystical as this sounds, and little as it agrees with our otherwise mechanical conceptions of the economy of life, it was until very recently a widespread view, although perhaps it is now abandoned by many who formerly held it, and has been imperceptibly modified into a quite different conception, for which the word 'rejuvenescence' is retained, but with the altered meaning of a mere 'strengthening of the metabolism' or 'of the constitution.' By many authors, indeed, the two meanings of the word are not clearly kept apart. I shall return later to the modified meaning of the word 'rejuvenescence,' and shall keep in the meantime to the original meaning of the word, which implies a renewal of life which would otherwise die out.

This meaning seemed to gain a firm hold, when, about fifteen years ago, the French investigator Maupas published his remarkable observations on the conjugation of Infusorians. These seemed to show that colonies of Infusorians which were artificially prevented from conjugating gradually died out; not of course at once, but after many, often several hundred, generations; ultimately a degeneration of all the animals in such colonies set in, and ended only with their utter extinction. Maupas himself interpreted this asa senile degenerationwhich took place because conjugation had been prevented, and he therefore regarded conjugation as a 'rajeunissement karyogamique,' a rejuvenescence, and therefore a means of preventing the ageing and final dying off of the individuals—of obviating, in short, the natural death to which in his opinion they would otherwise besubject. This conception was greeted with general approval, and there are many people who still regard conjugation as a process by which the capacity for life is renewed—a view which I must still dispute as emphatically as I did some years ago.

In the first place, the observations on which this theory is based admit of another interpretation, quite different from that which has been assumed to be the only possible one. Maupas prevented conjugation, not perhaps because he had isolated individuals and their progeny, but by exposing the whole colony of near relatives to unusual conditions when conjugation was just about to set in, namely, by supplying them with particularly abundant food. The need for conjugation then disappeared, as, conversely, it could be called forth at any time in a colony by hunger. But these are artificial conditions, and indeed the breeding of Infusorians for months in a small quantity of water on the object-glass certainly does not correspond to natural conditions. We must admire the skill of the investigator who was able to keep his colonies alive for months and years under such artificial conditions, but we may venture to doubt whether the fate of extinction which did ultimately overtake them was really due to the absence of conjugation, and not to the unnaturalness of the conditions.

In any case a repetition and modification of Maupas' experiments is very desirable, and would be of lasting value[15].

[15]Since the above was written Calkins has made a series of new experiments, the results of which differed in several respects from those yielded by Maupas' experiments. When his infusorian-cultures began to grow weaker, as happened frequently and at irregular intervals, he was always able to restore them to more vigorous life by a change of diet, and especially by substituting grated meat, liver, and the like for infusions of hay. Certain salts, too, had the same effect: the animals became perfectly vigorous again. Calkins believes that chemical agents, and especially salts, must be supplied to the protoplasm from time to time. He reared 620 generations of Paramœcium without conjugation. But the 620th was weakly and without energy. The addition of an extract of sheep's brains made them perfectly fresh and vigorous again. Further experiments in this direction are to be desired, but, according to those of Calkins, it is probable that Infusorians can continue to live for an unlimited time even without conjugation.

Let us, however, assume for the moment not only that Maupas' observations were correct, which I do not doubt, but also that they were rightly interpreted. Would they in that case afford a proof that amphimixis means a rejuvenescence of the power of life? To my thinking, not in the remotest degree.

It certainly seems as if this were true at the first glance; the colony which is prevented from conjugating goes on multiplying for a considerable time, often indeed for hundreds of generations, but this may be compared with sufferers from hunger, whose life does not cease at once if the feeling of hunger is not appeased.

It was certainly made evident by these experiments that Infusorians which were prevented from conjugating were incapable of unlimited persistence. But even this in no way proves that amphimixis has a power of rejuvenating life, but simply that these animals are adapted for conjugation, and that they degenerate without it, just as the sperm-cell or the ovum dies if it does not attain to amphimixis.

My opponents take it as axiomatic that the life-movement must come to a standstill of itself, and that it therefore requires help. Even so distinguished a specialist on the Protozoa as Bütschli argues that organisms are notperpetua mobilia, and when one remembers the physicist's theory of the impossibility of aperpetuum mobilethis looks at first sight like a formidable objection. But does the organism always remain the same as long as it lives, like a pendulum which friction causes to swing more and more slowly till ultimately it comes to a standstill? We know surely that the phenomena of life arise from a continual process of combustion, which is followed by a constant replacement of the used-up particles by new particles; we know that life depends on an unceasing metabolism, which brings about changes in the material basis of the organism every moment, so that it is constantly becoming new again.

I shall attempt to show later on that the cells cannot be the ultimate elements of the organism, but that the life-units visible with the microscope must be made up of smaller invisible units. These, therefore, undergo 'metabolism,' which conditions their multiplication and their destruction, and this 'metabolism' is not to be seen only in the building up and breaking down of 'albuminoid substances,' as the physiologists say, but in the alternation between the multiplication and the dissolution of these smallest vital particles. Therefore, it seems to me that the movement of life, whether in a single-celled or in a many-celled organism, is not to be compared to one pendulum, but to an endless number of pendulums which succeed one another imperceptibly in the course of the metabolism, always producing anew the same result, which therefore may continuead infinitum. Suppose, then, that we possessed our present conception of life as a process of combustion, and of metabolism as the agency which continually provides new combustible material in the shape of new vital particles, but that we knew nothing about multicellular organisms and their transitory existence, but were acquainted only with unicellular organisms and their unlimited multiplication by division. If we were then to make the observation that all multicellular organisms are mortal, subject to natural and inevitable death, it would at firstappear to us quite unintelligible, since we should be aware that in these also the fire of life is continually being fed by the supply of new combustible material. Not the potential immortality of unicellular organisms would then appear to us remarkable and surprising, but the limitation of the life of multicellular organisms—the occurrence of natural death. Who knows whether, in that case, many of those investigators trained in regard to unicellular organisms alone would not say just the opposite of what Bütschli has said, that there could be no natural death in many-celled organisms, since single-celled organisms prove to us that life is an endless chain of transitory minute vital units?

Furthermore, our physiologists are still far from being able to explain the natural death of many-celled organisms from below—I mean from a knowledge of its necessary causes; on the contrary, they argue from the known occurrence of natural death to the causes which underlie it; and thus they have arrived at the idea, undoubtedly correct, that the somatic cells of the body are gradually so altered by their own activity that they are ultimately unable to function any longer and must die off. Therefore, if we were unacquainted with death, we should not have been able to infer it from our physiological knowledge, and still less from our knowledge of the unicellulars.

As our insight has in point of fact grown by starting from the mortal many-celled organisms, and has only later penetrated down to the unicellular organisms, so we can understand the genesis of the conclusion, deduced from the mortality of the many-celled organisms, that unicellular organisms also are unable to continue without limit the renewal of material and of vital particles, and that consequently they would be subject to natural death if nature had not found in conjugation a 'remedy' for 'the physiological difficulties which ensue automatically and necessarily from the constitution and from the continual functioning' even of unicellular organisms.

But we ask in vain for a shadow of proof of this remarkable conception; it is an axiom deduced from our knowledge of natural death among multicellular organisms, and bolstered up by a mistaken application of the idea of 'perpetual motion.' Or may we regard it as a proof of this if it should be found that all unicellular organisms are adapted for conjugation?

We shall see later on that amphimixis has certainly quite a different and, undoubtedly, a very important effect, namely, that it increases the capacity of the species for adaptation; and a life-renewing effect in Bütschli's sense could only be ascribed to it in addition if the assumption of the necessity of a natural death inunicellular organisms were not directly contrary to the clear facts of the case; but this is just what it is.

We are acquainted with such contradictory facts, not perhaps among the unicellulars themselves, where it is difficult to procure direct proof, but in regard to the germ-cells of many-celled organisms which correspond to unicellular organisms. We know that under certain circumstances the ovum is capable of persisting by itself—in cases of parthenogenesis—how then can we conclude that amphimixis is in the case of Metazoan germ-cells the cause of their capacity for development? We can only conclude, it seems to me, that their power of developing is usually bound up with the occurrence of amphimixis. So we may conclude in regard to the unicellulars that their unlimited power of multiplication is bound up with the occurrence of amphimixis, but not that amphimixis is the cause of this power, or that it implies a rejuvenescence of life. If unicellular organisms could have been made immortal through amphimixis, then what I maintain would be proved—that they possess potential immortality; but if they did not possess it, no artifice in the world could give it to them; amphimixis could be at most only the condition with the fulfilment of which the realization of their immortality was bound up.

One may ask, How then can amphimixis be a condition of their survival? why should Infusorians which have not conjugated at the proper time be doomed to extinction? And from the standpoint of our present knowledge I am as little able to give a precise answer as my opponents. But I can give one in relation to the amphimixis of multicellular organisms, for in regard to these we know that each of the germ-cells—male and female—uniting in fertilization, is of itself incapable of development and doomed to perish, the sperm-cell because it is too small in mass to be able to develop the whole organism, and the ovum because, in order to become capable of being fertilized, it must undergo certain changes which make it incapable of independent development. We have seen that after the two maturing divisions in the egg-cell have been accomplished the ovum no longer contains a mechanism of division, as the centrosphere breaks up after the second division; embryonic development can therefore only begin when a new centrosphere has been introduced into the ovum, and this is normally brought about by fertilization, that is, by the entrance of the sperm-cell, whose nucleus is accompanied by a centrosphere.

Thus amphimixis is seen to be really a condition of development. But we now know that the ovum can emancipate itself from this condition,by only going through a part of the processes of maturation which are related to the subsequent amphimixis, and by thus retaining its own centrosome. Nothing is more instructive in this connexion than the cases we have already briefly discussed of facultative or occasional parthenogenesis. We have seen that in some insects, for instance in the silk-moths, there are sometimes, among thousands of unfertilized eggs, a few that develop little caterpillars. If we examine a large number of such unfertilized eggs we not infrequently find among them several which, although they have not gone through the whole course of development, have at least gone through the earlier stages, and others which may have advanced somewhat further and then come to a standstill; in short, we can see that several of these eggs were capable of parthenogenetic development, although in varying degrees.

The cause of this parthenogenetic capacity has not as yet been definitely determined by observation, but we shall hardly go wrong if we seek it in the fact that the centrosphere of the ovum does not always perish immediately and completely during maturation, and may persist, rarely in its integrity, but sometimes in a weakened state. Future observations will probably reveal some differences in the size or aster-forming power of the centrospheres of such eggs; in any case it is of the greatest interest that stimuli of various kinds—mechanical or chemical—can strengthen the disappearing centrosphere of the ovum, although as yet we are far from being able to say how this comes about.

The experiments already mentioned of Tichomiroff, Loeb, and Winkler give us at least an indication how we must picture to ourselves the origin of parthenogenesis, namely, through the fact that the breaking up of the apparatus for division, introduced for the sake of compelling amphimixis, is prevented. Minute changes in the chemistry of the ovum, similar to those caused artificially in the ova of the sea-urchin by the introduction of an infinitesimal quantity of chloride of magnesium (Loeb), in the ovum of the silk-moth by friction or by sulphuric acid (Tichomiroff), or in the sea-urchin ovum by an extract of the sperm of the same animal (H. Winkler), will effect this modification, and normal parthenogenesis is induced.

For the ovum, therefore, amphimixis is certainly not a life-renewing or rejuvenating factor; it only appears as such because the process has in the course of nature been made compulsory by making the two uniting cells each incapable of developing by itself. As we have seen, this is true also of the sperm-cell, for although it contains a centrosphere, and would be capable of division as far asthat is concerned, yet in almost all animals and plants it consists of such a minimal quantity of living matter that it is unable to build up a new multicellular organism by itself. Only in one alga (Ectocarpus siliculosus) has it been observed that not only the female germ-cell can develop parthenogenetically under certain circumstances, but that the male-cell may also do so. In this case, however, the difference in size between the two is not great, and it is noteworthy that the male plant, in correspondence with the smaller size of the zoosperm, tends to be a somewhat poorly developed organism.

If we are forced to the conclusion in regard to multicellular organisms that amphimixis does not supply the power of development to the ovum, but that, on the contrary, the power of development is withdrawn from the ovum, so that amphimixis can, so to speak, be forced, must we not assume something similar for unicellular organisms also? May not amphimixis be made compulsory in their case also, in that the Infusorians in preparation for conjugation go through changes which make their unlimited persistence possible only on condition that they conjugate? In my opinion the division of labour in the nucleus, which is differentiated into a macronucleus and a micronucleus, and the transitory nature of the former, may be regarded as an adaptation in this direction. In any case, it is striking that an organ which otherwise persists without limit among unicellular organisms, the nucleus, is here subject to natural death after the manner of the body of multicellular organisms, that it breaks up and must be reformed from the micronucleus which in this case is alone endowed with potential immortality. I am inclined to regard this as an arrangement for compelling conjugation, since it is only after conjugation that the micronucleus forms a new macronucleus, although the latter is indispensable to life, as we see from experiments in dividing Infusorians artificially.

Suppose we had to create the world of life, and it was said to us that amphimixis must—wherever possible—be secured periodically to all unicellular and multicellular organisms, what better could we do than arrange devices which should exclude individuals which, by chance or constitution, could not attain to amphimixis from the possibility of further life? But would amphimixis then be the cause of persistence or a principle of rejuvenescence?

I do not see that there can be any ground for such an assumption other than the tenacious and probably usually unconscious adherence to the inherited and deep-rooted idea of the dynamic significance of 'fertilization,' no longer, perhaps in its original form, which regarded the sperm as the vital spark which awakened new life in the deadovum, but in the modified form of the 'rejuvenating' power of amphimixis.

Quite recently an attempt has been made to modify the idea of the 'rejuvenating' effect of amphimixis so that it should mean only an advantage, not an actual condition of persistence. Hartog, in particular, admits so much, that the occurrence of purely asexual and purely parthenogenetic reproduction excludes the possibility of our regarding the process of amphimixis as a condition of the maintenance of life. But then we must also cease to regard the 'ageing' and dying off of Infusorians which have been prevented from conjugating as an outcome of the primary constitution of the living substance, and should entirely abandon the misleading expression 'rejuvenescence.'

If we fix our attention on the numberless kinds of cells in higher organisms and on multicellular organisms as intact unities, we see that they all die off, that they are subject to a natural death, that is, a cessation of vital movement from internal causes, yet no one is likely to refer their transitoriness to the fact that they do not enter into amphimixis. We find it quite 'intelligible' that the cells of our body should be used up sooner or later as a result of their own function, though we are very far from being able to demonstrate the necessity for this, and so really to 'understand' it.

It is only from the standpoint of utility that we can understand the occurrence of natural death; we see that the germ-cellsmustbe potentially immortal like the unicellular organisms, but that the cells which make up the tissues of the bodymaybe transient, and indeedmustbe so in the interests of their differentiation—often great and in one direction—which determines the services they render to the body. They required to become so differentiated that they could not continue to live on without limit, and they did become so differentiated because only thus could an ever-increasing functional capacity of the whole organism be rendered possible; but they die not because 'rejuvenescence through amphimixis is denied them, but because their physical constitution is what it is.' And we must explain the death of the whole many-celled individual in a similar way. When we were trying in a previous study to establish the unlimited continuance, the potential immortality, of unicellular organisms, we noted that an eternal continuance of the life of the body of multicellular organisms could certainly not be a necessity, since the continuance of these forms of life is secured by their germ-cells. A continuance of the body cannot even be regarded as useful from any point of view. And what is not useful for a form of lifedoes not arise as a lasting adaptation, which is of course not to say that an immortality ofmulticellular organisms, such as they are now, would even have been possible. If these organisms were to attain to such a high degree of functional capacity and of structural complexity as they now exhibit, they obviously could not also have been adapted at the same time to an eternal persistence of life.

This is in perfect harmony with our whole conception of the impelling forces in the development of the organic world; the ever-increasing functional capacity of the structure arose from the advantage which this afforded in the struggle for existence, in comparison with which the apparent advantage of the endless life of the individual was of no account whatever.

I will not here follow out this idea. I have merely touched on it in order to make clear that the death of individuals in all multicellular organisms gives us no ground for thinking of the unlimited life of the germ-cells as dependent on a special artifice of nature, such as amphimixis is often supposed to be. Let us always remember that there is parthenogenesis, and that there are unicellular germs (spores) which are never fertilized, and that the reproduction of many species of animals and plants occurs in this way without the intervention of amphimixis at all.

Attempts have recently been made to prove that parthenogenesis is a kind of self-fertilization, and these have been based on the observations of Blochmann and Brauer, which showed that in the bee and in the salt-water Crustacean,Artemia salina, the reducing second maturation division of the ovum-nucleus is not suppressed, but is regularly accomplished, and that the two daughter-nuclei which result from this division unite with each other subsequently. I have already noted that these statements do not hold true, at least with regard to the bee. In this case the second maturing division takes place without any subsequent fusion of the two daughter-nuclei. According to the observations of Dr. Petrunkewitsch, which I have already mentioned, and for the exactness of which I can vouch, the second maturation-spindle is unusually long, so that the two daughter-nuclei are pushed very far apart (Fig. 79,Rsp 2), and only the inner of the two nuclei (K 4) becomes a segmentation nucleus, while the outer undergoes a remarkable fate; it unites with the inner nucleus which results from the division of thefirst maturation cell(K 2), and from this union the primitivegenital cells of the animal appear to arise—an observation the eventual theoretical importance of which can only be estimated later.

Meantime all we can gain from it is a certain mistrust of the interpretation of the processes of maturation inArtemiawhich have hitherto been given; at least we are tempted to suppose that the copulation of two nuclei which Brauer observed inArtemiamay not have led to the formation of the segmentation nucleus there either, but may have had some other significance.

But, even if we leave this point entirely out of account, there remain all the cases of regular parthenogenesis in which this mode of reproduction occurs alone and not in alternation with the sexual mode. In these only one maturing division is undergone, and only one polar body is formed, and thus there can lie no possibility of supposing a self-fertilization of the ovum.

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