Cytology, with special reference to the metazoan nucleus

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CYTOLOGY

MACMILLAN AND CO., LIMITED
LONDON +- BOMBAY +» CALCUTTA * MADRAS
MELBOURNE

THE MACMILLAN COMPANY
NEW YORK + BOSTON + CHICAGO
DALLAS + SAN FRANCISCO

THE MACMILLAN CO. OF CANADA, LtD.
TORONTO

Cn OO GY

MERE VSPECIALSREF ERENCE TO, THE
METAZOAN NUCLEUS

BY

W. E. AGAR

PROFESSOR OF ZOOLOGY IN THE UNIVERSITY OF MELBOURNE

MACMILLAN AND CO., LIMITED
ST. MARTIN’S STREET, LONDON

1920

QH
ys)
A 32

COPYRIGHT

Pio ACE

For many years now it has been ‘apparent to biologists that the nucleus
- —and especially its most conspicuous constituent, the chromatin—has
- special claims to be considered the master substance of the organism,
not only determining its form and activities within the life-time of each
individual, but by means of the gamete nuclei bringing about that organic
continuity between parent and offspring which we call heredity. The
absorbing interest of this conception has attracted many workers to the
field of nuclear cytology, and consequently the advance in knowledge
and the output of hypotheses have been very great in recent years.
As must happen in any growing branch of science, the results of all this
research are scattered through many publications, and any one whose
duty it has been to lecture to students on this science must often have
felt difficulty in recommending to inquirers a concise course of reading
which will give a summary of the main results in this field of research,
and at the same time indicate where more detailed information can be
obtained. This book is an attempt to provide for this want.

The discovery of mitosis, and the recognition of all that is implied
by that process, has led biologists to lay more stress on the nucleus than
upon the cell, considered as units, and lately the ‘“ unit” has tended to
shift to certain constituents of the nucleus, namely, the chromosomes ;
still more recently the advance of knowledge has made it profitable to
of which the

a)

focus attention on the even more elementary “ units
chromosomes themselvés are composed. In this book, consequently,
problems concerning the organization and physiology of the cell as a
whole are scarcely touched upon, practically the whole space being
devoted to the nucleus and its constituent parts. Moreover, the field
has been still further restricted by confining attention chiefly to the
Metazoan nucleus. The nucleus of the Metaphyta is very similar, in
structure and behaviour, to the Metazoan nucleus, and only rarely needs

Vv

vi CYTOLOGY

special mention. With the Protista the case is different, but even these
forms have been treated in a very brief and eclectic manner. This is
partly because, like most cytologists, the author has unfortunately very
little first-hand knowledge of the cytology of this group, and partly
because Protistan cytology has not up to the present time helped us very
much to gain an insight into those aspects of the nucleus and its activities
with which we are here concerned.

As the science of Physiology is almost entirely founded on the physio-
logy of the higher Vertebrates, so is Cytology mainly the cytology of the
Metazoa and Metaphyta. In both cases the greater simplicity of the
technique of dealing with the larger forms has no doubt been partly
responsible for this state of affairs, but there is another and more
important reason. The functions of organs and the meanings of pro-
cesses are far easier to interpret when the organs are complicated and
the processes specialized than when they are simple or generalized. The
specialization of the nerve cells of the Metazoa for conduction of im-
pulses, of their muscle cells for contraction, and of their gland cells for
secretion has enabled physiologists to gain a far deeper insight into the
physiology of these processes than they could have obtained from the
study of the Protistan cell in which no one function dominates over
the others. Similarly, the high specialization of the mitotic processes
in Metazoa and Metaphyta has afforded cytologists an insight into their
meaning far greater than could ever have been obtained from the study
of the less specialized nuclei of most of the Protista. In order properly
to understand any process, however, it is necessary to know as many
variants of it as possible, as well as the steps by which it has been
evolved. For this reason a very brief account of some of the more
striking differences between the Metazoan and Protistan nuclei has
been given.

Each branch of Science presents its own special technical difficulties
toits students. Cytology has at least its full share of these. The objects
with which the cytologist has to deal are extremely minute, and indeed
his analyses are almost invariably limited merely by the imperfection of
his optical instruments and technique. However powerful the former
and perfect the latter, he is sure that below the limits of visibility there
exists a morphological complexity at least as great as that which he has
already revealed. At present he has no methods comparable with those
of the chemist and physicist for dealing indirectly with ultramicroscopic

PREFACE vil

complexity, though a promising start in this direction has been made by
Mendelian analysis and its correlation with hypothetical “ factors ”’
or “ genes” in the idioplasm. Even when dealing with objects slightly
above the limits of visibility, the difficulties of observation are often very
great, so that two cytologists examining an identical object will often
give a different account of it. Many examples will occur to any cytologist
—for instance, the different accounts given of the changes undergone by
the chromosomes when passing into the resting nucleus at telophase.
The appearance of the disintegrating chromosomes of the same organism
has been variously interpreted as (1) vacuolation, (2) a splitting into
two threads, (3) the formation of a single spiral thread, (4) the formation
of two intertwined threads.

Besides many other difficulties inherent in the nature of cytological
research, the science suffers especially severely from one of the difficulties
in the way of progress of all sciences. The researcher can only select
for study a minute fraction of the mass of objects presented to him, and
inevitably those objects appear to him significant, and therefore worthy
to be studied, which fit into his preconceived ideas. If a cytologist sets
out to study the gametogenesis of some animal, he will probably pass
under review through his microscope many hundreds of thousands of cells.
Out of these he can necessarily only select a minute proportion for detailed
study. The cells which he thus selects are, of course, those which seem
to him to represent stages in the process which he is endeavouring to
reconstruct. If he has already formed a theory regarding this process,
having a more definite mental image of the process as conceived by him
than of the possible alternatives he more readily picks out for study those
objects which appear to favour his theory than the others, which he
rejects (as he is bound to reject the great majority) as equivocal or of no
significance. This certainly appears to be the explanation of the partisan
nature of so much cytological work.

The student must not get the impression from the above that it is
hopeless to discover the truth in cytology. Gradually one or other of
conflicting views becomes recognized as being nearer the truth than its
rivals, or else some generally accepted principle is raised from the ashes
of them all—either by the gradual accumulation of evidence or by some
important discovery which is generally recognized as providing the key
to the problem. In this way a science of cytology has grown up, firmly
established as regards its main outlines.

Vill CYTOLOGY,

A glance through the pages of this book will show that it makes no
pretence to trace the historical development of the science, and conse-
quently the works selected for special reference are not always those
which contain the first, or even the most important, contribution to the
matter under discussion. In most cases they have been selected either
as giving a particularly clear account of it, or because they contain good
general discussions and literature lists which would be useful to the
student referring to them.

Since the book is intended to give a summary of the more important
results of cytological research, it follows that the great majority of the
figures are taken from the original works of other authors scattered through
various scientific journals. They have in nearly every case been redrawn
for the purpose by Miss Helen L. Ness, often with the omission of details
not required for the purpose for which they are used.

COME NTS

CHAPTER I

PAGE
NUCLEUS AND CYTOPLASM : : : : , I
A. THE CYTOPLASM - ; : : ; : I
1. The Cytoplasm proper . ; : : I
2. The Cell Membrane 3
3. The Centrosome : : : ; 4
4. Chondriosomes . : ; ; : ae 4
5. Metaplastic Bodies 4
B. THE NUCLEUS 4
C. MITOSIS 6

1. The Division of the Chromosomes, and their Relation to
the Resting Nucleus 13
2. Chromatin and Linin . : : 18
3. Nuclear Membrane and Karyolymph ; 19
4. Karyosomes and Prochromosomes ; ; : 19
5. The Achromatic Figure. 21
D. AMITOSIS j : 24

CHAPTER: ii

MEIOSIS : : ; : : ; § 26
A. MEIOSIS IN THE MALE . 31
1. Metosts tn Tomopleris onisciformts : , ; ei
2. Metosts tn Lepidostren paradoxa : : i | ey
3. Metosis in Certain Insects : 41
B. DIVERGENT VIEWS OF THE PROCESS OF MEIOSIS 43
1. Parasyndests and Telosyndests . ‘ : ; 43

2. The Mutual Relations of the Homologous Chromosomes
during Syndests ; 48
3. Meiosis with Tetrad Formation ; 49

1x

Dr CYTOLOGY

4. Which of the two Divisions of the Meiotic Phase effects
the Separation of the Homologous Chromosomes ?

5. Synizests

C. MEIOSIS IN THE FEMALE : 2

1. The Continuity of the Chromosomes

2. The Relation between the Chromosomes and the Nucleolt

3. The Connection between the Germinal Vesicle and Volk
Formation

4. Does any Stage comparable to the Germinal Vesicle occur

in Spermatogenests ? .

CHAPTER iit

SYNGAMY, EARLY DEVELOPMENT, PARTHENOGENESIS .

A.

Erie ZOn Gs

THE DEVELOPMENT OF THE SPERMATOZOON

SYNGAMY

GONOMERY

63

)

Go

. THE GERM-TRACK
. PARTHENOGENESIS

Obligatory Parthenogenests

Facultative Parthenogenesis

The Hlomology of the Metiotic Divisions in Obleebions ry
and Facultative Parthenogenesis

Artificial Parthenogenesis

CHAPTER IV

THE SEX CHROMOSOMES

- W&

6.

The Sex Chromosomes in Insects ;
Which of the two Meiotic Divisions acts as the Reduction
Diviston for the Sex Chromosomes ?

Various Forms of the X and Y Chromosomes

. Behaviour of the Sex Chromosomes during Syndesis and

the Metotic Prophase, and outside the Meiotic Phase
Sex Chromosomes in Animals other than Insects
Cases where the Differential Sex Chromosome is present
7m the Female
Some Special Life Histories :
The Relation between the Sex Chromosomes and the
Determination of Sex

PAGE

56

CONTENTS xl

CHAPTER V

PAGE
K THE CHROMOSOMES ; : : . ; 29 923
A. THE CONTINUITY OF THE CHROMOSOMES : >, 2a
B. THE COMPOSITION OF THE CHROMOSOMES OF SMALLER UNITS . 34
C. VARIATION IN THE NUMBER OF CHROMOSOMES , : oe rigo
1. Variation in Chromosome Number due to Fragmentation
or Linkage . : : . : 7 930
2. Variation in Chromosome Number due to Irregularities
of Mitosts. : : E : ona
3. Variation in Chromosome Number due to Multiplication
or Fusion : , . : : eer a7
CHAPTER. VI
HEREDITY AND MORPHOGENESIS : , . : oS
A. THE EQUALITY OF INHERITANCE FROM MALE AND FEMALE
PARENTS : : : ; ‘ : 54
B. THE PROCESS OF MITOSIS AND ITS IMPLICATIONS. THE
FUNCTIONAL DIFFERENTIATION OF THE CHROMOSOMES » 1162
C. THE PROCESS OF MEIOSIS : : ; . 2 £67
D. THE PARALLEL WHICH EXISTS BETWEEN CHROMOSOME BE-
HAVIOUR AND THE RESULTS OF BREEDING EXPERIMENTS . 168
1. The Genetics of a Tetraploid Plant ; ; Ee oo
2, Segregation and Parthenogenests : : Ne 0/4 |
3. Segregation and Bud-Variation : ' 72
4. The Interchange of Hereditary Factors between Homo-
logous Chromosomes . : ERA
5. The Cytological Basts of Mutation : : = OO
E. STERILE AND PARTIALLY STERILE HYBRIDS : : weLos
F. THE NUCLEUS IN MORPHOGENESIS : oF OF
G. CHROMIDIA AND CHONDRIOSOMES : ; . 190
1. Chromtdia . . ; : : .) “EOo
2. Chondriosomes . . , . : ». -1O5
3. The Relation between Chromidia and Chondriosomes . 200

xi CYTOLOGY

CHAPTER VII

THE NUCLEUS OF THE PROTISTA AND PLANTS : PAO
A. PROTISTA ‘ : u : 3 : 5 QO

B. ANIMALS AND PLANTS—HAPLOID AND DIPLOID CONDITIONS . 210
BIBLIOGRAPHY : : : ; ¢ . seat
222

INDEX

CHAPTERS |
NUCLEUS AND CYTOPLASM

In general a cell is composed of two principal morphological constituents,
the nucleus and the surrounding protoplasm, or better, cytoplasm, since
some authors use the former word to include both cytoplasm and nucleus.
This division of the cell constituents is quite sharp in all but the lowest
organisms, the nucleus being delimited from the cytoplasm by a membrane
except during a certain period of its division processes. In certain
unicellular organisms (Protista), however, the word “nucleus”’ is
inappropriate, since the material which composes this structure in the
higher organisms is scattered through the cytoplasm as minute granules,
or.chromidia (see Chapter VI.). In other Protista both nucleus proper
and chromidia are present (e.g. Difflugia), while certain Bacteria are said
to consist entirely of “‘ nucleus.’’ Even in multicellular organisms, both
in Metazoa and Metaphyta, there are frequently minute bodies in the
cytoplasm which are supposed by some cytologists to be derived from
the nucleus, and to consist of true nuclear material, and therefore to be
comparable to chromidia.

Ay THE CY TOPLAS

The cytoplasm surrounding the nucleus includes a number of different
structures of which the following are the most important :
(1) The cytoplasm proper.
(2) The cell membrane.
(3) The centrosome.
(4) Chondriosomes.
(5) Metaplastic bodies.

(1) The Cytoplasm Proper

The living cytoplasm itself consists of a viscid, nearly transparent
substance, which often is clearly not homogeneous. Great difference of
opinion exists as to its structure, chiefly owing to the fact that very little

I B

2 CYTOLOGY. CHAP.

organization can be made out in it in the living state (its constituents
having nearly the same refractive indices), while when “ fixed ” by one
of the various killing and hardening agents, and stained, the structure
which is thus made visible varies considerably with the reagents used for
fixation and the subsequent staining. It is not proposed therefore to do
more than mention the most important views, especially as at present we
have no means of correlating structure with function in the case of the
cytoplasm. In our present state of knowledge, to decide between these
various views is comparatively unimportant as regards general biological
problems, apart from biophysics, with which we are not here concerned ;
but as we shall see later, the matter stands otherwise with the nucleus,
where exact determination of structure and function is often of critical
importance for theories of heredity and other problems.

Views as to the structure of the cytoplasm can be arranged as follows :
(a) the reticular ; (0) the fibrillar ; (c) the granular; (d) the alveolar.

(a) The reticular theory. According to this, the cytoplasm consists
of a more solid constituent forming a reticulum or network, like a sponge,
containing in its meshes a more fluid substance known as the cell sap or
enchylema. In addition, a greater or smaller number of minute granules
or microsomes are embedded in the reticulum.

(6) The fibrillar theory. According to this view the reticulum is
not continuous, but is composed of disconnected threads embedded in a
matrix (Flemming,! 1882). ‘

(c) The granular theory. As developed by Altmann (1893) this
depends more upon theory and less upon observation than do the other
views. The cytoplasm is supposed to consist essentially of granules,
only the largest of which are visible through the microscope. The
microsomes mentioned above are examples of these. Each granule is
itself a living organism or bioblast and bears much the same relation to
the cell as the cell itself to the whole organism. As regards the inter-
granular substance, Altmann supposed that this is mainly composed of
granules below the limit of visibility. Any substance which may be
left over between these ultimate granules is non-living matrix. This
theory is mainly of historical interest.

(d) The alveolar theory. The alveolar theory of Biitschli (1892)
supposes the cytoplasm to possess a frothy structure similar to that
of the emulsion formed when two immiscible fluids are shaken together.
The cytoplasm therefore consists of minute drops of one fluid sus-
pended in a second, denser fluid, which, being generally small in bulk
compared with the included droplets, forms thin films surrounding them
like the films of soapy water surrounding the air in a foam of soap bubbles.

1 References to the exact source of all authorities quoted will be found at the end
of the book.

; CYTOPLASM 3

In optical section these films or lamellae surrounding the droplets are
easily interpreted wrongly as a reticulum. This theory is supported by
the behaviour of the artificial emulsions made by Biitschli, which exhibit
many striking resemblances to cytoplasm.

Granules or fibrillae (chondriosomes, chromidia, etc., see Chapter VI.)
may be suspended at the nodes of the apparent meshwork formed by the
denser fluid. Thus this theory is not incompatible with the fibrillar
theory, the alveolar structure applying to the matrix in which the fibrillae
are embedded.

The alveolar theory is the one that fits in best with the known
properties of the cytoplasm, and especially with its undoubtedly fluid
nature. For living cytoplasm is, physically, a fluid—often very viscid
indeed, but nevertheless fluid. During life streaming movements are
often observable in it. Moreover, bodies such as the nucleus, which are
very large relatively to the meshes or alveoli, can move through the
living cytoplasm. Examples of rapid change of position of the nucleus
in the cell are afforded by living Protozoa such as Amoeba, and by the
movements of the pronuclei in fertilization in the Metazoa (Chapter IIT.).
Many other proofs of the fluid nature of the cytoplasm could be cited,
such as the spherical shape assumed by vacuoles and by fragments of
cytoplasm extruded from a cell into water. These facts are impossible
to reconcile with the presence of a permanent supporting reticulum
forming part of the essential structure of the cytoplasm. In many
kinds of cells supporting reticula and fibrillae are indeed undoubtedly
present, but these are of a different order of structure and belong to the
architecture of the cell as a whole, and not to the structure of the cyto-
plasm, which lies within the meshes of the supporting framework.

(2) The Cell Membrane

With few exceptions (certain Protozoa, leucocytes) animal cells are
plainly delimited by an outer metamorphosed layer of cytoplasm, or by
a membrane secreted by the cytoplasm, the distinction between the two
being often very difficult to make and indeed unreal.

Sometimes, however, nuclear division is not followed by cell division
with development of a membrane between the two cells, and in this case
there arises a structure known as a syncytium, in which a number of
nuclei are embedded in a continuous mass of cytoplasm. Well-known
examples of syncytia are the plasmodia of Mycetozoa and the ectoderm
of Nematodes.

In certain cells the membrane attains a much greater importance,
and may lose its connection with the underlying cytoplasm which
secreted it, so that the latter comes to lie more or less freely within it
as if in a box with which it had no close organic connection. In such a

4 CYTOLOGY CHAP.

case of course the cytoplasm inside must form a new membrane round
its periphery. Examples of cell membranes which have become relatively
free from the cytoplasm by which they were secreted are the cellulose
cell walls of most plant cells and the vitelline membranes of many eggs.

(3) The Centrosome (see also p. 21)

As we shall see, during division of the nucleus the centrosomes are
the dynamical centres of the cell. Even when the nucleus is not actually
in process of dividing, the centrosome may exert a powerful influence on
the topographical arrangements both of the nuclear constituents and of
the cytoplasm. This is
specially well illustrated in
what is known as the
“bouquet” stage in gameto-
genesis (p. 33), where the
chromosomes are in the form
of U-shaped loops, so orient-
ated that their free ends are
directed towards the centro-
some and the apices of the
loops towards the opposite
pole of the nucleus. In
these cells the cytoplasm is

; heaped up round the centro-
Diagram of a Cell. a, alveoli of cytoplasm; c, chromatin

in the form of fine granules (chromioles) embedded in the linn SOME, which is embedded in

meshwork; ce, centrosome, .containing centriole ; ch, chondrio- 5

some; mb, metaplastic body; mm, nuclear membrane; p, @ Mass of chondriosomes,

plasmosome. ce A ON : 2
chromidia,’” etc., which it

appears to have attracted round itself (p. 191).

Fie. 1.

(4) Chondriosomes

These are minute granules or filaments embedded in the cytoplasm.
They are discussed in Chapter VI.

(5) Metaplastic Bodies
are non-living material included in the cytoplasm, such as yolk
granules, fat globules, excretory and secretory granules, etc.

By THE NUCEEUS

The study of the nucleus is in many respects easier than that of the
cytoplasm. There are several reasons for this, amongst which may be
mentioned the comparative coarseness of its structure, and the strong,
selective affinity for stains possessed by its constituents.

I NUCLEAR CONSTITUENTS 5

The structure of the nucleus varies profoundly according as to whether
it is going through the processes connected with nuclear division, or is
in the phase between two division periods. In the latter condition,
which we will consider first, the nucleus is said to be at “‘ rest.””’ It must,
however, be understood that this word implies merely that the nucleus is
at rest from division. The “ resting nucleus”’ is doubtless in the phase
of its greatest physiological activity.

A typical Metazoan resting nucleus consists of the following parts :
(1) chromatin ; (2) linin ; (3) wuclear sap or karyolymph ; (4) karyosome ;
(5) plasmosome—the two last mentioned both being known as nucleoll ;
they may or may not be present, and if present they may be multiple ;
(6) nuclear membrane.

The arrangement of these constituents varies greatly, the commonest
disposition being such that the linin is a faintly staining substance form-
ing a spongework stretching throughout the nucleus and containing
embedded in it the chromatin—a substance which colours intensely
with most stains. The meshes of the linin-chromatin reticulum—or
rather spongework—so formed are filled with the fluid karyolymph.
Karyosomes are larger aggregations of chromatin, but the term is incap-
able of exact definition. It is in practice restricted to comparatively
large chromatin masses occurring in nuclei in which the rest of the
chromatin (if any) is finely distributed. Nuclei of a coarser structure
may contain equally large aggregations of chromatin at the nodes of the
reticulum, though these are not generally called karyosomes.

Plasmosomes are composed of a substance called plastin, which is
different in nature from any of the above-mentioned substances. Many
nucleoli, however, are incapable of classification as karyosomes or
plasmosomes, since they partake of the nature of both, consisting of
plastin impregnated with chromatin. Examples of this kind of nucleolus
(sometimes called an amphinucleolus) are found in the “ karyosomes ”’
of many Protista, and probably in the nucleoli of certain Metazoan
oocytes. True plasmosomes disappear before nuclear division and are
reformed in the young daughter nucleus. They are doubtless of a
metaplastic nature.

Since it is impossible to appreciate the nature of the various consti-
tuents of the nucleus without knowledge of their behaviour during
nuclear division, further discussion of them will be postponed till after
we have studied this phase in the life of the nucleus.

A nucleus divides to form two nuclei in one of two ways, the one
being known as indirect division, mitosis or karyokinesis, and the other
as direct division or amitosis. The overwhelming majority of nuclear
divisions among the Metazoa and Metaphyta are of the first or mitotic
type, which we will now proceed to consider.

6 CYTOLOGY

CHAP.

C. MITOSIS

The essential feature of mitosis is the rearrangement of the chromatin
and linin to form a number of separate, thread-shaped bodies, the chromo-

i r A.
feat 3
{ Fe = . & ~
i, * ae %
A B

382

D E F

G H
IG. 2:

Diagram of Mitosis. The nucleus contains six chromosomes. A, resting nucleus; B, early prophase,
individual chromosomes not yet distinguishable, centrosome dividing; C, middle prophase, appearance of
spindle figure ; D, late prophase. The nuclear membrane has disappeared and the chromosomes are becoming

attached to the spindle fibres. E, metaphase; F, anaphase; G, telophase; H, nuclear and cell division
complete, and daughter nuclei reconstituted.

1 MITOSIS 7

somes, each of which subsequently divides into two daughter chromosomes.
The original series of chromosomes is thereby duplicated into two exactly

OO
|

5 G
Fic. 3.

Mitosis in Lepidosiren (mesenchyme cell). A, resting nucleus; B, very early prophase; C, D, middle
prophase; E, late prophase. The nuclear membrane has disappeared and the chromosomes are becoming
attached to the spindle fibres. F, metaphase (seen from above). Only about half of the chromosomes are
shown. G, anaphase; H, telophase, reconstruction of one of the daughter nuclei; I, two of the chromosomes
from H, in transverse section and under a higher magnification.

8 CY TOLOGY CHAP.

similar groups, from each of which groups a new nucleus is con-
stituted.

Certain cell structures —the centrosomes and spindle, forming
together the achromatic figure—though generally outside the nucleus,
are inseparably connected with mitosis and must be considered with it.

The process of mitosis is illustrated by the diagrammatic Fig. 2,
while Figs. 3 and 4 show how the principal stages actually appear under
the microscope. Fig. 3 shows a mitosis of a nucleus with abundant

Fic. 4.

The first cleavage mitosis in the egg of Echinus esculentus (micro-photographs by Professor T. H. Bryce).
A, late prophase, nuclear membrane breaking down; B, metaphase, C, early, and D, late, anaphase.

chromatin, but not very voluminous achromatic figure, while Fig. 4
represents a mitosis of a nucleus poor in chromatin, but provided with
a very well developed achromatic figure.

The sequence of events in mitosis is commonly divided into four
main phases, namely, prophase, metaphase, anaphase (Strasburger, 1884)
and telophase (Heidenhain, 1894). It must not be forgotten, however,
that these are arbitrary divisions of a continuous process.

The prophase consists essentially in the reconstruction of the chromatin
and linin of the resting nucleus into filaments, which by a process of

1 MITOSIS 9

condensation ultimately form the relatively short and thick chromosomes
of the later stages. Karyosomes, if present, since they are composed of
chromatin, disappear, being used up with the rest of the chromatin in
the formation of the chromosomes. If plasmosomes are present, they dis-
appear either before or after the disappearance of the nuclear membrane
(see below), apparently without participating in the formation of the
chromosomes or playing any further part in the life-history of the nucleus.

For some time after the thread formation, which starts in the early.
prophase, has proceeded or even been completed (by conversion of the
entire chromatin content of the nucleus into filaments), the length of the
threads is far greater than the circumference of the nucleus (Figs. 3, 6,
7, 8), and hence the nucleus is filled with a complicated tangle—the
spireme of Flemming—in which it is impossible to discern how many
separate filaments are present. There may indeed be no apparent
breaks in the thread, and when such do appear it is often difficult to
determine whether they are real interruptions of continuity, or merely
the optical effects of a sharp angle
in the thread, etc. This gave rise
to the old view that in the early
prophase there is but a single
greatly convoluted thread present,
and so this stage was known as

A
. the continuous or wunsegmented Fic. *5.
spireme, in contra-distinction to Meiotic prophase ( ¢) in Oenothera rubrinervis showing

: cohering chromosomes. (Gates, Botanical Gazette, 1908.)
the segmented spiveme of the A, uncut nucleus showing single thick spireme; B, later

stage showing the spireme segmenting into chromosomes.

later prophase, in which a num-
ber of separate threads is plainly present. However, careful examina-
tion, and especially comparison with forms in which the nuclei are poorer
in chromatin and the prophase filaments consequently less voluminous,
has led very generally to the conclusion that the conception of the con-
tinuous spireme as a constant stage in prophase is incorrect, but that at
all stages of the prophase the spireme generally consists of as many
separate segments as there will ultimately be chromosomes. In fact, the
spireme is a tangle of very long thin chromosomes. It is not, however,
uncommon for even fully formed chromosomes to cohere by their ends
(Fig. 5), and there is no doubt that this occasionally happens in the
case of the early prophase filaments also; in this way a continuous
spireme might be formed, but its continuity would be, so to speak,
accidental and not an essential feature of it.

A striking and important characteristic of the prophase threads or
chromosomes in many species is their duplicity. This can often be
observed from the very beginning of the prophase (Figs. 3, 8), and is
caused by the longitudinal division of each chromosome into two

Io CYTOLOGY CHAP.

daughter chromosomes, by which the division of the nucleus as a whole
is effected.

The remaining processes of mitosis are concerned merely with the
separation of the daughter chromosomes and the reconstruction of new
nuclei out of them.

In many species the division of the chromosomes is not apparent till
a later stage (metaphase), and indeed the moment at which division
occurs seems to vary greatly in the different cells of a single organism.
It must, however, be remembered that we are dealing with very minute
bodies, in which a narrow cleft may easily be obscured by the certain
amount of distortion (swelling, shrinkage, etc.) inevitable during fixing
and staining. The question of the division of the chromosomes will be
returned to again (p. 13).

Another striking and important characteristic of the prophase chromo-
somes in many animals and plants is their alternate expansion and
constriction, giving them the appearance of a string of beads. This is
also a very variable phenomenon, generally most conspicuous in early
or middle prophase. The beads are known as chromomeres, and are
discussed in Chapters V. and VI.

In the later prophase shown in Figs. 2, C, 3, D, the spireme consists of
obviously separate chromosomes. The nucleus, which has been steadily
increasing in size since the inception of the prophase, has now attained
its maximum volume, and the chromosomes are usually evenly spaced
out through it. This stage of the prophase is conspicuous enough to
have earned the special name of diakinesis (Hacker, 1897, 6).

Except in the rare cases where the whole mitosis takes place within
the nuclear membrane, diakinesis ends with the disappearance of this
membrane, so that the chromosomes lie naked in the cytoplasm.

During prophase the centrosome (in animal cells) divides, if it has not
already done so, and the two resulting daughter centrosomes move apart
to take up positions at opposite poles of the nucleus. As they separate,
connection is maintained between them by fine lines or fibres, the spindle
fibres, and at the same time similar fibres radiate out from the centro-
somes into the cytoplasm, the asters. The whole system of fibres (together
with the centrosomes in animal and certain plant cells) is often known as
the achromatic figure. After the dissolution of the nuclear membrane,
some of the spindle fibres grow in and attach themselves to the chromo-
somes (Fig. 2, D).

Soon after. the disappearance of the nuclear membrane the chromo-
somes typically become arranged in one plane, at right angles to the
spindle fibres, in the manner shown in Figs. 2, 3, 4. The plate of chromo-
somes so formed is known as the equatorial plate.

As we have already seen, each chromosome is already, or now becomes,

I MITOSIS II

longitudinally split into two daughter chromosomes. The metaphase in
| Strasburger’s sense is the rather ill-defined moment when the two daughter
chromosomes begin to move apart. Very often, however, the word is
used to cover the whole period in which the complete mitotic figure
persists, with the chromosomes arranged midway between the two
poles of the spindle and the daughter chromosomes not yet completely
separated.

At this stage it can be seen that each daughter chromosome is attached
by one or more spindle fibres to one (and only one) centrosome.

The anaphase is concerned with the final separation of the two groups
of daughter chromosomes, each of the latter travelling up the line of the
spindle fibres towards one of the poles of the spindle. It is generally
agreed that the fibres of the achromatic figure are the visible expression
of the forces by which the movements of the chromosomes are effected, —
but there is considerable difficulty in determining the nature of their
action (p. 23).

Often the separation of the daughter chromosomes takes place very
regularly, so that by the splitting of the individual chromosomes which
compose it the metaphase equatorial plate is divided into two daughter
plates, which gradually diverge from one another. In other cases the
movements of the chromosomes are not so regular, so that the separating
daughter chromosomes travel up to the poles more independently.

The telophase comprises the metamorphosis of each of the two clumps
of daughter chromosomes into a new resting daughter nucleus; the
details of this process are discussed below.

During telophase, or late anaphase, the cell body becomes constricted
between the two new nuclei, the constriction becoming deeper and deeper
till finally two separate cells are produced, each containing one of the
new daughter nuclei.

Each daughter nucleus thus contains one of the products of division
of each of the chromosomes in the mother nucleus. As regards chromo-
some constitution, the daughter nuclei are therefore of like constitution
with each other and with the mother nucleus.

A fact of fundamental importance for cytological theory, and one
that has been established by innumerable observations, is that, with
certain mostly well-understood exceptions which will be discussed in the
later chapters of this book, the number of chromosomes in the nuclei
of any given species is constant. Thus to take the species whose nuclei
are figured in this chapter, the number of chromosomes in the nuclei of
Lepidosiren is 38. It is indifferent in what tissue the nucleus is situated ;
whether it is a skin, nerve, muscle, connective tissue or other nucleus,
the number of chromosomes which it exhibits at mitosis is 38. In

12 CYTOLOGY. CHAP.

Allium cepa (the onion) the number is 16. The species Ascaris megalo-
cephala contains two varieties, one of which, bivalens, has four chromo-
somes, the other, wnivalens, has only two. This last example has the
smallest number of chromosomes yet recorded, or indeed conceivable,
for an animal reproducing itself sexually, as will appear directly. The
largest number of chromosomes so far accurately determined for any
species is probably the 208 found in the crustacean Cambarus tmmunis
(Fasten, 1914). Often nearly related forms differ widely from each
other as regards the number of their chromosomes.

Another extremely important fact is that in many species, both of
animals and plants, the chromosomes in any one nucleus are not all of
the same length, and, moreover, that the relative sizes of the chromosomes
are constant, in spite of the fact that the whole series of chromosomes may
be longer in some tissues than in others. The relative size differences
are also independent of the changes in length undergone by the chromo-
somes during mitosis, for these affect the whole series of chromosomes
alike and approximately simultaneously. Indeed, throughout the whole
mitosis, from the early prophase to the end of anaphase, the chromosomes
are almost continuously shortening and thickening.

The constancy of the number of the chromosomes and of their relative
sizes in individual organisms and species, together with many other
considerations discussed in Chapter V., has led to an almost general
agreement that the chromosomes, though not recognizable as such in
the resting nucleus, nevertheless maintain their continuity from one
mitosis to another, so that the substance—at least the essential living
substance—of each chromosome, though diffused in the resting nucleus
and indistinguishably intermingled with the substance of the other
chromosomes, is nevertheless condensed together again in the next
prophase. The same series of chromosomes that entered into the resting
nucleus at telophase reappears therefore at the next prophase, each
single chromosome of the one stage being continuous with one of the
other.

An examination of those species in which the size differences among
the chromosomes are strongly marked discloses at once the fact that
there are two chromosomes of each size. If the chromosomes of such a
form are designated, in order of magnitude, by the letters of the alphabet
A, B, C .. ., we find in each nucleus two chromosomes of each kind,
namely, A+A+B+B+C+C... We may here anticipate the later
chapters by explaining that this double supply of chromosomes is due
to the fact that in sexual reproduction the new individual is formed by
the union of two germ cells, one from each parent, and that each germ
cell has one complete set of chromosomes (A + B+C+ . . .), so that the
fertilized egg has the doubleset (A +A+B+B+C+C+ ...). Owing

I THE CHROMOSOMES 13

to the continuity of the chromosomes, this double series is perpetuated
_ throughout the nuclei of the growing embryo. Corresponding chromo-
somes, for example, the two A’s, are known as homologous chromosomes.

The above remarks on the continuity of the chromosomes and the
nature of the chromosome equipment of organisms are anticipatory
of the later chapters of the book, where these points will receive more
detailed consideration ; we will now return to certain problems of mitosis,
the main steps of which we have just outlined.

(1) The Division of the Chromosomes, and theiy Relation to the Resting
Nucleus

As we have seen, the prophase chromosomes are often from their
first appearance double, 7.e. split into two daughter chromosomes,
possibly signifying that the chromosomes, or rather the elements of
which they are composed, were already divided in the resting nucleus.
It is even possible that the actual moment of division may be during the
anaphase of the previous mitosis.

The problem of the mode and moment of division of the chromosomes
is therefore intimately bound up with the question of the exact processes
by which the compact chromosomes of the anaphase are changed into
the resting nucleus, and those by which they are condensed out of it
again in the following prophase. Moreover, a knowledge of these processes
is essential to a proper understanding of the relation between the structure
of the resting nucleus and that of the chromosomes.

Unfortunately the telophase and early prophase are two of the most
difficult stages of the whole nuclear life-history to interpret, and very
different accounts of them have been given by different workers, some
of whom have claimed a large measure of generality for their conclusions,
explaining the conflicting results of other researches by faulty fixation,
wrong interpretation, etc. The contradictory conclusions reached by
different cytologists must indeed be attributed to these factors to a certain
extent, since they often refer to the same object. This is very well
illustrated by the work on three forms which have been much studied
on account of the size and clearness of their cytological elements, namely,
the tissue cells of the larval salamander, the developing eggs of Ascaris
megalocephala, and the root tips of the onion (Alliwm cepa).

The egg of Ascaris is specially favourable for this study owing to its
small number of chromosomes, and to the fact that in the nuclear re-
construction at telophase the ends of the long chromosomes usually
form projections from the main mass of the nucleus, thus greatly facilitat-
ing the study of the changes taking place in a single chromosome (these
remarks refer to nuclei in the “ germ track ” only, see Chapter ITI.).

Figs. 6, 7, 8 show the process by which the chromosomes pass into the

14 | - CYTOLOGY eHAP:

resting nucleus at telophase, and reappear in the following prophase in
Ascaris, Salamandva and Allium respectively, as observed by different
workers.

The following are the principal views held regarding these three
objects :

(1) The telophase chromosomes undergo a process of vacuolation, by |
which each becomes converted into a spongy cylinder; this becomes
further decomposed into a loose spongework. The spongeworks formed

PIG. 6:

Blastomere nuclei of Ascaris megalocephala, showing the evolution of a single spiral thread from each
telophase chromosome, and its reappearance as the prophase chromosome of the following mitosis. (After
Bonnevie, A.Z.,1 1908.) A, B, telophase ; C, resting nucleus ; D, prophase.
by all the chromosomes become indistinguishably merged into one
another, forming a “ network of networks,” which is the constitution
of the resting nucleus. In the prophase a reverse process takes place,
each chromosomal spongework becoming concentrated first into a spongy
band, and then into a homogeneous thread. Division of the chromo-
somes into daughter chromosomes takes place in prophase. [Van
Beneden and Neyt (1887), Ascaris ; Boveri (1909), Ascaris ; Kowalski
(1904), Salamandra (Fig. 7) ; Grégoire (1906), Allium (Fig. 8).]

(2) The telophase metamorphosis consists essentially in the formation
of long threads from the chromosomes; the reticulum of the resting

a”)

1 For the abbreviations used in references to certain journals, see p. 217.

I THE CHROMOSOMES 15

nucleus is formed by the intertwining of these threads, which at the same
time become irregular and broken up (as regards the chromatin; the
linin basis of the threads remains continuous) and connected with each
other by anastomoses. Prophase consists of the reverse process. There
are two variations on this view :

(a2) Two threads are formed from each chromosome, and hence the
prophase chromosomes are from the first double, and even the resting
nucleus is duplex as regards its chromatin constituents. According to
this view, therefore, the real division of the chromosomes into daughter
chromosomes takes place not in prophase but in the previous telophase

Fic. 7.

Larva of Salamandva maculosa. (A, B, after Kowalski, L.C., 1904 ; C, D, after Schneider, Fest. fiir
R. Hertwig, 1910.) A, C, telophase; B, D, prophase.

or anaphase. This view is held by many workers, e.g. Schneider (gto),
Salamandra (Fig. 7); Dehorne (1911), Salamandva and (1911) Allium
(Fig. 8), with, however, a different interpretation as to the part played
by the anaphase division in the following mitosis ; Lundegardh (1913),
Allium ; Schustow (1913), Alliwm.

(6) Only one thread is normally produced from each chromosome in
telophase, the division of the chromosomes taking place in prophase.
[Bonnevie (1908), Ascaris (Fig. 6) and Allium (Fig. 8) ; Boveri (1909),
Ascaris, exceptionally ; Vejdovsky (1911), Ascaris. |

It is clear that where different workers base such contradictory
conclusions on identical material, the reason for their differences must
be sought largely in the difficulty of interpreting these confused stages,

16 CYTOLOGY CHAP.

so that the same microscopical picture is interpreted by one cytologist as
vacuolation, by another as the unravelling of a single twisted thread,
andby a third as the intertwinings of two threads. Thus, even in the
most obvious cases of thread formation, this is always accompanied by
irregularities in the thickness and in the distribution of the conspicuous

Fic. 8.

Root tips of Allium cepa. (A, B, C, after Dehorne, A.Z., 1911 ; D, E, after Bonnevie, A.Z., 1908 ; BAG:
after Grégoire, L.C., 1906.) A, D, F, telophase; C, E, G, prophase; B, resting nucleus.

chromatin along the inconspicuous linin basis of the thread, and also by
outgrowths and anastomoses, which are generally sufficient to conceal
entirely in the resting nucleus its essential construction out of compara-
tively few long threads. The same factors, acting in telophase at a still
earlier stage of thread formation, may easily conceal the true nature of
this process, and convert what is essentially an irregular and twisted
thread into the appearance of a reticulum. On the other hand, the early

I TEEOPELASE £7

stages of the conversion of a long cylindrical object (like the anaphase
chromosomes) into a spongework or reticulum is naturally the formation
of a row of vacuoles down its axis, and this is easily mistaken in optical
section for a splitting of the cylinder into two threads, or as the develop-
ment of a coiled thread. Both these explanations have been invoked
by cytologists to explain the different interpretations arrived at by their
fellow-workers. Thus, it would be impossible to decide whether the
structure of the telophase chromosomes of Fig. 3, H, as revealed by their
transverse sections in Fig. 3, I, has been derived from that of the compact
anaphase chromosome by its simple vacuolation, or by the formation
within it of a spirally wound beaded thread, or even of two such threads
intertwined.

The theory of thread formation in telophase is an attractive one,
for it is highly probable, firstly, that the physiological meaning of the
telophase metamorphosis of the chromosomes is the resulting increase
of surface, compared with volume, of the chromosomes, and secondly,
that the chromosomes, at any rate in prophase, consist of chromomeres
or other constituents arranged in a linear series. The simplest way of
conserving this linear arrangement, and at the same time increasing the
surface, is by the outgrowth of the compact anaphase chromosome into
a long thread ; this, moreover, explains the usually immense length of
the early prophase chromosome compared with the same chromosome in
metaphase or anaphase. Indeed, where the telophase metamorphosis of
the chromosome consists of vacuolation and reticulation we must suppose
that the linear arrangement is only obscured (as, for example, in the case
of a long string of beads which has become tangled into a knot) and
not lost, for it reappears in the following prophase (which we may compare
with the unravelling of the knot).

Summing up, we must take as provisionally established the following
propositions :

(1) The telophase transformation of the chromosomes consists
essentially of an increase of their surface relatively to their volume.

This may be effected either by the conversion of the compact anaphase
chromosome into a long thread, or by its vacuolation, reticulation or
other method ! of irregularizing its outline, with consequent temporary
loss of visible linearity of structure, though essentially this is retained.

(2) The division of the chromosomes into daughter chromosomes in
preparation for the metaphase may take place in the anaphase or telo-
phase of the preceding mitosis, or may not be demonstrable till the
prophase or even till the beginning of the metaphase itself.

1 For example, the curious forms assumed by the chromosomes in the germinal vesicles
of many animals, Fig. 23.

Cc

18 CYTOLOGY CHAP.

(2) Chromatin and Linin

Chromatin is distinguished from linin by its much greater.affinity for
most stains. It is from this feature that it gets its name, while the linin
is often called, in contradistinction, achromatin. By many cytologists
chromatin is believed to be composed of very minute granules, or
chromioles (see Heidenhain, rg11). The blocks of chromatin seen in
most resting nuclei, or the chromomeres of prophase chromosomes, are
aggregations of numbers of chromioles.

In many nuclei the meshes of the chromatin and linin spongework are
filled with a granular mass, as if the karyolymph had been precipitated
by the fixative. According to Heidenhain (see 1g11), however, the
granules are granules or chromioles of a substance allied to the true
chromatin, and known as oxychromatin, the chromatin proper then being
designated basichromatin. This terminology is based on the fact that
the chromatin in the usual sense of the term, 7.e. the basichromatin, has
a special affinity for the basic aniline dyes, while the oxychromatin stains
more readily with acid dyes. (These terms do not refer to the acid or
alkaline reaction of the solutions of the stains, but to their chemical
derivation.) Unless the contrary be stated, the word chromatin as used
in this book refers to the basichromatin.

The interpretation of the granular mass in the meshes of the true
chromatin spongework as composed of pre-existing granules and not
due to precipitation of the karyolymph by the fixative is made more
probable by the observations of Gross (1916), who was able to observe
these granules in the living nucleus.

There is some reason to believe that the two kinds of chromatin (ii
indeed the oxychromatin be entitled to this designation) are different
phases of the same substance, for they appear to be convertible into each
other—as, for instance, in the growth stage of the oocyte (cf. Jorgensen,
1913). Moreover, in the prophase of all mitoses the oxychromatin
disappears completely, either by solution, or by conversion into or absorp-
tion by the basichromatin. Hence a nucleus rich in oxychromatin
presents a characteristically different appearance in the resting and
prophase stages ; in the latter the spireme filaments stand out sharply
in a perfectly clear karyolymph, while in the former the basichromatin
spongework is partially obscured by the mass of faintly stained oxy-
chromatin. In the young daughter nuclei the oxychromatin is formed
anew, probably at the expense of the basichromatin. Doubtless in
correlation with its disappearance in mitosis, oxychromatin is generally
very scanty, or altogether absent, in nuclei which are undergoing rapid
multiplication.

It is generally supposed that the chromatin (in the form of chromioles)

I NUCEEAR CONSTITUENTS 19

is embedded in the linin, but a few cytologists (e.g. Grégoire and Wygaerts,
1904; Lundegardh, 19f2) hold the view that there is no distinction between
the two substances, and that the common aspect of the nuclear substance
as consisting of a deeply staining material superimposed on a much
finer and more weakly staining framework is not due to any chemical
difference between the two parts, but simply to the fact that the stain is
retained by the coarser masses (chromatin) but not by the very fine
strands usually interpreted as linin. This view, however, seems to have
little in its favour, and there are very great difficulties in the way of
accepting it. For instance, in the prophase the young chromosomes are
often connected by numerous fine transverse unstained threads (linin)
(Figs. 3, D, 16, G). The chromosomes are all approximately of the same
thickness, and the transverse threads, which are much finer than the
chromosomes, are also approximately equal in thickness to one another.
If these threads were merely thinner strands of the same material as
the very much thicker chromosomes, it is hard to understand why we
do not find all gradations in thickness between the chromosomes and
transverse threads.

Heidenhain (1911) considers the linin to be the contractile substance
by which the movements undergone by the chromatin in prophase and
telophase are brought about. It is probably closely similar to cytoplasm
in nature, though plainly of a firmer consistency.

(3) Nuclear Membrane and Karyolymph

The mode of formation and nature of the nuclear membrane is
uncertain. It is possible that it is formed out of the linin framework of
the nucleus, or, on the other hand, it may be a condensation or precipita-
tion of the cytoplasm where it comes into contact with the karyolymph
or nuclear sap which accumulates between and within the chromosomes
at telophase. The telophase nucleus may in this case be conceived of as
lying in a vacuole full of karyolymph, the cytoplasm round the circum-
ference, and therefore in contact with the nuclear sap, becoming hardened
to form the nuclear membrane. Whatever its mode of origin, the
membrane becomes an integral part of the nucleus.

As a rule, the nuclear membrane disappears in the late prophase, but
it may persist throughout the whole mitosis (e.g. many insects) or may
disappear in early prophase (e.g., Mesostoma, von Voss, 1914).

(4) Karyosomes and Prochromosomes

The term karyosome may be applied to any mass of chromatin large
enough to stand out conspicuously from the general nuclear groundwork.
| Thus in a finely reticulated nucleus a comparatively small aggregation of
chromatin may be alluded to under that name, while a nucleus with a

20 CY TOLOGY CHAP.

coarser structure may contain larger chromatin masses which would not
receive that title.

No general rule can be given as to the relation of the karyosomes of
the resting nucleus to the telophase or prophase chromosomes. In some
cases they are portions of the chromosomes which have failed to undergo
the telophase dissolution and remain as compact chromatin blocks. This
is well seén in Fig. 9 (Lepidosiven). Here the anaphase chromosomes
form a dense ring (daughter
plate), the apices of the V-shaped
chromosomes being on the inner
. circumference of the ring, while
the limbs radiate outwards. At
the end of telophase, when the
daughter nucleus has been re-
constructed,a ring of karyosomes
occupies the place previously
occupied by the apices of the
V’s. These bodies gradually get
dispersed through the nucleus
and. disappear, so that in the
middle of the resting period they
are absent.

On the other hand, in many
forms in which karyosomes occur
they are not traceable back to
the telophase chromosomes, but
are secondary formations, the
newly reconstructed nucleus
being without them (Allium,
Lundegardh, 1913).

As a rule, the ‘number of

Fic. 9. karyosomes in the nuclei of any
Telophases in mesoderm cells of Lepidosiren. organism is highly variable, but
A, side view ; B, polar view. y

in other cases the number is
found to be constant, and to be the same as the number of chromo-
somes in the species in question, as shown by Rosenberg (1904). More-
over, these karyosomes may act as centres of formation for the
chromosomes in prophase, for which reason they have received the
name of prochromosomes (Overton, 1906). They have been specially
studied in plant cells (Fig. 10).
The presence of “‘ prochromosomes ”’ in the resting nucleus has been
taken, with some justice, as additional evidence of the continuity of the
chromosomes from one mitosis to another. It must be remembered,

I THE ACHROMATIC FIGURE ZY.

however, that they only form a special case of karyosomes present in
varying numbers, and often not traceable into the prophase chromosomes.

(5) The Achromatic Figure

This is the name given to the centrosome and system of radiating
lines proceeding from it through the cytoplasm, which are plainly con-
cerned with the separation of the daughter chromosomes. The term
refers to the fact that (with the exception of the centrosome and centriole)
the substance of which the system is composed (sometimes known as the
archoplasm) has much the same weak staining reaction as the bulk of the
cytoplasm and the linin. The main features of its development during
mitosis and its general disposition have already been described (Fig. 2).

A complete achromatic figure at the metaphase of mitosis consists of
the following parts: (rz) A minute deeply staining centrosome occupies
the centre of the radia-
tions at each pole of the
figure, and may contain Oo SS A

(2) a still smaller central eNGy } AN AN i ,
granule, the centriole. ‘s | ~ eee ‘ay Se EN
The centrosome is the Ne ee ~F ee
point of insertion ! of the BAY Poy <4 ee
so-called fibres of the ah Zz ay SOS N
achromatic _ figure, sae, 7
namely, (3) the radiating Fic. 10.

fbrescomposing theaster, — _ Preckromosnnes in Pollen Moths raaioas By prophase
and the spindle fibres.

The latter are of two kinds,-(4) the mantle fibres, which are attached to
the chromosomes, and (5) the fibres of the central spindle, which run right
through from one centrosome to the other. In many forms, however, a
central spindle seems to be absent.

The terminology of these various parts is unfortunately in some
confusion, especially so far as concerns the centrosome and immediately
associated structures. This is largely because the centres of the system
are occupied by a substance arranged in concentric layers, and opinions
differ as to how much of this should be called centrosome. The centriole
is very often, indeed generally, indistinguishable from the rest of the
centrosome, and in this book the latter term is used to cover the centro-
some together with the contained centriole (when such is present).

Fig. 2 illustrates a case where the whole, or nearly the whole, achro-
matic figure arises from the cytoplasm outside the nucleus. Very often,
however, the spindle at least is of intranuclear origin, probably derived

1 The fibres cannot always be traced actually into the centrosome, but sometimes end in
a clear spherical mass of cytoplasm, called the centrosphere, surrounding the centrosome.

22 CYTOLOGY: CHAP.

from the linin; and finally the whole achromatic figure, including the
centrosomes, may be intranuclear (e.g., Ascaris megalocephala univalens,
Brauer, 1893).

In nearly all resting cells the achromatic figure disappears except for
the centrosome, and occasionally a mass of differentiated cytoplasm
surrounding it, called the attraction -sphere; this corresponds to the
central mass of the aster which surrounded the centrosome during mitosis.
Even the centrosome can only be demonstrated on favourable material.
In most resting cells both cytoplasm and chromatin seem to be disposed
without reference to the centrosome, but in others this body obviously
exerts a powerful influence on the disposition of the various cell con-
stituents. A good example of such a cell is afforded by the gametocytes
in the “ bouquet ”’ stage (Chapter IT.).

Though so minute, the centrosome is often a conspicuous body owing
to its intense affinity for certain common stains. In order to form the
spindle figure it divides by simple fission into two daughter centrosomes,
which separate from one another, spinning out the central spindle (when
such is present) between them, and each becoming the centre of an astral
radiation. These facts have led many cytologists to look upon the
centrosome as a permanent cell organ, comparable in autonomy to the
nucleus, and only arising by division of a previous centrosome. This
view, however, is beset with grave difficulties. There is, for instance,
strong evidence that a centrosome may arise de novo in the cytoplasm,
and thereafter behave in precisely the same way as a centrosome derived
by fission from a previous centrosome (p. 95). Moreover, in the higher
plants, which have an achromatic figure otherwise essentially like that
of animals, there are no centrosomes.

The division of the centrosome may, like that of the chromosomes,
take place in anaphase, telophase or prophase. In the two former cases
it is of course double in the resting cell.

It must be remembered that the division of the chromosomes is an
autonomous process independent of the achromatic figure, for it often
takes place before the spindle figure is formed or while it is still outside
the nucleus. For the separation of the daughter chromosomes, however,
a properly developed achromatic figure appears to be essential. Thus
Wilson (1901) found that in the eggs of the sea-urchin developing by arti-
ficial parthenogenesis (p.° 95), various abnormalities of the achromatic
figure often appeared. One such irregularity was the failure to form a
proper bipolar spindle, instead of which a single aster only was formed
(monaster, as opposed to the amphiaster of a normal bipolar figure). In
such eggs the ordinary nuclear cycle may be gone through many times.
At each mitosis the chromosomes divide, but the daughter halves do not
separate ; instead of forming two daughter nuclei, they enter into a

1 THE ACHROMATIC FIGURE 23

single resting nucleus again, and there is no cell division. Thus nuclei
are produced with three or four times the normal number of chromosomes.

The mechanism by which the achromatic figure brings about the
separation of the daughter chromosomes and subsequently cell division
is still imperfectly understood. Two main theories are held: one that
the “ fibres’ of the astral rays and spindle figure are actually what
they appear to be, namely, fibres or threads, and the other that they are
merely lines of force or stress. The first and simplest form of the fibrillar
theory supposed that the mantle fibres are contractile, comparable to
muscle fibres, inserted at one end into the centrosome and at the other
into the chromosomes. The centrosome being held in place by the
astral rays, contraction of the mantle fibres pulls the chromosomes

HiGsar te

Early and late anaphase in the formation of the first polar body in Echinus esculentus.
(Bryce, Q.J.M.S., 1903.)

towards the centrosome. This simple theory, however, is met by in-
superable difficulties. One of these is that in telophase of most mitoses
the chromosomes come very close indeed up to the centrosome, thus
demanding an apparently impossible amount of contraction on the part
of the fibres, while such a contraction would of necessity be accompanied
by a relatively enormous thickening of the fibres, which, however, is not
observed. Again, in the formation of the polar bodies during the matura-
tion of the egg, the spindle fibres appear to exert a pushing rather than
—or at any rate in addition to—a pulling action in bringing about their
extrusion from the surface of the egg (Fig. 11). These and many other
considerations have led to the further hypothesis that the separation
of the daughter chromosomes is aided by an elongation of the fibres
which connect the separating daughter chromosomes, together with
those of the central spindle, which pushes the centrosomes apart. How-

24 CYTOLOGY CHAP.

ever, no mechanical theory of contraction and expansion appears to be
capable of giving a satisfactory explanation of all the facts, and
consequently many cytologists look upon the rays of the aster and the
spindle “‘ fibres’”’ merely as the expression of lines of force emanating
from the centrosomes as centres. An analogy for this view is found in
the position taken up by iron filings in a bipolar magnetic field. A very
serious objection to this theory, however, is that the lines radiating
out from the two centrosomes frequently cross each other, and this
is incompatible with a system of lines of force. This is illustrated
incidentally in Fig. 33, D.

The mechanism by which the achromatic figure brings about the
movements of mitosis must therefore be for the present admitted to be
quite unknown. An account of the whole problem of the achromatic
figure, much fuller than attempted here, will be found in Wilson’s text-
book The Cell, and a discussion of the physical and mechanical problems
involved in mitosis is given by Meek (1913).

D. AMITOSIS

In amitosis or direct division, the nucleus, without departing from
the resting structure, divides by simple transverse fission. In the
simplest case it first changes from a sphere into a dumb-bell or hour-
glass shape (Fig. 12), and then becomes nipped across at the constriction
to form two separate nuclei. In other cases the nucleus produces lobes
which may become constricted off from the parent nucleus to form
independent nuclei. In this mode of nuclear multiplication there is no
chromosome formation, and apparently no mechanism to ensure that
the daughter nuclei are each supplied with one of each of the chromatin
elements of the mother nucleus, as is provided for by the longitudinal
division of the chromosomes in mitosis. Therefore if each product of
amitotic division, at least amongst the Metazoa and Metaphyta, were
able to form a complete set of perfect chromosomes, it is plain that we
should have to modify greatly the theory of chromosome continuity
and differentiation outlined above, or else postulate some at present
unknown mechanism for the precise partition of the chromatin elements
in amitosis. This would be especially the case if it were shown that
normal gametes could be produced from the descendants of cells which
have divided amitotically, though it is conceivable that tissue cells could
survive and multiply even though lacking some of the chromosomes or
chromosome constituents.

The mere occurrence of amitosis therefore is of little significance
unless it can be shown that nuclei produced in this way can afterwards
proceed to complete chromosome formation and normal mitosis. In

: AMITOSIS 25

many cases where amitosis has been described, no proof even that cell
division follows has been given, and in these cases there is no evidence,
even if mitosis does subsequently take place in the cell, that it is not
preceded or accompanied by refusion of the nuclear fragments. In the
spermatogonia of many animals the nuclei become deeply lobed or
constricted during. a prolonged resting period, and occasionally one or
more of these lobes becomes nipped off, producing a cell with two or
more nuclei, generally of unequal sizes. There is no evidence, however,
that cell division follows the nuclear fragmentation, nor do we find any
reason to believe that each of the nuclear fragments may proceed to a
separate mitosis within the same cell. On the contrary, there is every
reason for the belief that the lobing, or fragmentation, is merely a tempo-
rary phenomenon, probably correlated with the necessity for increase

Pe
_Kres :
aw
ian i
jon
> =e Pe Ry S OP one Pte I i iaT
Sasi oe ee
a 3 : 7 Y a

HiGs 12:

Four stages in “ amitotic ” division in a tendon of a new-born mouse. (Nowikoff, A.Z., 1910.)

of surface relative to volume, and that the lobes are withdrawn or the
fragments fuse together again before mitosis. In Lepidosiven the
spermatogonial nuclei during periods of nuclear inactivity become very
irregular and deeply lobed. Preparations for mitosis are only found,
however, in approximately spherical nuclei, implying that the lobes
have been withdrawn. Meves (1891, 1895) found that the spermato-
gonia of the salamander are lobed in the winter. In the spring the lobes
are withdrawn and the now regularly rounded nuclei proceed to mitosis.

In the cleaving eggs of Triton (Rubaschkin, 1905) the nuclei are often
completely divided into two, three or even more separate nuclei, though
it is probable that in this case they have not been produced by the
fragmentation of a single mother nucleus, but by the separation of the
telophase chromosomes into groups, from each of which a separate little
nucleus is formed. In prophase, chromosomes are formed in each one
of the separate nuclei, which remain isolated till the nuclear membranes

26 CYTOLOGY CHAP.

break down at the end of the prophase. A single metaphase figure is
then formed from the combined chromosomes of the various parts.

We therefore see that the fact that a nucleus is lobed, or divided into
separate parts, is no evidence that it is dividing, or has divided, into
daughter nuclei, each of which is capable of proceeding to a separate
mitosis. On the contrary, it is probably a temporary phenomenon
only, the lobes being withdrawn, or the fragments being fused to form a
single nucleus again, at mitosis.

Irregular or fragmented nuclei are known as polymorphic nuclei,
and are of fairly frequent occurrence. Besides the examples cited, they
are particularly characteristic of certain leucocytes.

Even when amitosis is followed by cell division, as appears to be
sometimes the case, it is very difficult to show that the nuclei can subse-
quently divide mitotically. Meves believes that normal mitosis may follow
a peculiar type of amitosis, accompanied by cell division, in the sper-
matogonia of thesalamander. The amitosis in these cases is not effected
by a simple nipping off of a lobe of the polymorphic nuclei described
above, but by a more complicated process in which the centrosome plays
an important part, and which has not yet been found to be of general
occurrence. The identification of nuclei in which true mitosis is taking
place as the descendants of nuclei which have divided amitotically is,
however, necessarily uncertain.

The amitotic multiplication of nuclei in the cleaving egg and germ
track of tapeworms was first described by Child (1904), but has been
contradicted by others (e.g. Richards, 1909; Harman, 1913). A con-
siderable controversy has grown up over this matter, for a guide to the
literature of which the reader is referred to Nakahara (1918).

An experiment by Nathansohn (1900), following one by Pfeffer, has
had a good deal of importance attached to it. So-called amitosis was
produced in the green alga Spirogyra by placing the living filaments in
I per cent ether solution. The process of nuclear division. was observed
under the microscope, and takes twenty-five to thirty minutes. The
nucleus, which contains (usually) one large nucleolus, becomes opaque,
then presently clears again ; two nucleoli are now found to be present.
Then the nucleus becomes constricted and divides, apparently by amitosis,
one nucleolus going to each daughter nucleus; cell division follows.
Nathansohn kept these cells under observation and found that mitosis
could take place in them, and even that conjugation occurred between
descendants of nuclei produced in this way. The above processes were
also examined in more detail in fixed and stained preparations.

Experiments by Hicker (1900), confirmed by Schiller (1909), on the
effect of ether on mitosis in the cleaving eggs of the crustacean Cyclops
have important bearings on the Spirogyra experiment. Living eggs of

= AMITOSIS 27

Cyclops were placed in weak solutions of ether in water, with the result
that, though amitosis was not produced, the normal course of mitosis
was superficially much altered. The anaphase and telophase especially
acquired a superficial resemblance to the later stages of amitosis, owing
to a tendency of the chromosomes to start their telophase metamorphosis
before they had completely separated in metaphase. The anaphase
thus consisted of two confused masses of chromosomes, which began to
draw apart while still connected with one another, forming an hour-
glass figure not unlike that formed by a nucleus dividing amitotically
into two. The modification of the normal course of mitosis thus induced
does not result in death of the nucleus, for eggs removed from the ether
solution into pure water resumed normal development.

Very similar results were obtained by Némec (1904) by the action of
chloral hydrate on the root tips of several of the higher plants.

It is probable, therefore, that the apparent amitosis observed by
Nathansohn was really a’ modified mitosis, i.e. a division preceded by
chromosome formation.

Proliferation by amitosis has often been described in pathological
growths, but here again there is no proof that normal mitosis may follow.

Summing up as regards the Metazoa and Metaphyta, it is extremely
improbable that normal mitosis ever takes place in nuclei produced by
true amitosis—that is to say, by direct mass division of the nucleus
without any sort of formation of chromosomes and their division into
daughter chromosomes for partition to the daughter nuclei.

A summary of the literature on the subject of amitosis in the Metazoa
and Metaphyta is given by Nakahara (1918).

The question of amitosis in the Protista must be reserved to
Chapter VII.

CHAPTER Wt
MEIOSIS

IN the life-cycle of the great majority of organisms there occurs a moment
when a new individual, the offspring, is formed by the fusion of two
reproductive cells budded off from the parents. The reproductive cells
are the gametes, and the cell formed by their union is the zygote. The
male gamete is the microgamete or spermatozoon, and the female gamete
the macrogamete or ovum.

This periodical fusion of cells at fertilization or syngamy involves the
fusion of their nuclei, and hence a mechanism must exist to prevent a
corresponding periodical doubling of the mass of nuclear constituents, or,
to put it from the point of view of the hypothesis of the continuity of
the chromosomes, we must look for a mechanism to prevent the doubling
of the number of the chromosomes at each act of syngamy. This
mechanism is found in the fact that each gamete is provided with only
one-half of the number of chromosomes characteristic of the ‘‘ species ”
(i.e. the zygote or ordinary individual).1 Thus the gamete is said to be
haploid and the zygote diploid in regard to their chromosome equipment.
The process of reducing the number of chromosomes to one-half is known
as meiosis.

The special cytological problems of the production of the gametes,
or gametogenesis, centre in the manner by which meiosis is brought about.
This always (in Metazoa) takes place in one of the last two mitoses
involved in the production of the gamete. Hence these two mitoses are
known collectively as the meiotic phase, though only one of them actually
effects the halving of the chromosome number, and is therefore, strictly
speaking, the meiotic division. In nearly all cases this division is the
first of the two mitoses of the meiotic phase—i.e. the penultimate mitosis
of that long series of divisions by which the gamete is produced from the
primordial germ cell. The second mitosis of the meiotic phase differs in
no essential from the ordinary mitoses of the body (somatic mitoses),
except that it takes place in a nucleus containing only half the number of

1 This applies to all Metazoa. In a few Protista, and in many Metaphyta, the haploid
individual is the characteristic representative of the species, the diploid phase being very

transitory. See Chapter VII.
28

CHAP. 1 MEIOSIS | 29

chromosomes. Hence the first mitosis of the meiotic phase—.e. the
| meiotic division proper—is often known as the heterotype, and the second
| one as the homotype, division. Before proceeding to a detailed considera-
tion of this important part of the life-cycle it will be necessary to give a
brief sketch of the course of gametogenesis.

The natural starting-point for this sketch is the primordial germ cell.
This is of course one of the products of those divisions of the unicellular
stage of the zygote by which it becomes transformed into the multi-
cellular adult, and it may be defined as the first of the mass of cells thus
produced to be dedicated to the formation of reproductive cells alone.
It is only in a few cases that this cell has actually been identified in the
developing embryo (see Chapter III.), but whether visibly recognizable
or not it is plain that such a cell (or cells) must occur in the development
of every organism.

In some cases, for example in Ascaris megalocephala (p. 80), the

nuclei and the character of the mitoses of the primordial germ cell and
its derivatives are distinguishable from those of the other tissues of the
organism by various features, but in most cases they are essentially
similar until the meiotic phase is ushered in by the prophase of the
penultimate division before the formation of the gamete.
S The general course of gametogenesis is very similar in the two sexes,
differences in detail being associated with the relatively enormous size
of the macrogamete. Correlated with this, only one of the four cells
resulting from the two divisions of the meiotic phase becomes, in the
female, a functional gamete, the other three forming the very minute
“ polar bodies.”

A diagrammatic scheme of the course of gametogenesis of both sexes
is given in the accompanying diagram (Fig. 13). A few words of general
description will suffice, as cytological details will be given in actual cases.

The period of multiplication really involves a much greater number of
divisions than shown in the diagram. The cells in this period are known
as spermatogonia and oogonia (or ovogonia). In some animals, the earlier
generations of these cells differ in certain visible characteristics from the
later ones, with the result that the former are often termed primary
spermatogonia (or oogonia) and the latter secondary.

The cells in which the first meiotic division occurs in the male are the
primary spermatocytes. This division gives rise to the secondary sperma-
tocytes. For the sake of brevity, these cells may be referred to as
Spermatocyte I. (primary) and Spermatocyte II. (secondary), etc. Simi-
larly, the two divisions of the meiotic phase may be referred to as
Meiosis I., Meiosis II., and the various stages of the two mitoses as
Prophase I., Metaphase I., Anaphase II., etc., according as they belong
to the one or the other of the two divisions.

\

30 CYTOLOGY aa

The spermatocytes II. give rise, by the second division of the meiotic
phase, to the spermatids, which are nothing else than young spermatozoa.

a

: polar bodies.

(Boveri, 1891.)

Secondary oocyte + first polar body.—
Ovum + first and second

Primary oocyte.

a
13.

BiG.

Diagram of the course of gametogenesis in the two sexes.

Primordial Germ Cell.

iN > Spermatogonia. Oogon ta_

Primary spermatocyte
Secondary spermatocytes.
Spermatids
[ young spermatozo

Porad uorpondiy Wnyy \pomag yymosy | aspyy 222029;

That is to say, each spermatid metamorphoses into a spermatozoon. Thus
four spermatozoa are produced from each spermatocyte I.

i MEIOSIS IN TOMOPTERIS 31

The fully formed spermatocytes I.—that is to say, just before the
meiotic mitosis takes place—are considerably larger than the later
generations of spermatogonia. Hence the interval between the last
spermatogonial mitosis and the meiotic division is called the growth
period. It is during this period that the changes of the long drawn-out
meiotic prophase are found.

In the female the growth period is much more conspicuous than in
the male, since it is during this period that the yolk, which is so abundant
in most macrogametes, is deposited, and hence the primary oocyte (or
ovocyte) is much larger than the primary spermatocyte. Meiosis I.,
instead of resulting in two similar oocytes II., is followed by a very
unequal cell division, resulting in one oocyte II., very nearly as large as
the parent cell, and a minute cell, the “ first polar body.” Meiosis II.
is followed by a similarly unequal cell division, resulting in the mature
ovum and “ second polar body.”

A. MEIOSIS IN THE MALE

The remarkable interest of the meiotic processes has resulted in a
great deal of attention being paid to this phase, and it has been investi-
gated in many species both of animals and plants. Unfortunately, the
difficulties of observation and interpretation are great, and have resulted
in a corresponding diversity of views as to the precise mode by which the
halving of the chromosome number is effected. .

In the first place we will describe the process as it occurs in the male
of the Polychaete worm Tomopteris (as described by A.and K. E. Schreiner,
1906 a), after which the more important variations on this scheme, or
different interpretations thereof, will be discussed.

(1) Meiosis in Tomopteris onisciformis (Fig. 14)

The spermatogonial or pre-metotic divisions present no special
differences from the somatic mitoses. The number of chromosomes is
eighteen. (It will of course be understood that the figures, being depic-
tions of actual sections, show only such chromosomes, or portions thereof,
as occur in the sections.) We may, therefore, begin our detailed description
with the last pre-meiotic telophase (Fig. 14, A, B). The daughter nuclei
reconstructed from this telophase are the primary spermatocytes. In
them the chromatin is arranged in a fine network, in which, however,
chromosome areas are said to be discernible in the form of parallel bands
along which the chromatin is more densely aggregated. A. and K. E.
Schreiner believe that by means of these bands the chromosomes of the
last pre-meiotic telophase can be traced continuously into the chromosomes
of prophase I. A conclusion of this nature, involving as it does the
negative demonstration that at no time between the two phases do the

32 CYTOLOGY CHAP.

chromosome areas lose their identity, is obviously a very difficult one to

Fic. 14.

Meiotic Phase in the male Tomopteris onisciformis. (After A. and K. E. Schreiner, A.B., 1906.)
A, B, telophases of last pre-meiotic (spermatogonial) mitoses; C, late telophase of same, passing into
primary spermatocyte ; D, leptotene stage; E, F, G, zygotene stage ; H, pachytene stage ; I, diplotene stage ;
J, diakinesis; K, nuclear membrane disappeared ; immediate prophase of meiotic I.; L, metaphase I.; M,
anaphase I.; N, prophase II.; O, metaphase II.; P, telophase II.

establish. Wilson, however (1912), who examined the Schreiners’ own
material, is inclined to agree with them on this point.

11 MEIOSIS IN TOMOPTERIS 33

An early stage in the preparation for the first meiotic division is
shown in Fig 14, D, where it can be seen that the chromatin is condensing
into fine threads, and also that (1) this condensation is most marked at
one pole of the nucleus (shown throughout the figure as the upper pole),
and (2) at this pole the chromatin threads converge in pairs. Antici-
pating, we may say, that each chromatin thread (of which there are
eighteen) is a chromosome, and that the pairing is not haphazard, but
that each pair consists of two homologous chromosomes in the sense
described on pp. 13 and 125.

In favourable preparations it can be determined that the centrosome
is embedded in the cytoplasm just outside that pole of the nucleus to
which the chromosomes converge.

Another important point to notice is that the condensed chromosomes
at the pole of the nucleus are not smooth, but resemble strings of
beads. These beads are the chromomeres, and will be further discussed
in Chapter V. This stage (Fig. 14, D), where the chromosomes are still
very fine, is known as the leptotene stage.

In the later stage, shown in Fig. 14, E, the condensation has spread
away from the pole along a further length of the chromosomes, and now
the homologous chromosomes which were paired in Fig. 14, D, are
beginning to approximate themselves still more closely, till they come into
actual contact. Like the preliminary condensation, this process begins
at the polar end of the chromosomes and spreads away from this point.
This coming together of pairs of chromosomes, which is of fundamental
importance, is often known as the conjugation of the chromosomes from its
resemblance to the conjugation of certain Protozoa, especially Infusoria
such as Paramecium. It is also known as syndesis, while the nucleus is
said to be in the zygotene stage.

Fig. 14, F, shows a more advanced stage of syndesis, and illustrates
also the fact that the process is not necessarily synchronous in all the
chromosomes. In the nucleus shown in the figure, three pairs of chromo-
somes are still in the leptotene stage.

In Fig. 14, G, syndesis is complete, and now instead of the eighteen
thin chromosomes of the leptotene stage we have nine thick chromosomes
formed by their fusion in pairs. Hence this stage is called the pachytene
stage. The chromosomes are now seen to be horse-shoe shaped, with their
ends directed towards the nuclear pole. A characteristic appearance
is thus produced in the pachytene nuclei of Tomopteris and of many other
forms which exhibit a similar polarization. This has earned for this
stage the further term of bouquet stage. In many species, however, such
a polar orientation is absent.

The chromosomes have by this time condensed and contracted

sufficiently to make it possible to count them, and it is found that there
D

34 CYTOLOGY CHAP.

are nine of the thick. bands, thus justifying the statement made above
that there were eighteen of the thin threads in the leptotene nucleus.

The nine thick chromosomes now present, having been formed each by
the conjugation of two homologous chromosomes, are said to be bivalent, in
contra-distinction to the separate, unconjugated or wnivalent chromosomes.

Fig. 14, H, is a later pachytene nucleus. In this the shortening and
thickening of the chromosomes, which proceeds throughout the meiotic
as throughout the somatic prophase, has progressed further.

At the stage shown in Fig. 14, I, a process the reverse of what
occurred in the zygotene stage is taking place, the bivalent chromosomes
splitting into their two constituents again. Hence this phase is called
the diplotene stage, or, since the members of each pair are often con-
spicuously twisted round one another, the strepsitene stage. The polar
orientation of the chromosomes is now less pronounced, and by the stage
shown in Fig. 14, J, it has quite disappeared. By this time the separation
of the two constituents of each pachytene bivalent has proceeded consider-
ably further, and indeed is complete in the case of some pairs. Others
remain attached at one end, giving rise to U-shaped figures, or at both
ends producing figures shaped (), or if twisted to figures of 8. Others,
again, may remain united at their middles and separate at their ends,
forming or \-shaped figures.

The nucleus has now commonly attained its greatest volume, and the
chromosomes are characteristically distributed round its periphery,
immediately beneath the nuclear membrane. This is the phase called
by Hacker diakinests, and is a conspicuous stage in most gametogeneses.
Soon after this the nuclear membrane disappears, and the chromosome
pairs lie in the cytoplasm (Fig. 14, K).

Thus the long series of changes which the nucleus passes through
from the first appearance of the leptotene threads to the point at which
we have now arrived, all take place in a long drawn-out mitotic prophase,
namely, the prophase of the first meiotic division, and are succeeded by
the metaphase and anaphase of this division (Fig. 14, L, M). In these,
the chromosomes which paired in syndesis and disengaged themselves
again in the diplotene stage are finally separated, one member of each
pair going into one daughter nucleus, and the other member into the
other. This division is therefore the true meiotic or reduction division,
since through its agency two secondary spermatocyte nuclei, each con-
taining nine chromosomes, are formed from one primary spermatocyte
nucleus containing nine pairs, or eighteen, chromosomes.

It will be noticed that each of the separating chromosomes in the
anaphase is itself split along its length. This is an exaggeration,
commonly found in meiosis, of the tendency (discussed in Chapter I.) of
chromosomes to exhibit already in the anaphase that division into two

I MEIOSIS IN TOMOPTERIS 35

daughter chromosomes, which will become operative in the succeeding
mitosis. In the present case, that mitosis is the second division of the
meiotic phase which follows immediately after the first without the
intervention of a resting stage. It is depicted in Fig. 14, N, O, P, and does
not differ essentially from a somatic mitosis except in having only half
the normal number of chromosomes. The last figure illustrates a not
uncommon minor feature of spermatogenesis, namely, that the meiotic
mitoses are not immediately followed by complete cell division, so that
for a time the four young spermatids are united in a four-lobed cell.

At this point we may leave the description of spermatogenesis in
Tomopteris, all the important nuclear phenomena being by now concluded.
The development of a spermatid into a spermatozoon is described in the
next chapter.

The fundamental fact in meiosis ts the segregation, into separate nuclei,
of the members of each pair of homologous chromosomes. This is brought
about in the metotic mitosis by the previous pairing of the homologues into
double or bivalent chromosomes, which take up a position on the spindle such
that the constituent chromosomes of each bivalent occupy the position taken
im an ordinary mitosis by the daughter halves of each single chromosome.
Thus the meiotic anaphase separates whole (homologous) chromosomes,
instead of the daughter halves of single chromosomes, and therefore the
daughter nuclei have only half the number of chromosomes present in the
previous cell generations. In other words, the pre-metotic nuclei have 2n
chromosomes, the primary spermatocyte has n double or bivalent chromosomes,
and the post-meiotic nuclet have n chromosomes.

The course of meiosis just described is schematized in Fig. 15, which
is a diagrammatic representation of its course in a species with four
chromosomes. Fig. 15, A, shows a pre-meiotic (spermatogonial) prophase.
The remaining figures illustrate the fundamentally important fact that
it is homologous chromosomes which pair together in syndesis, to break
apart again in the diplotene stage, and finally separate in the first meiotic
division. The proof of the statement that syndesis takes place between
homologous chromosomes is found in the large number of species in
which the chromosomes are of different lengths and even different shapes,
so that homologous chromosomes can be identified by their relative
sizes, just as they can be recognized in the diagram by their shading.
Species with chromosomes of varying lengths are very numerous and
will be met with frequently in the accounts in this book (see especially
p. 125). One of them, Lepidosiren, will be described immediately.

It is clear that, in the case illustrated, meiosis will result in gametes
having two instead of four chromosomes, and, moreover, that these
two will consist of one member of each of the two pairs present in the

36 CYTOLOGY | pares

pre-meiotic nuclei. As the chromosomes behave similarly in meiosis in
the female, the fusion of the male and female gametes results in the
reconstitution of a diploid nucleus with four chromosomes in two paits.
Thus, if we designate each of the differently shaded chromosomes of
the diagram by a different letter, the gametes contain chromosomes

Fic. 15.

Diagram of the principal stages of meiosis by parasyndesis. Two pairs of homologous chromoscmes are
shown, the members of one pair being stippled, and those of the other cross-striped. A, pre-meiotic
prophase, showing the four separate chromosomes; B, leptotene; C, pachytene; D, diplotene stages ;
E, diakinesis, showing the evolution of the definitive bivalents; F, meiotic metaphase; G, metaphase of
second division of the meiotic phase in the secondary spermatocyte formed from the upper daughter
nucleus derived from F,

A + B, and the zygote A+A+B+B. In syndesis pairing takes place
in such a way that the pachytene nuclei contain two bivalents, forming
the series AA + BB. We also see that one member of each bivalent was
originally introduced by the male gamete and the other by the female.

While the most important features of meiosis are all to be found

I MEIOSIS IN LEPIDOSIREN 37

in the above account of Tomopteris, various additional features and
minor modifications are commonly met with in other cases, as will be
illustrated by short descriptions of the meiotic phases in the lung-fish,
Lepidosiren paradoxa, and in certain insects.

(2) Metosis in Lepidosiren paradoxa (Fig. 16)

Lepidosiven is probably unsurpassed as an object for cytological
research, owing to the great size of its nuclei and the clear sharp outlines
of its chromatic elements as prepared for examination by the ordinary
cytological methods. Another great advantage which it possesses is
the fact that the chromosomes differ greatly from each other in size.
The number of chromosomes in the body tissues is thirty-eight, and
in the gamete nineteen.

The most important stages of the meiosis of this species are depicted
in Figs. 16 and 16a, in which a few pre-meiotic figures are also shown.
Fig.16, A, is a spermatogonial nucleus, taken from that part of the testis in
which active spermatogonial mitosis is proceeding. Its coarse structure as
compared with that of the spermatocyte I. nucleus is to be noted. This
is a common and conspicuous distinction between nuclei of these two
grades.

The spermatogonial prophases are of an extremely simple nature,
contrasting, therefore, strongly with the complicated series of events
which takes place in the meiotic prophase. The coarse blocks of
chromatin of the resting spermatogonial nucleus form themselves into
long threads by lengthening and fusion, and then these threads shorten
and thicken into the definitive metaphase chromosomes.

The further contraction of the chromosomes in anaphase (ig. 16, D)
brings to light a feature which is visible, though less conspicuous, among
the longer prophase or metaphase chromosomes, namely, that they are
of very different lengths (see also Fig. 65). Especially noticeable is the
pair of very long chromosomes which are bent (at this stage) into the
form of a V. As these chromosomes are about twice the length of the
next largest pair, they are easily identified whenever the chromosomes
are individually distinguishable.

As in Tomopteris, syndesis begins at the polar ends of the chromosomes
and spreads along them from this point. It will be noticed that polar
views of the nuclei are shown in this figure, while the corresponding
figures of Tomopteris represent the nuclei seen from the side.

The onset of the diplotene stage (Fig. 16a, H) is considerably obscured
in Lepidosiven by the simultaneous contraction of the greater part of the
chromatin elements into a compact mass, leaving the larger part of the
nuclear cavity free from chromatin. This is a frequent and extremely
characteristic feature of the meiotic phase, though it is not universal.

38 GYTOLOGY CHAP.

It is not found, for example, in Tomopteris. Owing to its conspicuous
nature and to the fact that it is confined to the meiotic prophase, it early
attracted the attention of cytologists, by many of whom it used to be
considered diagnostic of the fact that syndesis was in progress. In

Fie. 16:

Meiosis in Lepidosiren (male). (Agar, Q.J.M.S., 1912.) A, resting spermatogonial nucleus; B, C,
spermatogonial prophase; D, daughter plate from a spermatogonial anaphase; E, resting spermatocyte I.;
F, zygotene; G, pachytene nucleus.
consequence of this, the word synapsis proposed by Moore to cover the
whole of that period of meiosis in which syndesis occurs, has been applied
by many cytologists to its most conspicuous feature alone—namely, the
contraction just described. It has been thoroughly established, however,
that the contraction of the chromatin has no invariable relation to the

ic MEIOSIS IN LEPTDOSTIREN 39

J

conjugation of the chromosomes. Hence the word synizesis was proposed
for the contraction, and syndesis for the chromosome conjugation.

Fic. 16a,

Meiosis in Lebidosiren (male. (Agar, Q.J.M.S., 1911.) H, diplotene stage and beginning of synizesis ;
I, synizesis further advanced; J, synizesis breaking up; K, diakinesis, showing that the bivalents which
were formed in the zygotene stage are completely resolved into their univalent constituents; L, immediate

prophase of the meiotic division, showing the univalents pairing again; M, early anaphase I.
Unfortunately, however, cytologists are not agreed in their use of the
alternative words. Some continue to use the term synapsis for the

40 CYTOBOGY CHAP.

contraction, reserving syndesis for the chromosome conjugation. Others
use synapsis for the conjugation and synizesis for the contraction. In
this book the word synapsis is not used at all, on account of the confusion
as to which of the phenomena included in the original description it
should be reserved for. The term synizesis is employed throughout for
the contraction and syndesis for the chromosome conjugation.

That separation of the constituents of the pachytene bivalents which
occurs in the diplotene stage is carried to a much greater extent in
Lepidosiren than in Tomopteris, so that by the time diakinesis is reached
we frequently have all, or all except two or three, of the bivalents resolved
completely into their univalent constituents (Fig. 16a, K). Examination
of the stages leading up to this shows that in the beginning of the diplotene
stage the bivalents separate first in the middle, remaining attached ‘for a
time at their ends and thus form long oval rings. Subsequently these
rings break apart at the points of contact of the ends of their component
chromosomes. In diakinesis we thus find thirty-eight univalents, or at
least a majority of univalents with a few bivalents of which the com-
ponents have not completely separated.

Since in metaphase I. we again find nineteen bivalents, the chromo-
somes must reunite before this stage is reached. This second pairing
is apparently effected by the recently separated homologous chromosomes
coming into contact again, first by one end and later by the other, to
form closed rings similar to those present in the diplotene nucleus, with
the difference that the rings are now much smaller and thicker. In the
nucleus shown in Fig. 16a, L, there are fifteen complete rings, three pairs
of univalents are in contact by one end only (one of these being the
large pair) and two are still unpaired, accounting for the thirty-eight
chromosomes in all.

A conspicuous feature which now presents itself is the transverse
constriction or joint which is to be seen across each univalent chromo-
some. Sometimes (in the longer chromosomes) this takes the form of a
sharp angle in the chromosome, in others (the shorter ones) it is merely
a deep constriction making the chromosome dumb-bell shaped. The
result of each univalent being constricted across in this way is to make
each bivalent appear tetrapartite.

The development of these transverse constrictions can be traced by
gradual stages which space prevents from figuring here. It is not
apparent in the long chromosomes which are first formed by the disjunc-
tion of the bivalents in the diplotene stage, but as they contract it
gradually makes its appearance. An exactly similar process is to be
observed in the contraction of the long chromosomes of the pre-meiotic
metaphase, as they recede towards the poles in anaphase (Fig. 16, D).

I MEIOSIS IN INSECTS AI

Here again the joint takes the form of a sharp angle in the longer
chromosomes and of a deep constriction in the smaller ones. Evidently
the form it shall take depends upon the relative length and breadth
which a given chromosome has attained at a given moment.

As commonly happens in meiosis, there is usually no resting stage
between the two meiotic divisions in Lepidosiren. As soon as the chromo-
somes approach the poles in anaphase I. new spindles are formed and the
chromosomes, now longitudinally split, become arranged in the meta-
phase equatorial plates. Since each chromosome is still transversely
constricted as in the first division, figures very similar in appearance to
those of metaphase I. are obtained, although of course each tetrapartite
chromosome is now constituted out of two transversely constricted
daughter chromosomes, instead of out of two juxtaposed transversely
constricted whole chromosomes.

The pair of long chromosomes already alluded to should be noted in
Fig. 16a, K, L, M. From the study of favourable nuclei, it can be estab-
lished that these are formed by the breaking apart of one of the rings of
the diplotene stage. That is to say, they conjugated to form one bivalent
in syndesis, an example of the evidence mentioned above that conjugation
is not haphazard, but takes place between homologous chromosomes.

(3) Metosis in Certain Insects (Fig. 17)

The Insects are a group which have attracted much attention from
cytologists, the most important recent work on their meiosis being that
of Wilson (1912) on certain Hemiptera (Fig. 17).

The spermatogonial telophase (Fig. 17, A) passes into a confused
network (B) in which no chromosome limits are visible. The beginning
of the meiotic phase is marked by the appearance of a number of massive
chromatin bodies, of the diploid number (C). (The two denser bodies
visible at this stage, and more conspicuous in D-J, are the sex chromo-
somes, to be described in Chapter IV.)

The leptotene nucleus is derived from this stage by the resolution of
each one of the chromatin bodies into a spirally coiled filament, which
spreads out and interlaces with the other similarly formed filaments to
produce the leptotene nucleus. This is followed by synizesis, and this
by the pachytene stage. The mode of syndesis cannot be traced, but
since the leptotene threads are in diploid number and the pachytene
threads haploid, and since each pachytene thread splits longitudinally
into two in the diplotene stage (I), it is to be assumed that syndesis of
the type described for Tomopteris and Lepidosiren took place during
synizesis.

The fact is notable that neither the leptotene nor pachytene threads
are orientated to form a bouquet as they are in Tomopteris and Lepidosiren.

42 CYTOLOGY CHAP.

This absence of a bouquet is not, however, characteristic of insects in
general, for it is a conspicuous feature in many species.

Diplotene nuclei (I) show us that each pachytene thread proceeds to
split into a pair in the same manner as we saw in the two forms already

FIG. 17.

The formation of the meiotic bivalents in certain insects. (After Wilson, J.E.Z., 1912.) A-J, Oncopeltus ;
K, Anax; L-N, Protenor. A, B, spermatogonial telophase ; C, emergence of massive chromatin bodies in
spermatocyte I.; D, E, each chromatin body (with the exception of two, the sex chromosomes) is giving rise
to a single coiled thread ; F, the coiled threads of E have given rise to the leptotene nucleus ; G, synizesis;
H, the pachytene stage ;.the large, faintly stained body in this figure, and in figures I, J, is the plasmosome ;
I, diplotene stage ; J, ‘‘ confused stage”’; K, three contiguous cells showing the evolution of the coiled
threads from the massive bodies; L, M, N, evolution of the bivalent rings from the confused stage.

described. Instead, however, of these pairs condensing progressively
into the definitive bivalents of metaphase I., the early diplotene stage is
immediately followed by one which Wilson calls the “ confused stage ”’
(J), in which the double chromosomes lose their visible identity and
become merged into a vague, lightly staining reticulum. From this

II MEIOSIS 43

reticulum the bivalents presently condense out (L, M, N) in the form of
long loops, frequently twisted. :

These further condense into “ tetrad rods”’ or double dumb-bells,
very similar to many of the bivalents in Lepidosiven. Wilson, however,
believes that the condensation takes place in such a way that the trans-
verse joints of the bivalents correspond to the original cleft which
separates the two components of each diplotene bivalent.! If this be so,
these transverse joints represent the points of junction of the constituent
univalents of each bivalent, and hence they are of quite different origin
and significance from the transverse constrictions of the Lepidosiven bi-
valents. In accordance with this mode of formation, the transverse joint
is the plane of division in metaphase I., which therefore separates entire
univalent chromosomes as it does in the two cases already described.

B. DIVERGENT VIEWS OF THE PROCESS OF MEIOSIS

While it is generally accepted that meiosis consists in the segregation
of homologous chromosomes, it must not be supposed that all cytologists
agree that the accounts of the processes leading up to that segregation
which have been given above for Tomopteris, Lepidosiven and Hemiptera
are either completely correct interpretations of what occurs in these
forms or, even if this were granted, that the process occurs in essentially
the same manner throughout the animal (and vegetable) kingdom.
However, it is the general scheme of meiosis which is accepted in essentials
by more cytologists—at any rate, students of animal cytology—than
adhere to any other one scheme, and it is rapidly gaining new adherents.
We will now briefly discuss certain other schemes of meiosis that have
been proposed, together with a few general problems of this important
phase, leaving out of account a few special hypotheses which have been
put forward to explain the phenomena observed in certain individual
cases, and which can lay no claim to generality. We may also leave out
of discussion the pure—and, as it appears to the author, unjustifiable—
scepticism of certain cytologists (for example, Meves) who deny the
possibility of coming at present to any useful conclusion as to how the
number of chromosomes is reduced in meiosis.

(1) Parasyndesis and Telosyndesis *
These two schemes of meiosis have much in common, and a great
measure of generality is claimed for each by their respective supporters.

1 Wilson, however, believes that fusion in syndesis is complete, the bivalent chromo-
some undergoing internal reconstruction so that it is not possible to homologize the two
chromosomes which separate in the diplotene stage with those which united in syndesis.
See p. 48.

* Called parasynapsis and telosynapsis by cytologists, who employ the term synapsis in
the sense in which syndesis is here used (p. 39).

44 CYTOLOGY CHAP.

In many cases meiosis in one and the same species has been interpreted
by one worker as parasyndesis and by another as telosyndesis. The
point of difference between the two schemes lies in the observation and
interpretation of the zygotene, pachytene and diplotene stages.

Syndesis as described above in Tomopteris, Lepidosiren and certain
insects, and illustrated diagrammatically by Fig. 15, is parasyndesis, so
called because in the zygotene nucleus the homologous chromosomes lie
side by side and conjugate along their lengths. This interpretation of
the zygotene stage, with its consequent reading of the pachytene and
diplotene stages, was first given in its present form by von Winiwarter
(Ig01), and has since gained wide acceptance. To the Louvain school of
cytology is specially due the credit of having established the hypothesis
on a firm basis, both by new observation and the review of old work in
the new light (Grégoire, IgIo).

The theory now known as felosyndesis was first proposed by Mont-
gomery in 1903 for various Amphibia, and independently by Farmer and-
Moore in 1905 for several other forms. It must, however, be mentioned |
that Montgomery subsequently (1911) gave up his original view in favour
of the theory of parasyndesis.

According to the theory of telosyndesis the thin threads which are
seen joining together in pairs in the zygotene stage are not whole chromo-
somes conjugating to form a bivalent, but are the temporarily separated
daughter halves of chromosomes split for forthcoming mitosis. On this:
view, therefore, the duplicity of the chromosomes in Figs. 14, D, E, F, and’
16, F, is of precisely the same nature as the duplicity of somatic prophase:
chromosomes (cf. Figs. 3, 7, 8, etc.). The undisputed fact that the
number of thick bands in the pachytene nucleus is haploid is supposed to:
be due to the fact that each consists of two homologous chromosomes
joined end to end, as shown in the diagrammatic figure.

So far we have been dealing with what is mainly a matter of interpre-
tation and not of observation, but the next stage in the process involves
a difference of opinion on a matter more nearly approximating direct
observation. The telosyndetic view requires that the double chromo-
somes of the diplotene stage are not formed, as described above, by the
reopening of the space between two approximating chromosome threads
of the zygotene nucleus, but by the approximation of the limbs of the
horse-shoe shaped pachytene bands. They may then join at the free
ends to form rings instead of U’s, or break across at the point of junction
to form two separate but adjacent chromosomes, together constituting
a bivalent.

The constitution of the bivalents is now the same in both schemes
(Figs. 15, E, and 18, C’), and the further course of meiosis is described alike
in both cases. In Fig. 18 are shown three stages in the formation of the

we. Ty aS ao. ae =

1 PARASYNDESIS AND TELOSYNDESIS 48

efinitive bivalents out of the pachytene loops as depicted by Farmer and
oore. It will be noticed that the longitudinal slit, faintly visible here
nd there in the pachytene stage (A, and A’) appears from these figures
o be traceable into the slits occasionally indicated in each constituent
f the bivalents, and therefore represents the division plane of the second
ivision of the meiotic phase.

It would take too much space to discuss fully the relative merits of
he two theories, but the most important pros and cons can be briefly
ummarized, remembering that it is impossible to reconcile the divergent

Illustrating telosyndetic view of meiosis. (A-C, after Farmer and Moore, Q.J.M.S., 1905.) A-C, three
stages in the formation of the bivalents in Osmunda regalis. A’-C’, their interpretation according to the telo-
syndetic view. * Supposed points of junction of the longitudinally split constituents of each bivalent.

views by the simple assumption that parasyndesis holds for some species
and telosyndesis for others. For the disputed stages—zygotene and
diplotene—are too similar in many of the species which have been inter-
preted in opposite senses to have been brought about by such different
processes, and, moreover, in many instances opposite accounts have been
given of the same species.

1. The overwhelming balance of evidence of actual observation
appears to the author and many others (including the older cytologists
who were not interested in either theory) to favour the view that the
diplotene and diakinetic figures are produced by the reopening of the

46 CYTOLOGY CHAP.

slit within each bivalent pair in the zygotene nucleus. Such cases as
Tomopteris, where there is no synizesis to obscure any stage in the process, —
seem to be decisive. This fact alone appears to dispose definitely of the

theory of telosyndesis. 4

2. A considerable ‘degree of similarity patwech the duplicity of the
prophase chromosomes of many somatic mitoses and that of the zygotene -
pairs must be admitted. The threads which come together in the zygo-
tene stage are, however, more distinct than those of somatic prophases. —
In the leptotene nucleus, or earlier, they often show little or no sign of
paired arrangement, in this respect differing greatly from the duplex
chromosomes of somatic prophases, where the two longitudinal portions
of each chromosome are probably always closely approximated to one
another. Moreover, we do not find in somatic prophase that regular
fusion of the members of the pairs spreading away from their polar ends
which is such a characteristic feature of the zygotene nuclei in those
organisms which exhibit the bouquet orientation in this stage (e.g.,
Tomopteris, Lepidosiren). The fact, therefore, that the theory of
parasyndesis has to interpret the frequent duplicity of somatic prophase
chromosomes as caused by fission, and the superficially somewhat similar
duplicity of the zygotene nucleus as fusion, is no serious drawback to
that hypothesis—especially when it is remembered that, as will be
evident from the next paragraph, such a differentiation must in any
case be made between the double prophase chromosomes of Culex and
those of most other organisms.

3. There is commonly observed, even in somatic mitoses, a tendency
for homologous chromosomes to lie side by side, and this tendency can
be traced through all intermediate degrees up to its climax in Culex,
where even in somatic, spermatogonial and oogonial mitoses, homologous
chromosomes may be indistinguishably fused together, especially in
prophase and anaphase. It is instructive to compare Figs. r4, I, 17, I,
19, B, E, with Fig. 56, C, D, E, H, remembering that in the latter there is
no questioning the fact that the longitudinal components of the double
chromosomes are each an entire chromosome, the pairs being formed by
approximation of these, and not by fission of a single original one. Para-
syndesis is therefore but the climax of a widespread tendency of homolo-
gous chromosomes to apply themselves side by side.

4. As stated in Chapter I., a tendency for chromosomes to adhere by
their ends is sometimes observed in somatic mitoses. This may be taken
as favourable to the view that syndesis is effected in the same way, 7.e.
by telosyndesis.

5. There is no direct evidence that the pachytene loops consist of
two chromosomes united end to end. The fact that a break is often
observed in the loop, dividing it into two portions connected by an

I PARASYNDESIS AND TELOSYNDESIS 47

achromatic band, has indeed been cited in evidence (Montgomery, 1903 ;
Schellenberg, 1911). As, however, this break is frequently not in the
middle of the loop (Schellenberg), it cannot be taken as the point of
junction of the conjugating chromosomes, since the limbs of the diakinetic
and metaphase rings, loops, etc., are equal. There can indeed be little
doubt that these breaks are the transverse constrictions which develop
across the contracting chromosomes in Lepidosiven and elsewhere. It is

Fic. 19.

| A-C, parasyndesis in Planaria gonocephala, (After Schleip, Zool. Jahrb. Anat., 1907.) D-F, parasyndesis
jin Dytiscus marginalis (after Henderson, Z.w.Z., 1907) ; A, D, leptotene; B, E, zygotene; C, F, diplotene
stages.

characteristic that these transverse constrictions are often by no means
in the middle of the chromosomes (Fig. 65).

This very short and incomplete summary of the arguments for and
against the only two schemes of meiosis which can lay any claim to
generality must suffice for the present. It is clear that parasyndesis
is the hypothesis accepted in this book, and we shall find, especially in
Chapters V. and VI., that we continually meet with observations as well
as experimental results which are readily intelligible on the assumption
that conjugation of the chromosomes takes place by parasyndesis, but
are quite inexplicable on the theory of telosyndesis.

48 CYTOLOGY CHAP,

(2) The Mutual Relations of the Homologous Chromosomes during Syndesis

Three views are possible as to the mutual relations of the chromosomes
which unite in syndesis, and these views have, as a matter of fact, all
been upheld. They are:

1. Syndesis is a temporary fusion of the conibarine chromosomes,
comparable to that of conjugating Infusoria, and the chromosomes
separate again in the diplotene stage. (Many cytologists—e.g. Schreiner.)

2. The chromosomes fuse completely at syndesis, so that the two
chromosomes which separate in the diplotene nucleus are not the chromo-
somes which came together in the zygotene stage, but new products
formed by the longitudinal fission of the compound chromosome formed
in syndesis (Bonnevie, 1906 ; Vejdovsky, 1907, I91I ; von Winiwarter
and Sainmont, 1909; Wilson, 1912).

3. Syndesis is only a temporary approximation of homologous chromo-
somes, which never become organically continuous (Grégoire, 1910).

The distinction between the last view and the other two is a real one,
but the difference between the first two is largely a matter of words.
If two chromosomes fuse, a mutual influence, if not actual exchange of
substance, is implied ; whether or not the chromosomes which separate
afterwards are to be considered as the same as those that entered into
combination is a question of dialectics. Are two separating exconjugant
Paramecia, after exchanging micronuclei, the same individuals as those
which entered into conjugation ?

While there is therefore no need to attempt to decide between the
first two views, the question of whether organic continuity is or is not
established between the approximated chromosomes is a very important
one. As we shall see later, the possibility of an exchange of substance
between homologous chromosomes is an important—probably indeed
essential—supplementary hypothesis to the chromosome theory of
heredity.

As in so many cases, however, it is very difficult to arrive at a decision
by direct observation. The view that the conjugating chromosomes do
not enter into organic continuity is founded upon certain cases in which
a dividing line between them appears to be present throughout the
whole period of syndesis. It therefore really rests on negative evidence,
namely, that at no time during syndesis is the dividing line absent—an
observation which, as any cytologist will recognize, would be an exceed-
ingly difficult one to establish. There is of course no need to suppose
that fusion takes place simultaneously over the whole chromosomes.
Indeed it is certain that in many forms it begins at one end and
spreads thence to the other. The presence of slits here and there in
a zygotene chromosome is therefore no evidence that the constituents
are not fused at other points, or at other stages.

Wl MEIOSIS WITH TETRAD FORMATION 49

On the other hand, the view that organic continuity is established
between the two participating chromosomes is much more securely
founded on the very numerous cases where complete contact is observed
between the constituents of each pachytene thread, either over the whole
length of the thread at once, or now at one point and now at another.

(3) Meiosis with Tetrad Formation

Much error and confusion has been introduced into the study of the
meiotic phase by the failure to recognize the true nature of the transverse
constrictions of the meiotic chromosomes, and that they are often of
no direct significance in the process of reduction. It was for long supposed
to be a general rule that the quadripartite chromosomes, so commonly
formed in meiosis by the junction of two bipartite chromosomes, are
composed of four masses, one of which eventually reaches each of the
four spermatid nuclei. These “ tetrads,” as they were called, were
supposed to divide across one joint in the first meiotic division, giving
two ‘“‘ dyads ’’—one to each spermatocyte II. In the second division
the dyads were supposed to divide across at the remaining joint, giving
one “ monad ”’ to each spermatid. Thus one division of the tetrad was
said to be longitudinal and one transverse.

In the case of Ascaris megalocephala, the organism to which our
knowledge of cytology is due probably more than to any other one
species, this is indeed the fate of the tetrads. These are, however, pro-
- duced in this species in a different way from that described for Lepidosiren,
—as will be shown below. A similar partition of the four parts of the
_tetrads among the four spermatids has been frequently described for
insects also. Here again it appears that the apparently transverse
constriction has an origin different from that in the lung-fish (p. 43).

In other cases, however, notably the Copepoda, accounts of the
distribution of the four segments of each tetrad, one to each spermatid,
were based on faulty observation. In this group it has more recently
been shown (Lerat, 1905 ; Matschek, rgto) that the chromosomes which
separate in anaphase II. are not monads formed by division of the dyads
into their two parts, but are still bipartite, being formed by longitudinal
division of the dyads of anaphase I. Thus the transverse joints of the
Copepod tetrad are of the same nature as those in Lepidosiven, and do
not represent a division plane.

The false view of the composition and fate of what may be called the
Copepod type of tetrad was intimately connected with the older theories
of meiosis. At an early period of cytological theory (Roux, 1883 ; Weis-
mann) it was recognized that a chromosome consists of a number of
smaller dissimilar elements arranged in linear series (see Chapter V.).
Consequently, a longitudinal division of the chromosomes results in the

E

50 C¥TOLOGY CHAP.

division of each one of these smaller elements, and hence the two daughter
chromosomes derived by the longitudinal fission of the mother chromo-
some are identical. If, however, a chromosome were to divide trans-
versely, the two resulting chromosomes would be dissimilar, as each
would contain only a portion (half) of the smaller elements.

Now in a meiosis with tetrad formation of the Copepod type it follows
that if both the joints represent division planes, one division must be longi-
tudinal (or eguational, since the resulting daughter chromosomes receive
similar sets of chromatin elements), and the other division must be
transverse (or veductional, since each resulting daughter chromosome
receives only one-half of the set of chromatin elements). As we have
seen, however, it was by an error of observation that the transverse
joint in a Copepod tetrad was taken to be a division plane, and it is now
almost universally held that ‘“‘ reduction” consists in the separation of
entire homologous chromosomes, and not in their transverse division.
Indeed, while there are many cases, notably amongst insects, still in
need of elucidation, it is more than doubtful if transverse division of
chromosomes in the above sense ever occurs as the result of mitosis.

As mentioned above, the “ tetrads’”’ of Ascaris megalocephala consist
of four masses, one of which eventually reaches each of the four sperma-
tids. In other words, both joints of the tetrad are division planes.
An examination of the mode of formation of these tetrads reveals, how-
ever, that they are constituted differently from that which we have called
the Copepod type, as exemplified in Lepidosiren, etc.

Ascaris has been the subject of a very great number of cytological
investigations. The classical description of the meiotic phase (in the
male) is that given by Brauer (1893) for the variety A. m. bivalens, in
which, it will be remembered, the diploid chromosome number is four.

His description practically starts with synizesis, in which the chromatin
is contracted to one side of the nucleus in a fairly compact mass, from
which, however, chromatin threads project (Fig. 20, A). These threads
are at once séen to be double, and when cut in transverse section, or seen
in end view, they reveal themselves as quadruple, being divided longi-
tudinally by two division planes at right angles to one another (Fig.
20, B). These quadruple threads are to be interpreted as formed by
syndesis of two homologous chromosomes, each split longitudinally (in
preparation for the second division of the meiotic phase). Brauer,
whose investigations were carried out before the modern ideas as to
syndesis had been formulated, did not interpret the quadruple threads
thus, but as formed by the double splitting of a single thread. Later
work on meiosis in this animal, however (de Saedeleer, 1912), has revealed
all the principal stages as found in Tomopteris.

1 MEIOSIS IN ASCARIS 51

By the later stage shown in Fig. 20, C, synizesis has completely dis-
appeared, and all the chromatin is in the form of a long, doubly split
thread (only one split is visible in the plane of the figure). As there are
really two bivalent chromosomes present, these must be joined tempor-

ie (IPD
Y oye

by)

D E

: ve | |
. a1 oe
\ F G H
= & SR * @ e®°
i OG : \\ K =

Fic. 20.
Meiosis in Ascaris megalocephala bivalens. (A-M, after Brauer, A.m.A., 1893; N-P, after O. Hertwig, A.m.A.,
1890.) A, B,synizesis and syndesis ; C, D, E, formation of the definitive bivalents. In E the left-hand bivalent
is seen in side view and therefore only two of the constituents are visible; the right-hand bivalent is seen in
end view, and therefore its quadruple constitution is revealed. F, metaphase I.; G, H, anaphase I.; I,
preparation for second division; J, metaphase II.; K, anaphase II.; L-M and N-P, condensation of the
definitive bivalents in nuclei in which the four constituents of each are well separated.
arily end to end to form an “ unsegmented spireme ”’ as described on p. 9.
Later (D) this dissociates into its two components. Each of these is a
bivalent chromosome, longitudinally divided into four owing to the fact
that each constituent univalent is, as described above, itself longitudinally

divided. The quadruple nature of each bivalent chromosome is well

52 CYTOLOGY CHAP.

illustrated by Fig. 20, L-P, which show the condensation of the tetrads
in nuclei in which the parts of each bivalent are well separated.

Each bivalent tetrad therefore now consists of four parallel rods
produced by the longitudinal apposition of two homologous univalent
chromosomes each longitudinally divided into two daughter chromosomes.
Before the metaphase is reached each of the four rods has so contracted
as to be nearly spherical, and the tetrad now consists of four nearly
spherical masses, one of which, as is sufficiently explained by the figures,
is distributed to each of the four spermatids. Thus, as in Tomopteris
and Lepidosiren, one of the two meiotic divisions is reductional in the
sense of separating homologous chromosomes, and the other, like an
ordinary mitosis, separates the daughter chromosomes produced by
fission of the mother chromosome. While there is here no means of
deciding which division does which, it may be assumed from analogy
that the first division is the reductional one.

The resemblance between the “‘ tetrads’”’ of Ascaris and those formed
by some of the chromosomes in Lepidosiren or in the Copepoda is very
close, but obviously only superficial. In each case the tetrad is bivalent,
but in the one case (Ascaris) the joint in each univalent is really longi-
tudinal (as shown by its origin) and marks the plane of division of the
second meiotic division. In Lepidosiren and the Copepoda, on the other
hand, the joint in each univalent is a mere transverse constriction, and is
not operative as a division plane.

The accompanying diagram illustrates the structure of a meiotic
chromosome in Lepidosiven, Ascaris, and an insect such as Oncopeltus.
The linear series of dissimilar elements of which the chromosome is com- |
posed is represented by the letters of the alphabet, one chromosome being
represented by ABCD and its homologue by abcd. The diagram
illustrates the fact that the first and last stages (syndesis and gametes)
are alike in all three cases, and also that in all three it is the first meiotic
division which separates the homologous chromosomes.

The early appearance of the longitudinal fissure which becomes
operative in the second division, and which is responsible for the “ tetrad ”
form of the Ascaris bivalent, is acommon feature of meiosis. When both
this fissure and a transverse joint are present in each component of the
bivalent, the chromosome assumes an “‘octad” shape. This is well
exemplified in the bivalents of certain Copepods.

It would take us too far to consider other schemes of meiosis which
have been proposed, especially as none of them can establish any claim
to general application. A warning, however, is necessary against the
too ready acceptance of accounts in which no syndesis proper is said to

1 MEIOSIS 7 53

occur, the homologous chromosomes being supposed to come together for
the first time in diakinesis.

Probably such accounts have been due to failure to realize the com-
plete temporary disjunction of the ex-conjugant chromosomes which
may take place in the diplotene stage (cf. Lepidosiren). Hence the

LEPIDOSIREN. ASCARIS. ONCOPELTUS.
ABCD ABCD ABCD

SPOS” Sg. Ge bet Ad abcd abcd

ABCD

: ABCD | ABCD
The Definite Bivalents ab-cd abcd
abcd

eToAaA-yowp
pooAa-YoOWPr

| A A
| isp Mis}
A B-CD ABCD GG
AB-CD ABCD 1B 2)
Anaphase I) 2-, 0. +. (|= —— Eee
ab-cd | abcd d d
ab-cd abcd c c
joy)
aoa
ite | a ee oa re | A
ri i ea B B Beeb
Y Cc c C (€ C GC
ee ee esis
Anaphases II. ¢
e a a a a d d
ea oad pve ae
ri Le iy ol re c b b
Ke a | ' d | d a | a
Diagram of the constitution of a pair of homologous chromosomes in Lepidostren, Ascaris

and Oncopelius. The hyphens represent transverse joints in the chromosomes.

erroneous assumption is made that if the diploid number of chromosomes
is found in diakinesis, no previous syndesis has taken place. The classical
| description of diakinetic pairing without previous syndesis is that of
Korschelt (1895) for Ophryotrocha, but the further researches of A. and
K. E. Schreiner (1906 6), and Grégoire and Deton (1906), have disclosed
that stages of the usual type indicating parasyndesis precede diakinesis in
this animal also.

54 CYTOLOGY CHAP.

(4) Which of the two Divisions of the Meiotic Phase effects the
Separation of the Homologous Chromosomes ?

Korschelt and Heider, in their general account of meiosis (1903) give —
two subdivisions of the different modes of meiosis which they recognize.
These are called Pre-reduction and Post-reduction according as to whether
it is the first or second division of the meiotic phase which effects the
reduction (by separating homologous chromosomes).

In Tomopteris and Lepidosiren it is plain that it is the first division
which does this, and the great majority of modern accounts of meiosis agree
in this. They therefore fall into the category of pre-reduction. Most
of the earlier accounts, however, favoured post-reduction, but this was
mainly due to those errors regarding the composition and fate of tetrads —
of the Copepod type which have been explained above, and may therefore —
be ignored.

As will be readily appreciated, it is often very difficult to decide
which of the two divisions effects the reduction, especially in the case of
tetrads formed, as in Ascaris, by two longitudinal planes. Here it is a
question of tracing the two planes without break from their inception
into metaphase I. in order to see which plane it is that is operative in
this division.

Although pre-reduction appears to be by far the commoner, post-
reduction has been quite definitely described by competent workers,
especially in certain insects. Indeed in this group it appears that even
in the same animal some bivalents may divide reductionally in the first
and others in the second division (M‘Clung, 1914). In the case of certain
peculiar chromosomes, the “sex chromosomes’ (to be described in
Chapter IV.), no fact in cytology is better established than that these may
undergo pre-reduction in some species and post-reduction in others (p. 102).

(5) Syntizesis
The significance of this very characteristic stage in meiosis is quite |
obscure. The one thing certain is that it can have no direct necessary
connection with syndesis, since in many forms (e.g., Tomopteris) it is
absent. The fact that it is not found in certain species has even led some
cytologists, who happen to have worked chiefly with such species, to
doubt its natural occurrence at all, and to ascribe the presence of it in
forms used by other workers to their faulty methods of fixation. In
many organisms, however, synizesis occurs whatever method of fixation
is used, and the matter appears finally settled by the observation of
synizetic contraction in living and fresh tissues. A few examples out of
many such observations are those of Wilson (1909 0) in Anasa, Arnold
(1909) in Planaria, Schleip (1909) in Ostracoda.

1 SYNIZESIS 58

A comparison of the figures of Lepidosiren and Oncopeltus shows that
the intensity of the contraction varies. Oncopeltus forms an intermediate
stage between the extremely dense contraction in Lepidosiren and its
complete absence in Tomopterts.

The exact moment at which synizesis begins and ends also varies
considerably. In Lepfidosiven it begins with the onset of the diplotene
stage. In Oncopeltus it apparently corresponds with the zygotene stage
—at any rate it occurs between the leptotene and pachytene stages.
More frequently, perhaps, it sets in earlier still, when the leptotene threads
are emerging from the resting nucleus.

Finally, in many forms the telophase contraction of the last sperma-
togonial mitosis has been described as passing directly into synizesis
without an intermediate diffuse stage. As this conclusion is based upon
the negative evidence of failing to find diffused stages between the two,
these accounts should perhaps be accepted with some reserve. On the
other hand, there seems to be no theoretical reason to doubt them, and
so many different observers have given such accounts that it is difficult
to doubt their combined testimony. As examples may be quoted
Pertpatus (Montgomery, 1gor a), and Scolopendra (Blackman, 1905).

A few cytologists have described two synizetic contractions, separated
by a diffused stage. This observation has been upheld especially by
Farmer and Moore (1905) and by the adherents to their telosyndetic
scheme, in which the second contraction is supposed to bring about the
doubling over of the pachytene bands to form the rings, etc., of the
bivalents.

C. MEIOSIS IN THE FEMALE

So far we have confined our description of meiosis to that process as it
occurs in the male. The behaviour of the chromatin in oogenesis is
closely parallel to that in spermatogenesis, with, however, modifications
connected with the long-growth period of the oocyte and the compara-
tively gigantic size of the mature egg. The deposition of the yolk and
the growth of the oocyte I. take place between syndesis and metaphase I.
During this period, which may endure for months or years (mammals),
the chromosomes may not retain their chromosomal forms, but often
nearly, or quite, vanish into a peculiar form of resting nucleus known
as the germinal vesicle, to reappear immediately before the first meiotic
division. This special feature of oogenesis does not, however, destroy
the fundamental similarity between male and female meioses, and the
above discussions regarding syndesis, pre- and post-reduction, synizesis,
etc., apply as well to oogenesis as to spermatogenesis. Even the germinal
vesicle stage, as we shall see, is paralleled in the spermatogenesis of
certain animals.

56 CYTOLOGY CHAP.

Another difference between gametogenesis in the male and female
has already been alluded to, namely, the fact that in the female each
primary oocyte gives rise to only one functional gamete and three (or
two, if the first polar body does not divide) minute and functionless cells
(polar bodies) instead of to four functional gametes as in the male. This
is also obviously correlated with the necessity for the female gamete to

Fic. 21.

Early stages of oogenesis in the cat. (After von Winiwater and Sainmont, A.B., 1909.) A, young oocytelI.,
chromatin mostly very finely divided; B, C, development of leptotene stage; D, zygotene; E, pachytene;
F, diplotene stages ; G, H, development of ‘‘ germinal vesicle ”’ stage.
be very large and richly provided with reserve food material; the
achievement of this is materially assisted by the concentration of prac-
tically the whole of the reserve material into one macrogamete, instead
of its partition among four.

The correspondence between the mature egg with its polar bodies
and the four spermatids derived from one spermatocyte is of course only
complete in those cases where the first polar body divides into two, giving

nm MEIOSIS IN THE FEMALE 57

thus a set of four cells derived from the one oocyte I, Cases where the
first polar body does thus divide are very common, but the division does
not always occur, simply because the degeneration of the polar body as a
cell has often gone too far. Stages in its loss of power to divide can be
observed. In the sponge Sycon (Jorgensen, 1g10 a), for example, the first
polar body makes a beginning of a mitotic division which, however,
generally remains uncompleted.

As a rule, the polar bodies disintegrate and disappear soon
after the egg has been fertilized, but sometimes they can be traced,
adhering to the outside of the developing embryo, for a long time
(Ascaris).

An interesting confirmation of the essential homology between the
polar bodies and the ripe egg is provided by an observation of Lefevre
(1907) on Thalassema. The egg of this annelid, like that of so many
others, can be induced to develop without fertilization if placed in a
suitable excitant chemical medium (see Chapter III.). In some cases
not only was the egg induced by this means to start cleavage, but the
polar bodies also divided repeatedly, producing a morula-like cluster
of minute cells—in some instances as many as sixteen.

The later stages of oogenesis—7.e. the two actual mitoses of the
meiotic phase—are illustrated in Figs. 11, 32, 33, 82. As in the case of
the male meiosis, the important stages are to be sought in the long-drawn-
out prophase of the meiotic mitosis itself. The early stages of this
process in the cat are illustrated in Fig. 21.

It will be seen that up to the diplotene stage (F) the process is closely
parallel to the corresponding stages in spermatogenesis. Instead,
however, of straightway condensing into the definitive chromosomes of
the first meiotic division, the bivalents now lose their sharp contours,
diminish in staining capacity and become distributed in an irregular
fashion through the nucleus, while at the same time the nucleolus,
which was already present in the young oocyte, Increases in size. Thus
the nucleus passes into the germinal vesicle condition.

The manner in which the bivalents pass into the germinal vesicle and
re-emerge as the definitive chromosomes of the first meiotic division in
the dog-fish is shown in more detail in Fig. 22 (Maréchal, 1907). There
is a typical pachytene bouquet (A), the orientation of which is lost in the
diplotene stage (B). The chromosomes now lose their sharp outlines by
reason of the development of very numerous thread-like outgrowths
(Figs. 22, D; 23). These being arranged more or less at right angles to the
- central axis of the chromosome give the whole structure a characteristic
appearance which has often been compared to that of a cylindrical chimney
brush. Synchronously with the development of these outgrowths the
chromosomes as a whole lose their characteristic chromatin staining

=

58 CYTOLOGY CHAP.

reaction, appearing indeed to consist now entirely of achromatin or
oxychromatin. At the same time the number of nucleoli increases.

At the end of the growth period the chromosomes undergo a reverse
process of concentration, the filamentar outgrowths being apparently

Fic: 22.

Stages in the oogenesis of the dog-fish, Pristiurus. (After Maréchal, L.C., 1907.) A, pachytene stage,
nucleolar mass already conspicuous ; B, C, passage of the diplotene stage into the germinal vesicle. In C
the number of nucleoli has increased, D, early stage in the reconstruction of the chromosomes; E, F, later
stages in the condensation of the chromosomes into the definitive bivalents. These two figures are drawn
at the same magnification.

c, chromosome ; , nucleolar filament.

retracted on to the central axis. At the same time the staining capacity
of the chromosomes increases again, and they diminish enormously in
size. The nucleoli break up, and their substance shows evidence of
degenerative changes, forming small granules or droplets. Sometimes
these get arranged one behind the other into filaments superficially not
unlike chromosomes (Fig. 22, D) but having in reality no relation to these.

I MEIOSIS IN THE FEMALE 59

The particular problems raised by the germinal vesicle stage in
oogenesis are :

(x) The continuity of the chromosomes throughout this period.

(2) The relation between the chromosomes and the nucleoli.

(3) The connection between the peculiar germinal vesicle stage and
the synchronous enormous growth of the cytoplasm of the egg, together
with the formation of yolk.

(4) Does any comparable stage occur in spermatogenesis ?

(1) The Continuity of the Chromosomes

The conditions in the germinal vesicle have been urged against the
theory of the genetic continuity of the chromosomes, since in some species
the fully developed germinal vesicle—which it must be remembered is
interposed between syndesis and metaphase I.—shows no trace of

EN ui van a ig Ais sy has alls NS

awk

7) pe esi Mi Wn ii He ‘ ne ‘i

i?

Fic. 23.

A chromosome from the germinal vesicle of Pristiurus. (After Rickert. A.A., 1892.)

chromosomes. This condition occurs, for instance, in many Echinoder-
mata, whose fully developed germinal vesicle consists of an enormous
nucleolus suspended in a fine, very faintly staining, reticulum in which
no trace of individual chromosomes can be detected.

As in the case of the resting stage between two ordinary somatic
mitoses, however, we must ascribe the invisibility of the chromosomes in
such germinal vesicles to their extreme diffusion and loss of staining
power, and not to any loss of identity. This can be clearly determined
by a comparative study of this period of oogenesis. In the Copepoda
(a group which has been extensively studied in this connection) we
find a great range of variation in the degree of certainty with which
the chromosomes can be recognized throughout the growth period (e.g.
Matschek, 1910). In-Cyclops gracilis the chromosomes remain sharply
individualized throughout, as is also the case in Heterocope saliens
(Fig. 24). In Diaptomus castor, however (Fig. 25), the chromosomes
become very diffuse at the height of the germinal vesicle stage, and their

60 CYTOLOGY CHAP.

exact limits cannot be made out. Other species of Copepoda exhibit
intermediate conditions.

Hacker (1893) made the interesting observation that the degree of
diffusion of the chromosomes during the germinal vesicle stage may vary
in a single species. Cyclops and the allied genera of Copepoda mostly

om

=

Fic. 24.

The chromosomes during the oogenesis of Heterocope nee from the pachytene stage (A), through the
germinal vesicle stage (B, C) to the condensation of the definitive bivalents (D). (Matschek, 4.Z., rg10.)
lay their eggs in batches, of about ten to about a hundred at atime. The
eggs when laid do not leave the animal completely, but are cemented
together into masses, the so-called egg-sacs, which are carried about by
the animal until they hatch. A new batch of eggs is not laid till the

previous batch has hatched.
In females of Cyclops strenuus which have not yet laid any eggs, no

II MEIOSIS IN THE FEMALE 61

germinal vesicles with diffuse chromosomes are to be found, and it is
evident that the bivalents resulting from syndesis condense continuously

Fic. 25.

The chromosomes during the oogenesis of Diaptomus castor from the pachytene stage (A), through the
germinal vesicle stage (B-E) to the condensation of the definitive bivalents (F). (Matschek, A.Z., 1910.)
into the definitive bivalents of metaphase I. Animals, however, which
are carrying egg-sacs contain in their oviducts oocytes with well-developed
germinal vesicles with diffuse chromosomes. Hicker suggests that in

62 CYTOLOGY CHAP.

the latter case the oocytes are retained in the oviducts (waiting for the
previous batch of eggs to hatch) longer than in the former, and that this
delay accounts for the greater diffusion of the chromosomes.

This idea that the amount of diffusion of the chromosomes is in a
certain degree a function of the length of time which elapses between
syndesis and metaphase I. is supported by the observation of Matschek
(1910) that those Copepods in which the chromosomes undergo only a
moderate amount of diffusion in the germinal vesicle stage belong chiefly
to those species which lay comparatively few eggs at frequent intervals
(Cyclops gracilis, Heterocope saliens), while those in which diffusion is
carried to great lengths mostly lay numerous eggs at long intervals
(Diaptomus castor).

(2) The Relation between the Chromosomes and the Nucleoli

Many cytologists believe that the nucleoli of the germinal vesicle act
as temporary storehouses of chromatin, receiving this substance from the
chromosomes at the beginning of the growth period, and giving it back
to them at the end of it. This conclusion is based on the facts (1) that
in the beginning of the germinal vesicle stage the staining capacity of
the chromosomes diminishes, while the size and number of the densely
staining nucleoli increase; and (2) that at the end of this stage the
chromosomes regain their chromatic character, while the nucleoli break
up or give other evidences of degeneration—such as the development
of vacuoles.

Many other cytologists, however, deny any such direct relationship
between the nucleoli and the chromatin, and this is the view which
appears to be best supported by recent researches (for example,
Jorgensen’s comparative study of the nucleoli of the germinal vesicles
of a large number of animals, 1913). -

In any case, only very little of the nucleolar mass in the germinal
vesicle could contribute to the formation of the chromosomes, since
these in their final form are, in combined bulk, very much smaller than
the nucleolar mass (Fig. 26). The residue of this substance is thrown
out into the cytoplasm when the nuclear membrane is dissolved in
prophase I., and there degenerates.

In the descriptions of somatic mitoses and of spermatogenesis we
have generally avoided the use of the term “ nucleolus,’ substituting
either ‘“‘ plasmosome”’ or “ karyosome”’ as the case might be. The
germinal vesicle nucleoli do not appear to come under either of these
headings. Their chromatin staining reaction shows that they are not
merely plasmosomes, while on the other hand their relations—or rather,
lack of relations—to the chromatin structures of the nucleus prevent
one from calling them karyosomes. In many cases they are of a double

ae

I MEIOSIS IN THE FEMALE 63

nature (amphinucleoli), consisting of a plastin groundwork (plasmosome),
covered or impregnated with chromatin or a chromatin-like substance ;
or the two constituents may be separate, so that the nucleolus consists
of two parts, a chromatin and a plastin portion. These remarks refer
especially to the main nucleolus, which persists right through the growth
period. In many animals the secondary nucleoli which develop later

wie Dé
i? a ye
gee. °

iG 20:

Showing the fate of the nucleolus in the oogenesis of Daphnia pulex. (After Kuhn, 4.Z., 1908.) A, the
large central nucleolus is beginning to break up into much smaller bodies which are spreading over the thin
threads representing the chromosomes ; B, C, the continuation of this process, In C the nucleolus has completely
disintegrated into granules or droplets which conceal the chromosomes, D, the condensed chromosomes
(c) embedded in the disintegrated nucleolar mass; E, the meiotic division. Note the mass of nucleolar
granules left in the cytoplasm by the rupture of the nuclear membrane. A is drawn under a higher magnification
than the remaining figures, which are all to the same scale,

appear to be purely of the nature of plasmosomes
(Hacker, 1893).

e.g., Cyclops brevicornis

(3) The Connection between the Germinal Vesicle and Yolk Formation

The co-existence in the primary oocyte of two unique cytological
occurrences, namely, the germinal vesicle and the enormous growth of
the cell with its formation of reserve food material, naturally suggests a
causal connection between them. We have already drawn attention to
the fact that the diffusion of the chromatin in the ordinary resting
nucleus has the result of increasing its area in proportion to its mass,
and thus of favouring active metabolism, The excessive diffusion of

64 CYTOLOGY CHAP.

the chromosomes in the germinal vesicles of many animals has similarly
been held to be an expression of the intense activity required of them in
connection with the elaboration of the yolk. It has also been considered
that the enormous mass of nucleolar substance present at this stage
represents merely the accumulation of waste products of great metabolic
activity. Finally, many cytologists have described the actual extrusion
of chromatin from the germinal vesicle into the cytoplasm, either directly
from the chromosomes or indirectly by way of the nucleolus. The
extruded chromatin (chromidia) is supposed to take part in yolk forma-
tion, either by direct transformation into this substance, or by exerting
a formative influence on the cytoplasm. This matter of the extrusion
of chromatin is dealt with more fully in Chapter VI.

We are thus introduced to a body known as the yolk nucleus (not to
be confused with the em-
bryologist’s yolk nuclei of
Selachian, etc., embryos,
which are derived from
supernumerary sperma-
tozoa’; see 9p,77).. Dur-
ing the early growth
period, intensely staining
granules appear in the
cytoplasm of the oocyte.
These are variously inter-
preted as extruded chro-
matin, or as_ chondrio-

FIG. 27.
Yolk nucleus (y.”.) in the oocytes of (A) Anledon bifida (Chubb, Phil. somes (see Chapter VI.).
Trans., 1906), and (B) Paracalanus parvus (Moroff, A.Z., 1909). Sometimes, as in Echinus

(Schaxel, 191m a) and AHydractinia (Beckwith, 1914), they are
scattered uniformly through the cytoplasm. In other cases they are
concentrated into a more or less compact mass, often round the
centrosome as a centre, forming a conspicuous body in the cytoplasm.
Examples of such cases are found in Antedon (Chubb, 1906; Fig. 27),
certain Copepoda (Fig. 27), Amphibia, etc. The supposed connection
of these cytoplasmic bodies with yolk formation (whence their name of
“yolk nuclei ”’) rests chiefly upon the facts that their appearance
precedes yolk formation, and that as this proceeds they disintegrate
and finally disappear in the ripe oocyte, where no more yolk is being
deposited.

The term yolk nucleus has also been applied to what is probably an
entirely different structure, namely, the centrosome and surrounding
substance of the centrosphere, which sometimes forms a large conspicuous
body (e.g., Enchytraeus, Vejdovsky, 1907).

n MEIOSIS 6s

(4) Does any Stage comparable to the Germinal Vesicle occur in
Spermatogenesis ?

The undoubted correlation between the peculiar conditions of the
germinal vesicle and the long duration of the growth period in the female
meiotic phase, and probably also with the deposition of yolk, makes it

Fic. 28.

Germinal vesicle-like stages in spermatogenesis. A-G, Notodromas monacha (after Schmalz, A.Z., 1912) ;
H-N, Scolopendra heros (after Blackman, B.M.C.Z.H., 1905). In both cases the principal stages between the
pachytene nuclei (A and H) and the formation of the definitive bivalents (G and N) are shown.

a priovt improbable that a fully developed germinal vesicle stage should
be a normal occurrence in spermatogenesis, where the duration of the
growth period is comparatively short, and there is little or no deposition
of reserve food material. |
Nevertheless, stages obviously corresponding with the germinal
vesicle occur in the male meiotic phases of some animals. There is often
F

66 CYTOLOGY ie CHAP. II

a temporary lengthening out of the chromosomes in the diplotene stage
(e.g., Tomopteris, Fig. 14, H, I), which may perhaps be considered an
indication of a tendency to pass into a germinal vesicle condition. The
“‘ confused stage’ of certain insects (Fig. 17) represents a slightly more
marked, but still rudimentary, condition of the same stage. In some >
Ostracoda (Fig. 28) there is a stage in spermatogenesis closely corre-
sponding to the germinal vesicle of the oocyte, the resemblance even
extending to the formation of “‘ yolk nuclei’ in the cytoplasm (Schmalz,
1g12). In the Myriopod Scolopendra (Blackman, 1905) where the
primary spermatocyte undergoes an unusually pronounced growth, the
resemblance to the oocyte germinal vesicle is even greater (Fig. 28).

CHAPTER: Tt
SYNGAMY, EARLY DEVELOPMENT, PARTHENOGENESIS

THE ripe microgamete or spermatozoon is a very minute motile cell,
highly specialized for the purpose of conveying the chromatin and centro-
somes from the male parent into the macrogamete. While varying
greatly in form in different groups of the animal kingdom, by far the
commonest form for it is a relatively large head containing the nucleus,
to which is attached a flagellum or ¢ail. The latter is the organ by
means of which the movements of the microgamete in search of the
macrogamete are carried out. It is not attached directly to the head,
but through the intervention of the muddle piece, which contains the
centrosome (in many spermatozoa). As will appear directly, the sper-
matozoon also presents other structural features of some importance.

A. THE DEVELOPMENT OF THE SPERMATOZOON

It is necessary first to consider briefly the development of the sper-
matozoon from the spermatid, since this matter bears upon the interpreta-
tion of the role of the nucleus in heredity. Before we can understand
either the structure or development of the spermatozoon we must have
some knowledge of the cytoplasmic bodies known as chondriosomes.1
The nature and significance of these bodies are obscure, and are discussed
in Chapter VI., but they undoubtedly play an important part in the
structure of the spermatozoon.

If material for cytological study be treated by appropriate methods
of fixing and staining, the chondriosomes are revealed as very definite,
though minute, bodies in the cytoplasm. Their behaviour in a particular
case, the insect Blatta germanica (Duesberg, I9gII a), may be taken as
sufficiently typical of the general course of events (Fig. 29).

Starting with the resting spermatogonium, the chondriosomes are here
in the form of longer or shorter filaments distributed irregularly through-
out the cytoplasm. In many other species they are granular instead of

1 See note on terminology on p. 195.

67

68 CYTOLOGY CHAP.

i rr. L

Fic. 29.

Chondriosomes in the spermatogenesis of Blatta germanica. (After Duesberg, A.Z., 1911.) The material
is prepared by Benda’s method, and the chondriosomes are shown darker than the chromatin. A,
resting spermatogonium, filamentar chondriosomes distributed through the cytoplasm; B, telophase of
a spermatogonial division; chondriosomes mostly congregated in a bundle which is just cut across; C,
young spermatocyte I. ; D, pachytene stage, chondriosomes temporarily conceutrated “outside pole of nucleus ;
E. later prophase I. ; F, metaphase I. ; G, young spermatid ; chondriosomes massed into the very conspicuous
“* Nebenkern ” ; H, chondriosome mass divided into two; I, the two chondriosome masses growing out into
the tail of the spermatozoon, and clasping between them the axial filament; J, the chondriosome mass now
forms a sheath round the axial filament, as shown in K, transverse section of the tail: L, ripe spermato-
zoon under a lower magnification.

qa.f, axial filament of tail; ch, chondriosome mass ; n, nucleus.

111 CHONDRIOSOMES 69

filamentar. During cell division they congregate between the separating
daughter nuclei. Finally, when cell division is almost complete, they
occupy the bridge of cytoplasm that connects the two nearly separated
daughter cells. When this is broken through, the bundle of chondrio-
somes is also broken across, and thus a portion of the chondriosome mass
is left in each daughter cell.

In the young primary spermatocyte they are again scattered through
the cytoplasm, but at the beginning of the growth period they concentrate
round the centrosome, forming thus a cap at the pole of the nucleus.

They remain in this position throughout the growth period, but in
the later prophase again become scattered through the cell and form
during mitosis a mantle round the spindle figure. Their distribution
between the two secondary spermatocytes is thus insured,

In the second division they behave in the same way, and thus each
spermatid receives a share of the chondriosomes present in the primary
spermatocyte.

In the spermatid they fuse together into a compact mass which may
be as large as, or larger than, the nucleus. This is the supplementary
nucleus, or “‘ Nebenkern,” of older cytologists (though this term has also
been applied to structures of different natures).

Next, the “‘ Nebenkern”’ divides into two, and elongates with the
lengthening tail of the spermatid, the two portions clasping between
them the axial filament of the tail. As the spermatid continues to
elongate, its parts become more and more attenuated, the ‘“‘ Nebenkern ”’
keeping pace with this elongation, and undergoing various minor changes.
In the adult spermatozoon it forms a sheath for nearly the whole length
of the tail, only a very short stretch of the latter projecting from the end
of the sheath.

In the spermatids of many animals the chondriosomes are scattered
instead of being concentrated into a ‘‘ Nebenkern.”’ In the spermatozoon
they are found in a variety of positions, but apparently are never absent
from the ripe spermatozoon.t This fact has led certain cytologists to
assign a very important function to the chondriosomes (Chapter VI.).

We are now in a position to study, very briefly indeed, the general
development of the spermatozoon from the spermatid. The development
of the mammalian spermatozoon as worked out for the guinea-pig by
Meves (1899) and Duesberg (1911 a) will serve as a type (Fig. 30). Very
briefly, the course of development is as follows: After the second
meiotic division the spermatid consists of a cell containing (1) the nucleus ;
(2) chondriosomes (here in the form of scattered granules) ; (3) two centro-
somes ; (4) the idiosome. The last-mentioned body corresponds more
or less closely to the attraction sphere of other cells. In the spermatid

1 An exception has been described in the case of Peripatus (p. 197).

D

ar

Fic. 30.

Metamorphosis of the spermatid into the sperma-
tozoon. Diagrammatized and simplified trom the
accounts of the process in the guinea-pig by Meves
(A.m.A., 1899), and Duesberg (A.Z., IgII). a, acro-
some; a.f, axial filament of tail; c, centrosome; ch,
chondriosomes ; #, head (nucleus) of spermatozoon ; 1,
idiosome; m, middle piece of spermatozoon with two
centrosomes and spiral chondriosome band; n, nucleus;
r, cytoplasm of spermatid which is thrown off; #, tail
of spermatozoon,

CYTOLOGY

CHAP.

this body is separated from the
centrosomes, and comparatively
large.

The changes undergone by the
nucleus during the development of
the spermatozoon are, so far as can
be seen, little more than a progress-
ive concentration.

The idiosome applies itself to the
end of the nucleus farthest from
the centrosomes, and there becomes

Set eee

ee

eg ON ON 9 TO

Fig, 31.

A,
D, Homo;

(After Retzius, 1909.)
C, Turdus ;

Various spermatozoa.
Notodromas; B, Locusta ;
E, Galathea.

m1 SYNGAMY a

metamorphosed into the acrosome which forms the anterior tip of the
ripe spermatozoon ; it appears to act as a spear-head by which the
spermatozoon perforates and penetrates into the egg.

In the spermatid shown in Fig. 30, A, a fine thread, is already growing
out from the distal centrosome (i.e. the centrosome farthest from the
nucleus). This is the beginning of the axial filament of the tail. Later,
the two centrosomes move down towards the nucleus, coming into contact
with one another at the same time, so that the base of the tail filament
is now continued past the distal into the proximal centrosome, This
comes into close contact with the surface of the nucleus and undergoes
certain metamorphoses not shown in these figures. The distal centrosome
parts company with the proximal one, grows into a ring surrounding
the tail filament, and travels down it for a certain distance. The portion
of the mature spermatozoon in which the centrosomes are lodged is called
the middle piece.

The chondriosomes, which in the guinea-pig, as in other mammals,
are scattered through the spermatid instead of being collected into a
““ Nebenkern,”’ become concentrated round the tail filament between the
two centrosomes, and there form a granular sheath for the proximal
part of the filament, the granules being often specially dense along a
spiral band round the middle piece (Fig. 30, D).

By far the greater part of the spermatid cytoplasm is not used up in
the process of forming the spermatozoon, and is cast off about the stage
shown in Fig. 30, C.

A few representative types of other spermatozoa are shown in Fig. 31.

B. SYNGAMY

With certain exceptions which will be described later on in this chapter,
neither the male nor the female gamete is of itself in a position to start
development. Before this can occur, syngamy, or fusion of a male and
female gamete, must take place. The zygote thus formed is capable ot
development into a newindividual of the species, and, in the great majority
of cases, this development starts immediately after the zygote has been
constituted. Owing to the fact that the zygote differs superficially but
little from the ovum, except in its power of development, the process
of syngamy in the Metazoa is generally known as the fertilization of the
ovum.

The details of the process of syngamy vary but little, the essential
features being the penetration of the motile spermatozoon or micro-
gamete into the immotile egg or macrogamete, and the fusion of the two
gametic nuclei to form the zygote, or cleavage, nucleus ; this proceeds to
mitosis under the influence of the centrosome introduced by the male

72 CYTOLOGY. CHAP.

gamete. The general course of events is illustrated by the diagrammatic
Fig. 32 and is sufficiently explained by the legend of that figure. Fig.
33 on the other hand shows the process as it actually occurs in the Annelid,

Bic. 3%.

Diagram of fertilization. A, entry of spermatozoon into primary oocyte; B, the 9 nucleus has
moved to the surface and is in metaphase I.; C, 9 nucleusin telophase I. The tail of the spermatozoon
has broken off and is degenerating. The head of the spermatozoon has rotated so that the two centrosomes
which have disengaged from the middle piece now precede the g nucleus as it travels towards the centre of
the egg. D, @ nucleus in telophase II.; first polar body dividing; E, approach of the ¢ and ? nuclei; F,
the two nuclei, now of approximately equal size, and in early prophase, are in contact ; G, the two nuclei have
fused into a zygote nucleus: formation of spindle figure out of centrosomes and asters introduced by the
spermatozoon; H, telophase of first (cleavage) division of the zygote nucleus ; I, 2-celled stage.

h, head of spermatozoon (g nucleus); m, middle piece; .b.1 and p.b.2, first and second polar bodies ;
t, tail of spermatozoon.

Chaetopterus. The chief variations from the examples depicted concern
the moment of entry of the spermatozoon, and the condition of the
gametic nuclei (often called the male and female pronuclet) at the moment
of karyogamy or nuclear fusion.

3 olen moe

ut SYNGAMY 73

The case illustrated in the diagram is one where the oocyte I. proceeds
with the usual course of meiosis up to the end of the growth period, and
then pauses with its nucleus either in the “ germinal vesicle ”’ stage, or in
diakinesis, for the first meiotic division. The subsequent completion of
meiosis is dependent upon the entry of a spermatozoon into the oocyte.
Should this not occur, the oocyte perishes without further development.
The entry of a spermatozoon, however, immediately sets the meiotic pro-
cesses in operation again, and the nucleus moves towards the surface of
the egg, undergoes its two meiotic divisions to produce the first and second
polar bodies, and then returns to the centre of the egg to meet the male
nucleus. Examples of eggs in which fertilization is of this type are A sterias
(many workers) and Fasciola (Schellenberg, Igtt). Rarely the sperma-
tozoon enters the oocyte I. at a much earlier stage, e.g. in Saccocirrus
(Buchner, 1914), where it enters at the beginning of the growth period
and lies among the yolk during the whole of the long time which elapses
before the meiotic divisions of the egg take place and consequently
karyogamy becomes possible. Much more often, however, the sperma-
tozoon enters at a later stage. for instance, the pause in the meiotic
phase for the entry of the spermatozoon takes place in metaphase I. or
anaphase I. in Ophryotrocha (Korschelt, 1895), Chaetopterus (Mead, 1808 ;
Fig. 33), Thalassema (Griffin, 1899), Physa (Kostanecki and Wierzejski,
1896). It occurs in metaphase II. or anaphase II. in Amphioxus (Sobotta,
1897), the axolotl (Fick, 1893), and in the mouse and guinea-pig (Lams and
Doorme, 1908). In other cases the egg completes the process of meiosis,
forming both polar bodies, and the mature egg nucleus passes into the
resting condition to await the entry of the spermatozoon. Examples
of this type are the sea-urchins (many workers), and the sponge Sycon
(Jorgensen, IgIo a).

As regards the fusion of the two gametic nuclei, the diagram shows
each nucleus in the resting condition at the moment that karyogamy takes
place. In certain cases, however, the two nuclei do not fuse to form a
resting zygote nucleus, but they may fuse when in prophase for the first
cleavage division. In other cases the nuclei do not fuse before the
first cleavage mitosis at all, but each gamete nucleus forms its chromo-
somes while still independent, and the chromosomes are placed on the
spindle in two separate groups. In these cases the chromosomes derived
from the male and female parents are not united into a zygote nucleus
until the telophase of the first cleavage mitosis. Examples of karyogamy
of this type are Ascaris megalocephala (cf. Fig. 70) and Ophryotrocha
(Fig. 34). Indeed, it not infrequently happens that the complete fusion
of the gamete nuclei is postponed to a much later stage (p. 77).

The male nucleus, which is derived from the head of the spermatozoon,
is at first very much smaller than the female. This is obviously due

74 CYTOLOGY CHAP.

merely to the fact that the chromatin is more concentrated in the former,
for as it approaches the female nucleus it grows larger, and at the same
time becomes looser in texture, till ultimately the two gamete nuclei
are generally of the same volume and structure.

Since each gamete nucleus is haploid, it is obvious that the zygote
nucleus is diploid again.

As we have seen, a typical spermatozoon consists of three main
portions—head, middle piece, and tail. The fate of the head or nucleus

ig Pl

FrGs(33:

Syngamy in Chaetopterus pergamentaceus. (After Mead, J.M., 1898.) A, soon after entry of spermato-
zoon; ? nucleus in anaphase I.; ¢ aster developing ; B, 9 nucleus in anaphase II.; C, ? nucleus in form
of karyomeres (see p. 137) ; 9 achromatic figure has disappeared ; D, approach ; E, fusion, of gamete nuclei.

P.b.1 and P.b.2, first and second polar bodies.
we have already followed. The tail apparently takes no part in fertiliza-
tion. (For the fate of the chondriosomes, which are usually situated in
the tail, see p. 198.) Sometimes it does not even enter the egg, being
broken off from the middle piece as soon as this has penetrated into the
egg (sea-urchins). When the tail does enter the egg it breaks off from
the rest of spermatozoon soon after entry, and lies in the egg cytoplasm
for a time, and then degenerates.

There remains to be considered the middle piece, with which the
centrosome, as the development of the spermatozoon showed us, is
generally related. Owing to the mode of entry of the spermatozoon—
head first—the middle piece is at first behind the head. After entry,
however, the head and middle piece rotate so that the latter is in front
of the former during its journey towards the centre of the egg. The

—_—

a

il SYNGAMY 75

centrosome soon becomes visible in the middle piece, and an astral
radiation appears round it. It next divides into two (if it has not already
done so in the spermatid), a spindle figure being spun out between the
two daughter centrosomes, and thus a complete achromatic figure is

Li
Dee foe HN Rays
0 ELF FIRM NEN DS vi
PPD ITAN

FIG. 34.

Syngamy in various animals. A, Physa fontanalis (after Kostanecki and Wierzejski, A.m.A., 1896) just
after entry of spermatozoon; B, the mouse (after Lams and Doorme, A.B., 1908) spermatozoon in the act of
entering the egg; C, D, successive stages in the fusion of the gamete nuclei in Ophryotrocha puerilis (after
Korschelt, Z.w.Z., 1895); E, F, the penetration of the amoeboid spermatozoon in Ascaris canis (after Walton,
J.M., 1918).

c, centrosome in middle piece; /, head; ¢, tail of spermatozoon.
formed under the influence of the sperm centrosome. This is the
achromatic figure of the zygote nucleus, for after the formation of the
second polar body the entire achromatic figure of the egg, including the
centrosomes, disappears. Thus the centrosome, and hence the whole
achromatic figure of the zygote, is entirely derived from the spermatozoon.

This conclusion is based on numerous observations, and also is well

76 CYTOLOGY CHAP.

grounded on such experiments as those of Boveri (1907), who found that
sea-urchins’ eggs into which, from pathological causes, two spermatozoa
had entered possessed four centrosomes, with a four-poled first cleavage
mitosis (tetraster) ; if three spermatozoa had entered the egg, six centro-
somes were found to be present.

The important part played by the centrosome of the spermatozoon
introduces us to the thoroughly well established view that fertilization
has two functions—(r) the stimulation of the egg to develop, and (2) the
fusion of the gamete nuclei. These two processes are not necessarily
interdependent. Thus an egg may be stimulated to development by
the entry of a spermatozoon which belongs to such a distantly related
species that nuclear fusion between them is impossible (p. 160), or the
stimulus may even be provided by physical means without the inter-
vention of a spermatozoon at all (artificial parthenogenesis, p. 94).

Since an egg under certain circumstances may develop without fusion
of its nucleus with a microgamete nucleus, it is certain that karyogamy
is not a necessary condition of development. The function of the fusion
of the gamete nuclei must be looked for in its ulterior effects on variation
and heredity. The incapacity of either gamete to develop, under ordinary
circumstances, by itself, has indeed been looked upon as an adaptation
to ensure that karyogamy shall take place, the loss of power to develop
independently being attained in the case of the spermatozoon by its
general specialization and the reduction of its cytoplasm to a minimal
quantity, and in the case of the egg by the degeneration of the centro-
somes and rest of the achromatic figure after the completion of the meiotic
divisions. It is indeed difficult to believe that the developmental stimulus
given by the spermatozoon is not connected with the introduction of the
active male centrosome. It must however be remembered that in the
case of artificial parthenogenesis no new centrosome is introduced from
without. Moreover, in many eggs the pause in the meiotic processes in
which the egg awaits fertilization takes place in metaphase I. or II., that
is to say, at a time when its centrosomes and achromatic figure are fully
developed and active. Thus here, as in so many other biological problems,
a simple, almost mechanical, explanation is found to be inadequate to
cover all the facts.

As a rule, only one spermatozoon enters the egg. Even when, as in
the great majority of cases, the egg is surrounded by an enormous number
of spermatozoa seeking to enter it, the penetration of a single spermatozoon
usually immediately confers on the egg the power of resisting the entrance
of any more. The means by which this resistance is effected is not fully
understood, though in many eggs the exclusion of supernumerary
spermatozoa is certainly aided by the secretion of a membrane

ul SYNGAMY a7

round the egg almost instantaneously after the entrance of the first
spermatozoon.

In some eggs, however, more than one spermatozoon always enter at
fertilization (polyspermy), though in no case does more than one normally
fuse with the egg nucleus. In some cases the supernumerary spermatozoa
merely degenerate and are absorbed (Axolotl, Fick, 1893) ; in other cases
they maintain themselves for a considerable time in the egg cytoplasm,
even forming nuclei which may increase in number by amitotic fragmenta-
tion (Triton, Braus, 1895). In yet other types the supernumerary sperma-
tozoa metamorphose themselves into nuclei which multiply by mitosis
(exhibiting of course the haploid number of chromosomes) and may persist
for along time in development. In the case of the Elasmobranchs, where
this type of polyspermy has been most thoroughly studied (Rickert,
1899), the ultimate fate of these nuclei is not known, but it is extremely
improbable that they take any part in the formation of the embryo.
In the pigeon, the nuclei derived from the extra spermatozoa, which
also multiply by mitosis, disappear much earlier, namely, about the 32-cell
stage (Blount, 1909).

Cases such as the above, where several spermatozoa normally enter
the egg (though only one fuses with the egg nucleus), are said to exhibit
physiological polyspermy. This is specially characteristic of large, heavily
yolked eggs (Insects, Elasmobranchs, Amphibia, Reptiles, Birds). In
other cases, however, the entry of more than one spermatozoon into the
egg is pathological (pathological polyspermy) and leads to abnormal
development, owing to the multiplication of centrosomes and to the fact
that more than one of the microgamete nuclei enters into relation with
the female nucleus. If two spermatozoa enter the egg of Ascaris megalo-
cephala or of Echinus, both male nuclei form chromosomes and both give
rise to a pair of centrosomes. Thus there are three sets of chromosomes
(two ¢ and one ?) and four centrosomes. A four-poled spindle figure
is thus produced, and at the first cleavage division the egg divides simul-
taneously into four blastomeres instead of two, the 3x chromosomes being
irregularly distributed among the four nuclei. These special cases are
more fully described later on (p. 162).

C. GODNOMERY

We have seen that the gamete nuclei may fuse in the resting condition,
or may carry through the prophases of mitosis while still separate, in
which case the chromosomes of the two gametes come together for the
first time on the first cleavage spindle. Sometimes, however, the two
nuclei retain their individuality much longer (Fig. 35). In various species
of Cyclops, for instance (Riickert, 1895 ; Hacker, 1895), the resting nuclei

78 CYTOLOGY CHAP.

of the early cleavage stages are double, each portion being the direct
descendant of one of the gamete nuclei. Each constituent of such a
double nucleus is called a gonomere. In prophase each gonomere forms its
chromosomes separately from the other. The two groups of chromosomes
thus formed are usually indistinguishable from one another after the
break-down of the nuclear membrane, but in telophase they become
recognizable again owing to the fact that the group of chromosomes
derived from each gonomere again forms a nucleus distinct from, though
closely applied to, that formed by the other group. Occasionally, however,
the two groups are distinct during metaphase and anaphase as well
(Fig. 35, A). In later cleavage stages the chromatin derived from the two
gamete nuclei gradually mingles more and more, and double nuclei
become consequently rarer. In Cyclops brevicornis double and bilobed

Fic. 35.

Gonomery in Cyclops strenuus. (After Riickert, A.m.A., 1895.) A, 2-4-cell stage, the groups of chromo-
somes derived from ¢g and ? gametes quite separate; B, 4-cell stage. In the nucleus in prophase the two
groups of chromosomes are seen. C, 32-cell stage. Gonomeres indicated in most of the nuclei.
nuclei are still common in the 64-cell stage, and in later stages bilobed
nuclei with a nucleolus in each lobe are still to be found, as well as spherical
nuclei with two symmetrically placed nucleoli, which Hacker interprets
as the last remaining indication of gonomery. In the germ-track (see
p. 79) evidences of gonomery can be found at a much later stage of
development than in the somatic cells (Hacker, 1903).

A remarkable instance of gonomery is to be found in the Protozoan,
Amoeba diploidea (Nagler, 1909). This animal possesses two nuclei, in
close apposition to one another (Fig. 36), exactly like the double nuclei
of early Cyclops embryos. The life history shows that these nuclei are
the direct descendants of the two gamete nuclei—+.e. they are gonomeres.
During the asexual reproduction of the animal the two nuclei divide
separately, but simultaneously, so that each daughter cell again receives
a double nucleus. Sexual reproduction begins by the coming together
of two individuals which enclose themselves in a common cyst. Now
in each individual the gonomeres for the first time fuse into a zygote

I GONOMERY 79

nucleus, so that each of the conjugating amoebae has now a single
nucleus. These nuclei undergo a process of meiosis, comparable to the
formation of the polar bodies of a Metazoan egg, converting each amoeba
into a single gamete. The two gamete cells fuse together to form a zygote,
their nuclei, however, remaining unfused. Thus the binucleate condition
is restored, to be retained through an indefinite number of cell divisions

oO
a

Fic. 36.

Amoeba diploidea. (After Nagler, A.P.K., 1909.) A, the animal in its active phase, showing the double
nucleus (gonomeres); B, C, division stages showing simultaneous division of the gonomeres ; D, two indi-
viduals encysted preparatory to conjugation; E, in each individual the gonomeres have fused into a single
nucleus ; F, conjugation has taken place, and the zygote with the two gamete nuclei (gonomeres) is emerging
from the cyst, thus bringing the life cycle back to A again.
during asexual reproduction, the two gonomeres fusing together for the

first time immediately before gamete formation.

D. THE GERM-TRACK

One more feature of early development remains to be mentioned. In
a large number of animals the primitive germ-cells—those cells, that is to
say, that will eventually give rise to gametes—are visibly marked out
from the remaining or somatic cells at a very early stage of development.
The distinguishing marks may be features either of the nucleus or cyto-
plasm. The best-known case is that of Ascaris megalocephala (Boveri,
1899, 1904, IQI0).

Nothing remarkable is to be observed in the first cleavage division,

80 CYTOLOGY CHAP.

but when the nuclei of the 2-cell stage are preparing for the next

FIG. 37.

Differentiation of the germ-track in Ascaris megalocephala univalens. (After Boveri, Fest. f. Kupffer, 1899,
and Ergebnisse, 1004.) A, 2-cell stage. Diminution taking place in the cell S,, chromosomes intact in P}.
B, passage of the 2-cell into the 4-cell stage. In 5S), numerous small chromosomes formed by the frag-
mentation of the two large ones. The broken-off ends of the latter are seen outside the equator of the
spindle. C, 4-cell stage. InSj)(A) and S,(B) the cast-out ends of the chromosomes are seen in the cytoplasm.
D, mitosis which will give rise to the 8-cell stage. Diminution taking place in So, chromosomes intact in Po».
E, later embryo, process of diminution now complete. The two original chromosomes are still intact in Py
only.

mitosis, one of them is marked out from the other by the fact that its

MT GERM-TRACK 81

chromosomes have each broken up into a number of small pieces (Fig. 37, A).
The ends of the chromosomes break off as comparatively large club-
shaped pieces, and the central portions become divided into a great
number of much smaller, more or less spherical, fragments. The larger
pleces derived from the ends of the chromosomes are cast out into the
cytoplasm, where they degenerate and disappear. For this reason the
phenomenon is known as the diminution of the chromatin.

The cell in which chromatin diminution has taken place is now left
with a large number of small chromatin granules, and each of these acts
in future mitoses as a single chromosome. The embryo at this stage
(prophase of second cleavage division) consists therefore of two cells,
the one with two large chromosomes (in A. m. wnivalens, four in A. m.
bivalens), the other with a large number (about sixty in A. m. univalens)
of very small ones. The total chromatin content of the latter nucleus
is much less than that of the former, owing to the loss of the large pieces
from the ends of the original chromosomes.

All the descendants of the cell which has undergone diminution have
the same nuclear composition as their parent cell, the mitosis of the
numerous small chromosomes apparently taking place regularly, and
there being no further loss of chromatin. It is not so, however, in the
case of the cell with the two original chromosomes intact. For three
more successive mitoses this cell divides into one which undergoes
diminution and one which does not, so that in the 16-cell stage we have
one cell with the original chromosomes intact, one in which diminution
is in progress and fourteen with the diminished amount of chromatin
and numerous small chromosomes. After this, there is no further
diminution, so that after the next cleavage there are two cells with the
original chromosomes intact, and the remainder with the numerous
small chromosomes, and in all future mitoses the daughter nuclei remain
of the same composition as their mother nucleus. The two cells with
tniact chromosomes are the primitive gonad ; the remaining cells will
develop into the soma. Thus all the somatic cells of Ascaris megalo-
cephala have the numerous small chromosomes and lack the large portion
of the chromatin which was thrown out from the ends of the original
chromosomes, while all the germ-cells contain the original chromosomes,
with all the chromatin, intact (Fig. 38). The line of cells with intact
chromosomes leading from the undivided zygote to the gametes is called
the germ-track.

Phenomena, similar in principle but differing in detail, have been
observed in other species of Ascaris. In A. lumbricoides, however
(Bonnevie, 1902), only the ends of the chromosomes break off (and are
got rid of); there is no fragmentation of the middle portions of the
chromosomes. In A. canis (Walton, 1918) the chromosome ends are

G

82 CYTOLOGY CHAP.

thrown out and the middle pieces break up, as in A. megalocephala, except
that only two instead of about thirty small chromosomes are produced
from each original one.

In many animals the germ-track is marked out from the undivided
egg onwards by characteristics of the cytoplasm instead of the nucleus.
The fresh-water crustacean Cyclops furnishes an example. The germ-
track in this animal was first worked out by Hacker in C. viridis (1897 a),
his results being confirmed in all essentials by Amma in Ig11, who also

Soma

Pe PEs
Germ Cells
Fic. 38.

Scheme of the cleavage divisions in Ascaris megalocephala. (Boveri, Ergebnisse, 1904.)
fhe uppermost cell is the fertilized ovum.
@ Cell in which chromatin diminution has not taken place.
‘O° Cell in which chromatin diminution takes place.
© Cell with diminished chromatin.
discovered the same process in several different species of Cyclops and
in the allied genera Diapiomus and Canthocamptus. The account given
by the latter author for Cyclops fuscus will serve for an example (Fig. 39).
In the prophase of the first cleavage division one attraction sphere
is distinguished from the other by a group of granules which surrounds
it, these being completely absent from the other sphere. Consequently,
at cell division all the granules pass into one of the first two cells or
blastomeres and none into the other; nor do they ever appear in the
descendants of the latter cell. In the case of the blastomere containing
the granules the process is repeated in the following mitosis. After
the first cell division is completed the granules become clumped together

— een

————

/

m1 GERM-TRACK 83

and gradually disappear, and a new set of granules makes its appearance
in the next prophase. As the granules of the preceding mitosis have not
quite disappeared by the time the new set develops, the cell is never
altogether without them, and this fact makes its continuous identification
possible. The new granules appearing at prophase are again concen-

:
3
D

om

Differentiation of the germ-track in Cyclops fuscus (A-H), and Diahtomus coeruleus (1). (After Amma,
A.Z., 1911.) A, prophase of first cleavage mitosis, granules congregated round one attraction sphere; B,
same mitosis, metaphase; C, 2-cell stage, resting nuclei (note gonomery) ; D, prophase of second cleavage
division; E, 16-cell stage. All the nuclei have completed their division, and entered into the resting stage,
except that of the granule cell which is still in anaphase. F, division of the granule cell into the two
primitive germ-cells; G, H, I, later stages.

g, primitive germ-cells.

trated round one attraction sphere only, and thus again pass into only
one of the daughter cells. A differential cell division of this sort takes
place four times, so that the cleaving egg up to the end of the r16-cell
stage contains one, and only one, cell with granules, or granule cell. The
nucleus of this cell does not as yet differ markedly from those of the other
cells, except that it constantly lags a little behind the others in mitosis.

84 CYTOLOGY CHAP.

By the time that the 16-cell stage is reached this lagging has gone
so far that, when the remainder of the cells divide to form what
should be the 32-cell stage, the granule cell fails to divide at all, so that
the blastula at this stage contains only thirty-one cells.

After the fourth division of the granule cell—i.e. in the 16- and
31-cell stages—the granules are no longer confined to one attraction
sphere, but are scattered throughout the whole cell. Consequently, when
the granule cell divides next time, which it does at the close of the
31-cell stage, it produces two similar granule cells. These cells, which
remain without further division for a considerable time, are the primitive
germ-cells, from which the right and left gonads develop respectively.

Thus the germ-track in Cyclops and the allied genera is quite as clearly
marked out as in Ascaris. At first sight it might, however, appear that
the two processes were very distinct, the one being a case of nuclear, and
the other of cytoplasmic, differentiation. Nevertheless, while we do not
know the significance of the diminution of the chromatin and fragmenta-
tion of the chromosomes in the somatic cells of Ascaris, there is little
doubt that in both cases it is the nature of the cytoplasm of the cell which
determines whether it shall be a somatic or a gonadic cell. This is
fairly plain in the case of Cyclops, where it is clear enough that the
granules are of cytoplasmic origin. Amma gives reasons for the belief
that they are temporary metabolic products of a special portion of the
cytoplasm. We may conceive of this special cytoplasm as concentrated
in the undivided egg near one of the centrosomes, and consequently
passing into only one of the daughter cells, until, after four such divisions,
the cells have become so much reduced in size that now this substance
occupies the whole, or nearly the whole, of the cell instead of one pole
only of it. Henceforth division of this cell, or of its descendants, must
result in the passage of this substance into both daughter cells.

By the study of the diminution process in dispermic Ascaris eggs,
Boveri (1910) has shown that in this species also it is the nature of the
cytoplasm which determines whether diminution shall or shall not take
place in a particular cell. The eggs in question are the very rare abnormal
cases where two spermatozoa have entered the egg. Both centrosomes
introduced by the spermatozoa divide, and then form a quadripolar
spindle figure (cf. Fig. 74). On this spindle the 3, or 6, chromosomes
derived from the female and the two male gamete nuclei take up their
position (the account deals with A. m. bivalens). As a rule the first
cleavage mitosis of such an egg divides it simultaneously into four
blastomeres, and the twelve daughter chromosomes of the original six
are distributed among the four nuclei in a most irregular manner; for
example, one nucleus may get only one chromosome, another three, and
the other two four each, etc.

m1 GERM-TRACK 85

Now in the normal monospermic egg all the daughter chromosomes
of the zygote nucleus which pass into the one blastomere (designated by
Boveri, S) undergo diminution, and all that go into the other (P) undergo
their next mitosis intact. Thus, if the inducement to diminution were
furnished by the chromosomes themselves, we would have to suppose
that every chromosome of the normal zygote nucleus divides at meta-
phase into two daughter chromosomes, one of which is predestined to
undergo diminution and the other is not. If this were the case in the
dispermic egg, it would follow that six of the daughter chromosomes

Ee F
FIG. 40,

Diagram illustrating the part played by the cytoplasm in determining the diminution of the chromatin
in Ascaris megalocephala. (After Boveri, Festschr. f. Hertwig, 1910.) The shaded portion represents the

“vegetative ’’ cytoplasm. In every case the chromosomes remain intact in the cells containing this

substance, and undergo diminution in the cells which lack it. A, undivided egg; B, C, D, stages in the
production of the 4-cell stage in the normal, monospermic egg; E, F, G, results of the first cleavage of
dispermic eggs. According to the orientation of the spindles with regard to the egg axis, one of the three
types shown is obtained.

produced by the metaphase of the first cleavage mitosis were predestined
to undergo diminution, while their six sister chromosomes were not.
Moreover, it would often result from the unequal distribution of the
daughter chromosomes among the four primary blastomeres, that the
same nucleus would contain a mixture of chromosomes predestined for
diminution and of those predestined to remain intact. As a matter
of fact, neither of these expectations is realized. Diminution may take
place in one, two or three of the four cells formed by the first cleavage
division. All the chromosomes in any one nucleus behave alike, and the
number of chromosomes which escapes diminution, instead of being
always six, varies from two to twelve in different eggs.

86 CYTOLOGY cHaP.

The reasonable explanation which Boveri offered is that the uncleaved
Ascaris egg, like the eggs of so many other animals, possesses an internal
cytoplasmic differentiation into an upper, or “ animal,’ and a lower,
or “‘ vegetative,” pole. In the normal monospermic egg the first cleavage,
being horizontal, divides it into “‘ animal”’ and “ vegetative’ blasto-
meres, the former being less rich in yolk than the latter. The
chromosomes in the “‘ animal ’’ blastomere (S) undergo diminution, those
in the ‘‘ vegetative” blastomere (P) do not. The second cleavage divides
both blastomeres into two, and again the chromosomes in the cell nearest
to the “ vegetative ’’ pole alone remain intact.

Dispermic eggs, as stated above, divide simultaneously into four cells,
and this may take place in one of three ways, according as to how the
spindles are arranged in relation to the polarity of the cell—namely,
one P and three S cells, two P and two S cells, or three P and one S cell,
as illustrated in Fig. 40. Chromatin diminution takes place in all the S
cells but not in the P’s, that is to say, it takes place in all cells which
fail to contain a portion of the cytoplasm from the vegetative pole of
the egg.

A fair number of animals are now known in which the germ-track
is thus visibly marked out, the distinguishing marks being of the most
varied, and sometimes surprising, nature, though probably all ultimately
of cytoplasmic rather than nuclear origin. Thus in the crustacean
Polyphemus pediculus (Kuhn, tg11), when the egg is laid it has attached
to it the remains of one to three “ nurse cells ’’ (oocytes which have failed
to develop into eggs but have been absorbed by other oocytes). When
the vitelline membrane is secreted it is formed in such a way as to include
this little mass of nurse cell remains within the egg, and it soon becomes
embedded in the egg cytoplasm. As long as the included mass les
passive, it follows that it can only be present in one of the blastomeres,
and consequently at the 16-cell stage it is found in one blastomere
and is absent from the other fifteen. At this stage the little mass breaks
up and becomes scattered through the blastomere in which it lies. Conse-
quently, at the next division of this blastomere, both its daughter cells
contain portions of it. These two cells are the primordial germ-cells.

A useful summary of the various forms of germ-track differentiators
at present known is given by Hegner (1914 and 1915). There appears
to be nothing corresponding to the animal germ-track in the higher
plants (see, however, footnote to p. 144).

E. PARTHENOGENESIS

It is plain that the development of the egg without fertilization must
involve some modification of the general rule that the mature egg has

ul PARTHENOGENESIS 87

only half the number of chromosomes present in the other tissues of the
body. Otherwise this number would be halved in each generation, and
very soon brought to vanishing point. This eventuality is avoided by
one of two methods.

(1) The halving of the chromosome number in oogenesis is omitted,
and the ripe egg is diploid. This is usually accompanied by the formation
of only one polar body instead of two, and is the commonest form
of parthenogenesis, It is found in the Cladocera, Ostracoda, Aphids,
etc., amongst Arthropods and in Rotifers, etc. Occasionally, however,
two polar bodies are formed, without reduction of chromosomes (the
Hymenoptera Nematus and Rhodites).

(2) True meiosis takes place, the resulting ripe egg being haploid,
but reduction is omitted when the haploid individual developing from this
egg forms its own gametes. This form of parthenogenesis has been
found in several Hymenoptera. In all cases so far known, the haploid
individual thus produced is a’male. This, it will be observed, is in
accordance with the fact that in the great majority of animals in which a
difference in the chromosomes of the sexes has actually been observed
the male lacks the second sex chromosome, or has it replaced by an inert
chromosome (Chapter IV.). For it is obvious that a haploid animal
can have only one of each kind of chromosome, including the sex
chromosome.

The eggs of type (1) being diploid are incapable of fertilization, and
therefore committed to develop parthenogenetically ; they are said to
exhibit obligatory parthenogenesis. The haploid eggs of type (2) differ
in no way from eggs destined for fertilization. They seem indeed to
be equally capable of fertilization or parthenogenetic development. If
fertilized they give rise of course to an ordinary diploid individual, which
in all cases known is a female. If they are not fertilized they develop
parthenogenetically into a haploid male. Parthenogenesis in these cases
is therefore said to be facultative.

Further, the eggs of many animals which normally develop only
after fertilization can be induced to develop parthenogenetically by the
application of appropriate stimuli. Such eggs produce either haploid
or diploid individuals according as to whether meiosis had or had not
taken place before the parthenogenetic development was induced. This
phenomenon is known as artificial parthenogenesis.

The cytology of these three types of parthenogenesis will be considered
in order.

(1) Obligatory Parthenogenesis

By far the greater number of these cases are accompanied by the
formation of only one instead of two polar bodies, and the chief interest

88 CYTOLCGY CHAP. |

centres in the details of the meiosis, as we may call it, though it does
not result in a reduction of the chromosome number.

A puzzling feature about the changes undergone by the nucleus in
preparation for the single maturation division is their extraordinary
similarity to the typical phases of a true meiosis resulting in chromosome
reduction. The definitive chromosomes of the single maturation division
are also strikingly similar to those found in true meiosis, though the
former are univalent and the latter bivalent (Figs. 41, 42).

Thus, in both sexual and parthenogenetic Ostracods (Schleip, 1909)
there occurs a synizesis from which in the former the haploid, and in
the latter the diploid, number of chromosomes emerges. These chromo-
somes are remarkably alike in appearance in the two types of eggs, being
conspicuously double in both (Fig. 41). In the one case, however, the
duplicity is due to bivalency, in the other to the prophase division of

ee ere univalents. Kuhn
ASL iti (x908) found in par-
fi hae a) | Rian thenogenetic Clado-
byt ae es “we scera a stage with
ll os z oy 3% o conspicuous dupli-
\\aeg fe ia 7} ) Vinee gy ng city of chromatin ~
A ie x } v B c _ threads, strongly
Wo A suggesting syndesis
Bic. 47. (Big: Az)ty, Epis

A, parthenogenetic oocyte of Daphnia pulex during the growth stage stage, however, is
(after Kuhn, A.Z., 1908); B, C, chromosomes of the maturation divisions
of two Ostracods. B, Notodromas monacha (sexual); C, Cypris fuscata both preceded and
(parthenogenetic) (after Schleip, A.Z., 1909). ;
followed by one in
which the chromosomes are obscured by their relation to the nucleolus,
thus making correct interpretation difficult.

The similarity between the meiotic processes of sexual and partheno-
genetic species has indeed been cited by some cytologists as reason for
denying altogether the connection of the ordinary meiotic phenomena
(zygotene stage, etc.) with the reduction of the chromosomes. This is
undoubtedly going to an unjustifiable length, for (1) none of the animals
which exhibit parthenogenesis are really favourable objects for cyto-
logical study. They cannot be compared in this respect with, say,
Tomopteris, Lepidosiren, or the Amphibia, and consequently the details
cannot be said to be satisfactorily known ; and (2) the fact that the
diploid chromosome number appears in late prophase and metaphase
in parthenogenetic eggs is no proof that syndesis did not take place in
earlier prophase. We have only to remember the complete dissociation
of the ex-syndetic homologous chromosomes in the spermatogenesis of
Lepidosiven and in many oogeneses to agree with this proposition. It
is not impossible that, in the examples of parthenogenesis now under

ul PARTHENOGENESIS 89

discussion, syndesis takes place in the usual manner, and that the
homologous chromosomes become completely, instead of partially, dis-
sociated in the diplotene stage. Instead of pairing again to form the
bivalents of the meiotic metaphase, each chromosome behaves from now
onwards as it would in a somatic mitosis.

Strasburger (1907, 1909) was the first to elaborate this view in the
case of plants, and has in fact described the dissociation of the haploid
bivalents into diploid univalents in the parthenogenetic (‘‘ apogamous ”’)
development of the macrospore of the cryptogam Marsilia drummondit.

For animals, however, it still remains nothing more than a conjecture.

Although in the parthenogenetic species considered so far the meiotic
prophases are scarcely distinguishable from those of sexual forms (except
for the diploid chromosome number and the occurrence of only one instead
of two maturation divisions), there exist other forms where the meiotic
prophase stages are markedly different in allied parthenogenetic and
sexual species. This again was first described in plants (Urticaceae)
by Strasburger (1910).

In animals, Fries (1910) found in the crustacean Branchipus (Fig.
42), which reproduces sexually, all the ordinary phases of meiosis, namely,

' a leptotene stage followed by synizesis, during which the leptotene

threads arrange themselves in parallel pairs; these apparently fuse to
form pachytene bands which on the dissolution of synizesis are found
in the haploid number. In the nearly allied but parthenogenetic Artemia
salina, however, there is no synizesis, nor parallelization of leptotene
threads, nor fusion of these to form pachytene bands. On the contrary,
each leptotene thread condenses into a single chromosome, and conse-
quently these are present in diploid number. Again, however, we find
the definitive chromosomes—in the one case bivalents, and in the other
split univalents—surprisingly alike in appearance in the two genera.

Morgan (1915 a) also found in the Aphids Phyllaphis and Phylloxera
that in the ovaries of sexual females the meiotic prophases include a
synizesis, into which the full number of chromosomes (6) enter, and from
which three bivalents emerge. In the ovaries of the parthenogenetic
members of the same species, the six chromosomes of the early meiotic
prophase contract continuously into six univalents without ever being
condensed in a synizetic contraction.

A very few cases of obligatory parthenogenesis are known in which
two meiotic divisions occur as in sexual reproduction, but without
reduction of the chromosome number. The mature egg is therefore in
the same condition as in the ordinary cases of obligatory parthenogenesis
with only one meiotic division. This very puzzling phenomenon was
first (with the exception of the special case of Artemia) described by

go CYTOLOGY CHAP.

Doncaster (1907) for the parthenogenetic eggs of the saw-fly, Nematus
vibesit, and later by Schleip (1910) for Rhodites rosae, a gall-fly.

A very peculiar phenomenon observed by Brauer (1894) in Artemia
also falls into this category. As we have already seen (p. 89), the normal
procedure is for only one maturation division to take place, without

FIG. 42.

Meiotic prophase in the sexual egg of Branchipus (A-G), and in the parthenogenetic egg of Artemia (H-J).
(After Fries, A.Z., 1910.) A, young oocyte I.; B, synizesis and leptotene stage ; C, zygotene, D, pachytene
stages ; E, F, G, contraction of the chromosomes into the definitive bivalents ; H, young oocyte I. ; I, late
prophase, chromosomes longitudinally split ;_ J, definitive chromosomes of the maturation division.
reduction of chromosome number. As a rare exception, however, Brauer
found that a second division took place, again without reduction. The
two nuclei resulting from this second mitosis must be regarded as equiva-
lent to the nuclei of the mature egg and second polar body. The latter,
however, is not extruded from the egg, but remains close to the egg
nucleus and moves with it to the centre of the egg. Thereafter these
two nuclei act like the male and female gamete nuclei in fertilization,

ul PARTHENOGENESIS gl

and fuse together. The egg behaves, indeed, as if it had been fertilized
by the second polar body, except that, since no halving of the chromosome
number has taken place in either nucleus, the resulting zygote nucleus
has double the normal diploid number, that is to say, 168 instead of 84.

It should be noted that Brauer found this mode of meiosis much
rarer than the ordinary mode with only one maturation division, and that
Fries did not find any instance of it among his material.

(2) Facultative Parthenogenesis

The second type of parthenogenesis, where the egg matures in the
ordinary way and develops with the haploid number of chromosomes,
has been described in a few Hymenoptera. The classical example is
the honey-bee (Apis mellifica). It has long been known that the fate of
the bee’s egg depends upon whether it is fertilized or not. The queen
bee is impregnated by the drone during the nuptial flight, and the sper-
matozoa are stored up in her receptaculum seminis. As the eggs pass
down the oviduct they pass the mouth of this receptacle, and according
as to whether a spermatozoon issues from it or not, the egg is or is not
fertilized. If fertilized, the egg develops into a female (either a queen or
a worker according to the food supplied to the larva) ; if not fertilized
it develops into a male.

The cytology of the bee has been thoroughly worked out by Meves
(1907) and Nachtsheim (1913). The diploid chromosome number is
thirty-two, though, as in many other animals (e.g. Ascaris, p. 81), this
number is greatly exceeded in the somatic tissues outside the germ-
track, and may reach as high a number as sixty-four. On the other hand,
in the oogonia the chromosomes tend to come together in pairs, giving
sixteen double chromosomes, much as in some Diptera! (p. 126). Thus
the determination of the chromosome number is fraught with some
difficulty, but it is revealed by the number in the gamete, which is
sixteen, and in the female embryo, where it is thirty-two.

No difference is to be expected, nor as a matter of fact was observed,
between the meiotic processes of those eggs that are to be fertilized and
those that are not. After the second meiotic division the egg nucleus
leaves the surface of the egg (in which position, as usual, the maturation
divisions take place) and travels towards the centre. If the egg has been
fertilized it there meets with the male nucleus and an ordinary zygote
nucleus is formed. If it has not been fertilized, the egg nucleus continues
to travel right across the egg to the opposite side, as if in search of the

1 A simiiar but less strong tendency to pair is observed in the spermatogonia (Nachtsheim)
which is particularly noteworthy, because these nuclei are haploid. Moreover, the sixteen

double chromosomes in the oogonia unite into eight (tetravalent) chromosomes in the meiotic
prophase. See p. 92.

92 CYTOLOGY CHAP,

male nucleus, and there forms the first cleavage spindle of the developing
embryo.

Great interest attaches to the cytology of the spermatogenesis of the
individual developing from these eggs, for since it is already haploid
a further reduction of chromosomes must be avoided. How this is
accomplished has been worked out by Meves.

The main features of the meiosis in the drone are shown in Fig. 43.
None of the usual meiotic prophase stages, such as leptotene or zygotene
nuclei, are found; no essential stages seem to intervene between that
shown in Fig. 43, A, and that of Fig. 43, B, by which time the definitive
chromosomes have appeared. There are sixteen of these as in the
spermatogonia, each being conspicuously split longitudinally. An
intra-nuclear mitotic figure is formed and the chromosomes congregate
at the equator as if for an ordinary metaphase. This, however, is not
consummated ; the daughter chromosomes do not separate, but the
mitotic figure degenerates and the chromosomes become clumped together
again, generally at one pole of the elongated nucleus. Although the
nucleus does not divide, cell division proceeds, with the result that one
of the daughter cells lacks a nucleus. The non-nucleated cell is very
much smaller than the other, and of course takes no further part in
gametogenesis. Meanwhile the nucleus of the other cell prepares for the

A ntl al

second meiotic division, sixteen chromosomes again appearing as in the ©

abortive first division. The second division is carried through in the
normal way in so far as the nucleus is concerned, each chromosome
dividing into two daughter chromosomes which separate in anaphase.
Curiously enough, however, the cell again divides very unequally, though
this time both daughter cells receive a nucleus. These of course each
contain sixteen chromosomes (though here again they tend to come
together in eight pairs—see footnote to p. gI).

In spite of their unequal size, both spermatids begin the series of
changes which should convert them into spermatozoa, but it is probable
that only the larger one completes the process.

The essential feature of this spermatogenesis is of course the omission
of the true meiotic division, by which a further halving of the already
haploid group of chromosomes is avoided. In conformity with this, no
such stages as leptotene or zygotene nuclei have been described in the
male honey-bee. That these stages are really absent is made still more
probable by Armbruster’s (1913) observation on the spermatogenesis
of the solitary bee Osmia cornuta. He states that though he specially
looked for these stages he failed to find them; the telophase of the
last spermatogonial division appears to pass without important inter-
mediate stages into the diakinesis of prophase I.

The unequal cell division of the secondary spermatocyte in the honey-

ur PARTHENOGENESIS 93

bee is puzzling, and does not appear to have any significant connection

Fic. 43.

The meiotic phase in the drone of the honey-bee (Apis mellifica). (After Meves,°A.m.A., 1907.) A,
primary spermatocyte; B, prophase I.; C, abortive metaphase I.; D, E, degeneration of the mitotic figure,
beginning of cell division; F, prophase II. The cell has completed its division into a larger nucleated and
a smaller non-nucleated portion. G, metaphase II.; H, anaphase II.; I, cell divided into two unequal
spermatids. The non-nucleated cell derived from the first cell division is still attached.

with the peculiarities of the first division. In the hornet (Meves and

94 CYTOLOGY CHAP,

Duesberg, 1908) the first division is abortive as in the bee, but the
second is normal, resulting in two equal and similar spermatids.

(3) The Homology of the Metotic Divisions in Obligatory and Facultative
Parthenogenesis

The above account of the spermatogenesis of the bee shows that
it is the first division of the meiotic phase which is omitted, and this
is in accordance with the consensus of recent opinion that it is this
division which usually brings about the segregation of the homologous

chromosomes. It would be of great interest to determine which of the —

two divisions it is which is omitted in the maturation of eggs preparing
for obligatory parthenogenesis, if indeed the single maturation division

of these eggs can be homologized with either of the divisions of an_

ordinary sexual meiosis. It used generally to be assumed that it is the
second division which is omitted, but there is little evidence either Way.

What there is, is perhaps in favour of this view. Weismann and

Ishikawa (1887) found that the single polar body produced by the

parthenogenetic eggs of Cladocera frequently divides into two or more —
cells—a feature perhaps more characteristic of the first than of the

second polar body of an ordinary oogenesis. Again, Brauer’s observation
that in Artemia, after the first polar body has been formed in the usual
way, a more or less abortive attempt is in rare cases made to form another,
probably indicates that the single maturation division of the majority
of eggs corresponds to the first one of a sexual meiosis. As, however, the
second division is still unaccompanied by reduction of chromosome
number (like the two divisions without reduction in Nematus and Rhodites})
it is possibly useless to attempt to homologize the two divisions with the
first and second divisions respectively of an ordinary meiosis.

Even though it were shown that the single division of obligatory
parthenogenesis has all the characteristics (except that of halving the
chromosome number) of the normal first meiotic division, this need
cause little surprise. For few things are better established in cytology

than the fact that in the case of one type of chromosome—the sex

chromosomes (Chapter IV.)—the first is the reduction division in some
animals and the second in others, even nearly allied species differing in
this respect.

(4) Artificial Parthenogenesis

It has long been known that eggs which normally only develop after
fertilization may be induced by certain agents to develop without fusion

1 It is perhaps significant that there is found a variation in the chromosome number both
in Nematus and Rhodites somewhat similar to that in the bee (p. 91).

EE ee

III PARTHENOGENESIS 95

with a spermatozoon. The accurate study of this phenomenon dates
from the experiments of O. and R. Hertwig on Echinoderm eggs (1887).
The methods have been specially elaborated by Loeb and others, and
consist, so far as the eggs of marine animals are concerned, in placing
them in sea water of which the chemical composition has been altered
in various ways; for details the reader is referred to any of the works
cited below.

From the point of view of cytology, two points claim special attention
—the achromatic figure and the nucleus. As we have already seen, in
the mature egg the centrosome and other parts of the achromatic figure
apparently disappear ; a new centrosome, which in turn gives rise to
the rest of the figure, being provided for the zygote by the spermatozoon.
Eggs which have started development under the stimulus of chemical
reagents are, however, provided with typical centrosomes and spindle
figure, etc. Two alternatives as to the origin of these are possible: (1)
that the old egg centrosome does not disappear entirely, but is merely
rendered latent and is reawakened to activity by the stimulus which
induces the parthenogenesis ; or (2) that the centrosomes arise de novo
in the stimulated egg. Although the first alternative may be true in
some cases, it has been demonstrated beyond question that the latter
may occur also. Wilson (1g01) showed this for Toxopneustes. If the
eggs of this sea-urchin are subjected to sea water to which MgCl, has
been added, a large number of division centres (cyfasters) may appear
simultaneously in the cytoplasm. Each centre is provided with a
centrosome and rays, exactly as in a typical aster. These asters divide
again like normal asters, before nuclear division, their division being
preceded by that of the centrosome. Both Wilson and M‘Clendon (1909)
even obtained cytasters in eggs from which the nucleus had been
removed. Such enucleated eggs of <Astevias segmented for several
hours, dividing into a large number of irregular blastomeres by the
action of these cytasters. We are therefore compelled to conclude that
the centrosome and its derivatives, though normally a permanent cell
structure derived by division of a previous one, can arise de novo in the
cytoplasm.

As regards the nuclear cytology of artificial parthenogenesis, we
must distinguish between the types of eggs mentioned on p. 73, accord-
-ing as to whether maturation normally takes place before or after
entry of the spermatozoon or, in the case of artificial parthenogenesis,
the stimulus which takes the place of this.

The simplest case is afforded by those eggs which mature before the
entry of the spermatozoon ; for example, those of the Echinoidea. These
eggs mature in the ovary, and therefore when, after being laid, they are
subjected to the stimulus which is to cause them to develop, they are

96 CYTOLOGY CHAP.

already haploid. In such eggs, if caused to develop parthenogenetically,
the haploid egg nucleus acts in cleavage exactly like the zygote nucleus
in a fertilized egg. The resulting embryo is in consequence haploid, as
has been demonstrated by many observers, though originally Delage
erroneously supposed that the diploid number was restored by an act
of auto-regulation (Wilson, Toxopneustes, 1g01; Hindle, Strongylocentrotus,
EOLE, ete.).

Eggs which do not normally mature until the spermatozoon has
entered vary in their reaction to the artificial developmental stimulus.
Some, such as the annelid Thalassema (Lefevre, 1907), on being subjected
to the appropriate chemical stimulus undergo maturation, throwing out

"Ae 2
os \
\\, é.
Wg 27
[soto
~}2 ps} oak ie
{} ¥ ae -2\
= =
ay <3 ; SI
\wat, 2
A B !
Fic. 44.

Artificial parthenogenesis in Asterias. (Buchner, A.Z., 1911.) A, telophase of second maturation division.
The second polar body nucleus, instead of being thrust out of the egg, remains close to the egg nucleus.
B, the egg and second polar body nuclei have come into contact. Achromatic figure developing. C, the two
nuclei have fused.

p.v.1., first polar body.
two polar bodies, as if they had been properly fertilized. The haploid
egg nucleus then proceeds to divide as in Echinoids, and a haploid embryo ~
results. In Asterias, on the other hand (Buchner, Ig11I), while the first
maturation division is carried through normally and the first polar
body is cut off, the second division only proceeds normally as far as
telophase. Instead of the outer telophase group being extruded from
the surface of the egg in the second polar body, it is retained within the
egg and there forms a nucleus lying close to the inner group or egg nucleus
(Fig. 44). The egg and second polar nuclei now approach each other
again and fuse precisely as if the latter were the male gamete nucleus.
Thus the diploid number of chromosomes is restored. The resemblance
between this process and the rarer method of maturation of the partheno-

genetic egg of Artemia described by Brauer (p. go) is striking.

Hi " PARTHENOGENESIS 97

The immediate cause of the retention of the second polar nucleus
within the egg seems to be that the tendency of the chromosomes to
form karyomeres (see p. 131) in Echinoderm eggs is accentuated, or
rather accelerated, so that the chromosomes begin their telophase
metamorphosis before the daughter plates of the second meiotic division
have fully separated. Consequently the two daughter nuclei come to lie
close together.

The fertilization of Amphibian eggs by spermatozoa which have
been injured by radium emanation or by the action of certain poisons,
such as strychnine and nicotine, is in a sense intermediate between
artificial parthenogenesis and natural fertilization. G. Hertwig (1913)
established the, at first sight paradoxical, result that whereas if the eggs
of the common toad are fertilized by the spermatozoa of the frog, very
few zygotes result and these never develop so far as gastrulation, yet if
the frog spermatozoa be injured by subjection to radium emanation
before being added to the toad eggs, a large proportion of embryos pass
the hatching stage. The interpretation put forward is that in the first
case development is prejudiced by the incompatibility of the chromatin
of the two animals, while in the second case the frog spermatozoa are
so weakened that, though they enter the eggs, they have not the power
to fuse with the female pronucleus. Their entry, however, stimulates the
eggs to develop. This explanation is borne out by the further investiga-
tions of O. Hertwig (1913), who counted the haploid chromosome number
in newt larvae which had developed from eggs fertilized by spermatozoa
previously subjected to treatment with radium.

These experiments are closely comparable to the cross-fertilizations
between Echinoderms, Molluscs, etc., described on p. 161.

Nearly related to artificial parthenogenesis is the phenomenon of
merogony, the term applied to the fertilization of an egg fragment con-
taining no nucleus. Echinoderm eggs will survive being broken into
|, fragments, one only of which of course can contain the nucleus. The
| non-nucleated fragments can be fertilized by spermatozoa and then
develop into dwarf and haploid, but otherwise normal, larvae. This
phenomenon is discussed in greater detail in Chapter VI.

CHAPTER: ay
THE SEX CHROMOSOMES

(1) The Sex Chromosomes in Insects

In 1891 Henking, working on the spermatogenesis of the Hemipteran
insect Pyrrochorts, discovered that, contrary to the general rule in other
groups of the animal and vegetable kingdoms, the diploid number of —
chromosomes as determined in the spermatogonia is an wneven one,
namely, twenty-three.

Consequently, instead of all the spermatozoa having eleven chromo-
somes as if the diploid number were twenty-two, or all having twelve
as happens when it is twenty-four, half the spermatozoa have eleven
chromosomes and half have twelve. Taking a large stride in the
historical development of the case, we now know that the female Pyrro-
choris has twenty-four as its diploid number, and hence all its eggs
have twelve (Wilson, 1909 a).

Now it is obvious that if an egg is fertilized by one of the
spermatozoa containing eleven chromosomes, the resulting zygote will
have twenty-three, which as we have seen is the diploid number of the
male. If on the other hand an egg is fertilized by a spermatozoon with
twelve chromosomes, the resulting zygote will have twenty-four, the —
number of the female. Thus it appears that in these insects the sex of —
the individual is determined by the nature of the spermatozoon which
fertilizes the egg—the eggs being all alike, or indifferent, and the sperma-
tozoa of two kinds in equal numbers, namely, male-producers with
eleven chromosomes, and female-producers with twelve chromosomes.

As the odd chromosome of the male was first supposed to be an
additional one, it was for a time known as the “ accessory chromosome ”’
(M‘Clung). It is now known, however, that the female has one more ©
chromosome (in these forms) than the male, so that the unevenness of
the number in the male is not due to the addition, but to the subtraction
of a chromosome. Hence the term accessory is obviously unsuitable.
Other terms have been proposed, such as heterochromosomes (Montgomery)
and heterotropic chromosomes (Wilson). The now generally accepted

98

CHAP. IV THE SEX CHROMOSOMES 99

term of Sex Chromosomes is, however, a sufficient and obvious description
of them, and by this name they will be called here. Owing to the
comparative ease and certainty of the observations, and to the interest
due to their relation to sex, a great mass of knowledge of these sex chromo-
somes has been accumulated in recent years, American cytologists
having been particularly active along these lines.

FIG. 45.

The chromosomes of Protenor belfragei. (A, I-K, from Morrill, B.B., 1910; B-E, H, after Montgomery,
Trans. Amer. Phil. Soc., 1901; F, G, from Wilson, J.£.Z., 1906.) A, spermatogonial metaphase group.
Twelve ordinary and one sex chromosome. B, prophase I.,¢; C, metaphase I.,g; D, anaphase I.,¢; E
early anaphase II. in the. Sex chromosome passing undivided to one pole. F, G, polar views of the
chromosome groups at each pole of the spindle in anaphase II. One group with the sex chromosome, the
other without. H, late anaphase II., showing one spermatid with the sex chromosome, the other without
I, oogonial metaphase group, twelve ordinary and two sex chromosomes; J, chromosome group from a male
embryo; K, chromosome group from a female embryo.

X, the sex chromosome.

As a typical example of an animal with sexual dimorphism of
chromosomes we may take the Hemipteran insect Protenor belfraget
(Fig. 45). Its chromosome cycle has been worked out by Montgomery
(rgor 6), Wilson (1906 8, etc.) and Morrill (1910).

The spermatogonial chromosome groups (A) show thirteen chromo-
somes, one being more than twice as large as any of the others. This

roo CY LOLOGY CHAP.

is the odd sex chromosome, and on account of its large size it is easily
identified at all stages.

B is a late prophase I., showing six bivalents and the sex chromosome,
which, being without a homologue, must remain univalent. Correspond-
ing with the difference in valency, the behaviour of the sex chromosome
in the two meiotic divisions is remarkably unlike that of the other
chromosomes. In metaphase I. the bivalents separate into their
constituents in the usual way. The unpaired sex chromosome, on the
other hand, divides longitudinally as if the mitosis were somatic, thus
anticipating the normal division of the chromosomes in metaphase II.
Each spermatocyte II. therefore contains a similar chromosome group
of six ordinary chromosomes and one sex chromosome. In metaphase II.
the ordinary chromosomes divide longitudinally in the usual way, but
the sex chromosome, which has already undergone this division in
metaphase I., does not divide again, but passes intact to one or other
pole of the mitotic figure (E). Hence the two spermatids formed by
each secondary spermatocyte differ in their chromosome equipment,
one (G, and upper cell in H) containing the sex chromosome, and the
other (F, and lower cell in H) lacking it.

The female Pyrotenor has fourteen chromosomes instead of thirteen,
there being two of the large sex chromosomes instead of only one.
Hence all the mature eggs will have 6, or 5+ X (X standing for the sex
chromosome). If now an egg is fertilized by a spermatozoon of the
composition shown in F—+.e. without the X chromosome—the resulting
zygote will have 13 chromosomes thus :

3 3
(6+ X)+6=12+X (4, Fig. J).

If fertilized by the spermatozoon shown in Fig. G the result will be:

3
ee eee (9, Fig. K).
Protenor is an example of the simplest case known, in which the male
differs from the female in possessing one chromosome fewer. Several
more complicated conditions than this are known, however.

For instance, in Lygaeus, another Hemipteran, the male has the same
number of chromosomes as the female, each having a pair of sex
chromosomes, but while in the female these are equal, like any other pair
of homologous chromosomes, in the male they are unequal, one being
much smaller than its mate; this latter is of the same size as those of
the female. The relation between the male chromosome groups in
Lygaeus and Protenor may therefore be expressed by the statement
that in Lygaeus one of the pair of sex chromosomes is reduced, while
in Protenoy one is absent. Or, using the convenient notation now in

IV THE SEX CHROMOSOMES IOI

general use, X standing for the large sex chromosome and Y for the
small one, the chromosome formulae of the two species is as follows

(Lygaeus having like Protenor six pairs of ordinary chromosomes or
“ Autosomes ”’) :

DIPLOID. HAPLOID.
Protenor 9 12+XX _~ eggs all6+X.
6 12+X spermatozoa 6+ X or 6.

Lygaeus 9 12+XX_~ eggs all6+4+X.
6 12+XY spermatozoa 6+X or 6+Y.

The cases are seen to be exactly parallel. There are two classes

LR@E>* (AR
APG

| | | erat
Se ian ».4
Cc D

H J J K L
Fic. 46

The chromosomes of Lygaeus turcicus. (After Wilson, J.E.Z., 1905 and 1912.) A, spermatogonial
telophase; B, later telophase ; C, emergence of the massive chromatic bodies in the primary spermatocyte ;
D, leptotene stage; E, “ confused stage”; F, evolution of the bivalents; G, metaphase I.; H, I, daughter
chromosome groups of anaphase I.; J, metaphase II., X and Y, now paired to form a bivalent; kK, L, daughter

chromosome groups from anaphase II.
of spermatozoa in Lygaeus—one, the 6 + X form, will on fertilizing an egg
produce a female, the other (6+Y form) will produce a male. The
process of meiosis in the male Lygaeus is illustrated in Fig. 46. This
figure demonstrates the important fact that the sex chromosomes of the
male often (though not in all cases; see below) remain compact throughout
the whole meiotic prophase, including the leptotene and zygotene stages.
It will be noted in Fig. 46, G, that X and Y are not paired to form

102 CYTOLOGY. CHAP,

a bivalent like the other chromosomes, but are quite separate from one
another, and that each is constricted preparatory to division, as if it
were a somatic mitosis. Thus we now find six bivalent ordinary
chromosomes and two univalent sex chromosomes (only two of the
bivalents are shown in this figure). Hence each anaphase group contains
both X and Y. In prophase II. X and Y conjugate to form the unequal
bivalent shown in Fig. 46, J (metaphase II.), which results in the two
kinds of spermatid chromosome groups shown in K and L.

In still another Hemipteran (Oncopfeltus, Wilson, 1912) the X and Y
chromosomes are so nearly equal that in many individuals no inequality
could be demonstrated, though in others a distinct size difference was
detected. Even where they are equal the two sex chromosomes are
nevertheless easily identified by their compact form throughout the
meiotic prophase. This compact phase is, however, by no means a
universal feature of the sex chromosomes, and hence the possibility is at
once suggested that forms exist in which there are X and Y chromosomes
differentiated physiologically, but not visibly distinguishable from each
other or from the other chromosomes. Hence the sexual differentiation
of chromosomes, which has been demonstrated for a comparatively small
number of animals, so far from being peculiar to them, may be a universal
characteristic revealed by the lucky accident that such differentiation is
in some animals visible by ordinary methods of microscopic technique.

We will now consider some other features of the sex chromosomes.

(2) Which of the two Meiotic Divisions acts as the Reduction Division for
the Sex Chromosomes ?

It is a surprising fact that, in the cases just described, the sex
chromosomes divide longitudinally in the first meiotic division, while
the second is the actual reduction division, separating the X from
the Y (Lygaeus) or sending the single sex chromosome into the one
spermatid and leaving the other spermatid with no sex chromosome
(Protenor). In these cases, therefore, the reduction division for the
ordinary chromosomes is the first, and for the sex chromosomes the
second, meiotic division. What is perhaps even more surprising is that
the behaviour of the sex chromosomes varies in this respect in different
forms, and sometimes in nearly related species. An example of an insect
in which the first division is the differential one is given in Fig. 47.

The following table, compiled from the exhaustive summary of the
numbers of chromosomes in the Metazoa given by Harvey (1917), shows
how the orders of insects vary in this respect. It will be noticed that
most of the species within any order are alike, but in most orders there
are one or two exceptions to the general rule. In compiling this table

IV THE SEX CHROMOSOMES 103

The chromosomes of the beetle Blattella germanica. (Stevens, Carnegie Inst. Pub., 1905.) A, primary
spermatocyte; B, prophase I.; C, metaphase I., polar view, showing twelve chromosomes, X not distinguish-
able from the others in this view; D, portion of metaphase I., side view, showing X passing undivided to one
pole; E, late anaphase I.; F, telophase I., X in one daughter nucleus only ; G, prophase II. of the secondary
spermatocyte which contains the X chromosome; H, polar view of metaphase II. of secondary spermatocyte
lacking the X chromosome; I, similar figure of secondary spermatocyte containing the X chromosome ;
J, side view of metaphase II., showing the X chromosome dividing.

cases which are doubtful or which present any special complication have
been omitted.

|

| | Number of Species in which the Reductional Division for
the Sex Chromosomes is the
GROUP. |
a eee a” es -
| First Division. Second Division.
—— a = = _ P
| Neuroptera . ; a | I I |
Orthoptera : S| 88 fo)
| Coleoptera. . =| 38 2 |
Diptera . ‘ : *| II oO
Hemiptera heteroptera . 2 68
| Hemiptera homoptera_ . | 43 I
= : eet
Total . | 183 72

104 CYTOLOGY CHAP.

The two different ways of obtaining the same end result—the pro-
duction of the two types of spermatozoa in equal numbers—may be
represented in a diagram as follows :

First division equational. First division reductional.
Second division reductional. Second division equational.
Spermatocyte I. (xy) XY

Spermatocyte II. (xy) XY x Go)
Spermatid. (x) @) X (vy) (x) (x) (y) &

(3) Various forms of the X and Y Chromosomes

A frequent feature of the X chromosome is that it is compound,

Fic. 48.

The chromosomes of Gelastocoris. (Payne, B.B., 1909.) A, 2 diploid group, 30+2X,=38 chromosomes ;
B, 6 diploid group, 30+X4+Y=35 chromosomes; C, metaphase I.,g, 15+X4+¥Y-=20 chromosomes; D,
metaphase II., g ; the sex chromosomes are in the middle of the ring, the four components of the X chromo-
some in a group opposed to the single Y chromosome which is partly underneath them; E, metaphase II., g;
F, anaphase II., ¢ ; G, H, polar views of anaphase II., g—G with the Y chromosome, H with the X group.

IV THE SEX CHROMOSOMES 105

consisting of two or more components. These are separate in the somatic
or premeiotic nuclei of both sexes, and in all nuclei of the female (with
the probable exception of Phylloxera caryaecaulis, see p. 117); in the
meiotic phase of the male, however, they commonly become associated
in a degree varying from merely a more or less close grouping (Acholla)
to an actual junction (Syromastes) (Figs. 48, 49, 50). The Y chromosome
is always simple, even in those species where the X is compound. The

oh o@ ® O@s
C & °@
S20 6.29 e8%ee
@@ 00 y eee
B = D
A
ma i e°*,
DG rN e@e
Ns eee A 7% ge
is @ os e pie
‘@ @ @ | - " H
° & [,| | ae
@%e0 0, as
x" @e 9° nan ee
ae ong d 2N 00%
X E s ei he <i x

Fic. 49.

o

The chromosomes of Syromastes (A, B) and Acholla multispinosa (C-1). (Wilson, B.B., 1909 a, and Payne,
B.B., 1910.) In A and B the X chromosomes are blackened. A, ¢ diploid group, 20+X.=22 chromosomes; B,
@ diploid group, 20+2X2=24 chromosomes: C,@ diploid group, 20+2X;=30chromosomes. The three pairs
of small X elements are easily seen, the other two pairs are not at this stage distinguishable. D, ¢ diploid
group, 20+X;5+Y=26 chromosomes; E, metaphase I.,g, polar view. The Y element is characteristically
in association with the two largest of the X elements. F, G, anaphase I., side view cut in two sections. The
section in F contains the Y and the two large X elements, and that in G the three small X elements. UH, I,
anaphase II., showing the results of the segregating division,

following examples illustrate the range of variation in the constitution
of the sex chromosome (Fig. 51) :

(A) No Y chromosome present.

I. X Chromosome single (Protenor).
2. X » double (Syvomastes).
35 ¥ pentad (Ascaris lumbricotdes).

(B) Simple Y chromosome present.
1. X Chromosome single (Lygaeus, Oncopeltus).

2X 3 double (Fitchia).

3. os - triple (Prionidus).

4. X 55 quadruple (Gelastocoris).

5. x re pentad (Acholla multispinosa).

106 CYTOLOGY CHAP.

The last case is perhaps the most remarkable, the X group consisting
of two large and three very small constituents (Figs. 49, 50).

The chromosome equipment of the various examples is as follows
(omitting Protenor and Lygaeus, which have already been dealt with) :
n’ stands for the haploid number of ordinary chromosomes, and the

Anaphase I Two classes of | Reduced number Male and female
in d. spermatozoa. of chromosomes chromosome
in the egg. groups.
O 052
oF ~ Bee Soe SO
2 Oh Ot UCL ete tengo onwae

000615 9% 09915 3=eg Q30

oO
Oyo du ©) ome) 15 G50 26

FIG. 50.

Diagram of the results of fertilization in Acholla multispinosa. (Payne, B.B., 1910.)
he sex chromosomes shown in black.

number of components of the X chromosome is represented by the
suffixed numeral.

MALE. FEMALE.
Diploid Group. Gametes. Diploid Group. Gametes.
ae n’ +X, J
Syromastes (Wilson, 1909 a), n’=10 2n’+X, { n’ Te ane eee n’ +X,
Ascaris lumbricoides (Edwards, \ Fane pn’ +X; eh ier ee ;
1910), n’=19 j 2n’ +X, Ln! 2n'+X,X. n’ +X;
Peas (n’ +X, ; z
Fitchia (Payne, 1909), n’=12 2n’ + X.Y \ n+ 2 + KX, n' + Xo
i « Se - n’ +X. :
Prionidus (Payne, 1909), n’=11 2n’'+ X.Y { a, is Yy. 2n’+ XX, n’ +X,
: z n’+X . s
Gelastocoris (Payne, 1909), n’=15 2n’+ X,Y { nie. y an’ + XX, n’' +X,
, , r n’ + X, , ‘a ,
Acholla (Payne, 1909), n’=10 2n’ + X.Y { ee 2n’+ XX; n’ +X,

Thus in Acholla the female group has four more chromosomes than
the male, the actual numbers being 30 and 26.

(4) Behaviour of the Sex Chromosomes during Syndesis and the Meiotic
Prophase, and outside the Meiotic Phase

As was illustrated in the case of Lygaeus, the sex chromosomes in
the male retain a compact form, while the other chromosomes are in

IV THE SEX CHROMOSOMES 107

the linear or diffuse stages characteristic of syndesis and other phases
of the meiotic prophase.

Since where Y is absent it is obvious that the X chromosome, having
no mate, cannot go through the process of syndesis like the other chromo-
somes, the natural conclusion is that the visible difference between the
behaviour of the ordinary and sex chromosomes is the expression of the
fact that the latter is not taking part in syndesis. Even when a Y
chromosome is present, both it and the X chromosome often remain

x & a) led
Protenor Syromastes Ascaris
Anasa lumbricotdes

iN oO 0. 0.
. 9 a 3 :

Nezara Euschistus Nezara
viridula Coenurus hilaris Thyanta

oll () ( ()

* @ Vy) a8 LJ

ee
PRocconota Prionidus Gelastocoris Acholla
Fitchta Sinea mult ispinosa
Fic. 51.

Diagram of the relations of the sex chromosomes in various animals. (After Wilson, A.m.A., I91T.)

compact throughout the meiotic prophase, and no evidence of syndesis
between them can be found. The absence of conjugation is not surprising
in view of the physiological differentiation between the X and Y chromo-
somes which must underlie the frequent difference between them in
regard to size and composition. Moreover, as we shall see later, the facts
of sex-linked inheritance (p. 179) lead to the conclusion that the Y
chromosome is inert.

If the absence of syndesis be the explanation of the compactness of
the X and Y chromosomes during the zygotene stage in the male, it

108 CYTOLOGY CHAP.

follows that in the female, which has two equivalent X’s, these should
participate in syndesis, and that therefore they should not have the
compact form noticeable in the male at this stage. While the female —
meiotic phase has not been so carefully studied as the male, the facts, so —
far as they are known, are in general accordance with this hypothesis. In
the female syndetic and meiotic prophase nuclei in, e.g., Anasa, Harmostes, —
Alydus, Euschistus, Coenus and Podisus among Hemiptera (Wilson,
1906 b) and Ancyracanthus in Nematodes (Mulsow, 1913), the sex chromo-
somes are as extended and diffuse as the others, though they are compact
in the corresponding stage in the male meiosis of these species.

Another fact bearing out the same view is, that the X and Y chromo- ©
somes are not usually compact in the oogonial, spermatogonial and
somatic prophases, even in those species where they are so in the male
meiotic prophase, e.g., Anasa, Harmostes, Alydus, Euschistus, Coenus
and Podisus (Wilson, 1906 6), Archimerus, Anasa, Protenor, Chelinidea
(Morrill, 1910), Euschistus (Montgomery, IgIT).

A more striking example is afforded by the hermaphrodite generation
of Ascaris nigrovenosus (p. 113). This is of the female build of body,
but produces both eggs and spermatozoa in its reproductive organ.
The diploid group is 2n’ +XX, where n’ =5. The primitive germ cells, at
first all alike, differentiate later into oogonia and spermatogonia. In
the oogenesis the XX pair acts exactly like the other bivalents. In the
male meiotic prophase, however, one of the X’s may be said to take on
the characteristics of a Y chromosome. This XY pair condenses out
sooner than the other chromosomes, though in the female meiosis, where
both sex chromosomes retain their X character, they do not do so.

In some species (Aphis, Fig. 53) the X chromosome is filiform like the
others during syndesis, although, there being no Y chromosome, it cannot
be participating in this process. Even here, however, the X chromosome
is clearly distinguishable from the others in the diplotene and later
stages, being single or inconspicuously split for the second meiotic
division, unlike the conspicuously double bivalents formed by syndesis
of the other chromosomes (Fig. 53, E, F, G).

Taking everything into consideration, therefore, it is hard to escape
from the conclusion that in these species (by far the majority) where
the sex chromosomes remain compact during syndesis and the rest of
the meiotic prophase in the male, this is an expression of the fact that
they are not themselves engaged in the act of conjugation. It must be
admitted, however, that compactness at a time when the other chromo-
somes are linear or diffuse is not always due solely to the fact that the
one set is engaged in syndesis and the other not, for in some species the
same difference between the consistency of the sex and ordinary chromo-
somes is found outside the meiotic phase. The case of Apis also shows

IV THE SEX CHROMOSOMES 109g

that the failure to conjugate does not necessarily result in the sex chromo-
somes remaining compact at this period.

Wilson (1912) has made some interesting observations which possibly
indicate a slight tendency to syndesis on the part of the sex chromosomes
of the male, which, however, does not culminate in actual conjugation.
In the case of Oncopeltus he examined a hundred nuclei in syndesis.
In seventy-five of these X and Y were entirely separate ; in twenty-five
they were side by side, just in contact. In not one case, however, out
of hundreds examined, were they fused or even flattened together. In
Lygaeus the tendency for X and Y to come together is stronger, for
out of a hundred synizetic nuclei forty-five showed them separate and
fifty-five showed them in contact (thirty-six times end to end, and
seventeen times side by side), often pressed together, but never fused
into a single body. Again, in Ascaris nigrovenosus (p. 113) Schleip
found that in the metaphase I. of the spermatogenesis in the hermaphro-
dite the XY pair were usually separate, but in one individual were
always, and in others rarely, united into a bivalent like the other
chromosomes.

Another very common, but not universal, feature of the sex chromo-
somes is their tendency to travel to the poles of the spindle either in
advance of, or more often behind, the other chromosomes in the anaphase
of the reduction division. Here again this distinction is not found in
the corresponding phase in the female, except in the special case of the
male-producing parthenogenetic egg of Phylloxera (p. 119). Examples
of forms in which the X chromosomes lag in the male meiosis but not
in the female are Anasa, Archimerus (Wilson, 1905; Morrill, 1910) ;
Aphis and Phylloxera (Morgan, 1909; von Baehr, Ig12).

(5) Sex Chromosomes in Animals other than Insects

So far, except for occasional references, we have confined ourselves
to the consideration of the sex chromosomes of insects, since this group
by itself serves to illustrate all the main variations. They have, however,
been found in many other forms, in some of which they can be observed
as clearly as in the insects already described, while in others the evidence
for their presence is not so satisfactory.

Nematodes provide some very clear cases, the simplest being
Ancyracanthus cistidicola, a parasite in the swim-bladder of various
fresh-water fish (Mulsow, 1913). The male diploid group is eleven (10 + X)
and the female twelve (10+ XX). During the diffuse stages of the male
meiotic prophase the X chromosome retains its compact character.
Metaphase I. effects the differential division, the secondary spermatocytes
having six and five chromosomes respectively. In the second meiotic

II0 CYTOLOGY . CHAP,

division X divides equationally in those nuclei where it is present. The
four young spermatids formed from each primary spermatocyte remain
attached together, and it is easy to verify the fact that two have five
chromosomes and two have six. The chromosomes remain individually
distinguishable even in the ripe spermatozoon, so that fertilization of -
the eggs by the two different kinds of spermatozoa can be traced. The

whole case can be followed as clearly as in a text-book diagram. |

Similar simple conditions (presence of a single X chromosome, no Y
chromosome) have been described by Gulick in five species of Heterakis and —
Strongylus (1911).

Conditions are more complicated in the genus Ascaris. In A. lum-
bricoides Edwards (1910) found that the X element consists of a group
of five chromosomes which pass undivided to one pole in anaphase I.
There is no Y chromosome, and two classes of spermatozoa are thus
formed, one with nineteen and the other with twenty-four chromosomes, —

In A. Canis (Walton, 1918) the X group consists of six chromosomes, ~
the two types of spermatozoa having respectively twelve and eighteen —
chromosomes ; all the mature ova have eighteen. |

The problem of the sex chromosome in A. megalocephala has been
attacked by several workers. In this species the X chromosome appears
to be single, and there is no Y. Asa rule the X element is attached to
one of the larger chromosomes, and hence difficult or impossible to
recognize. In rare individuals, however, it is a separate element. In
the @ the two X’s can also be recognized, again generally, but not
always attached to the larger chromosomes. For further information
and literature in regard to this species the reader is referred to Frolowa
(1913). .

It is noteworthy that in Nematodes the division which is differential
for the sex chromosomes varies as it does in insects. In most species
so far described this is the first division, but in A. nigrovenosus (described —
below) it is the second.

Among Vertebrates, sex chromosomes have been studied principally
in Birds and Mammals.

Sex chromosomes have been described in many species of the latter
group, but in most cases the evidence cannot be considered quite
conclusive. The clearest example is perhaps the Opossum (Jordan,
1912). A summary of the work done on mammalian sex chromosomes

will be found in Jordan (1914).

1 The most circumstantial account of sex chromosomes in a mammal is probably that
of Wodsedalek (1913) for the pig. Until, however, some means is found of reconciling the
extraordinary discrepancy between this author’s account of the chromosomes of the sperma-
togonia of this animal and that given by Hance (1918 4), it is difficult to appraise the value

of the evidence.

IV THE SEX CHROMOSOMES IIl

(6) Cases where the Differential Sex Chromosome ts present in the Female

In all the cases described so far it is the spermatozoa which carry the
determining factor for sex; the male produces two kinds of gametes,
male- producing and female-producing, and is said therefore to be
heterozygous for sex. The eggs, on the other hand, are all similar, or
indifferent, and so the female is said to be homozygous. So far there is
comparatively little cytological evidence of the condition of the sexes
being reversed in this respect. In 190g Baltzer described such a case
in Echinoderms, but in 1913 he withdrew the statement, and meantime
Tennant (1g11) found that the sea-urchin Hipponoe conforms to the
usual rule, that the male produces the two kinds of sex-determining
gametes, while the female gametes are all alike.

The Lepidoptera and Birds present specially interesting features in
this connection, since experiments on sex-linked inheritance (see p. 180)
require that the female should be heterozygous and the male homozygous.

Unfortunately neither of these groups are favourable objects for
cytological study. In the Lepidoptera the number of chromosomes is
usually very high, and in Birds the conditions for observation seem
especially difficult, chiefly owing to a pronounced tendency of the
chromosomes to become agglutinated together.

In the latter group Guyer (1909) described an X chromosome in the
male fowl and guinea-fowl, resulting in the formation of the usual two
classes of spermatozoa, and thus apparently indicating male heterozygosity
in birds as in insects. These observations have been questioned by
Boring and Pearl (1914), but Guyer’s subsequent observations (1916)
confirm his original description and at the same time show that it may
not be incompatible with homozygosity of the male and heterozygosity
of the female. It appears that in the somatic tissues of the male fowl
there are eighteen chromosomes, of which sixteen are rod-shaped and
two are U’s. The latter are the sex chromosomes, the chromosome
formula being therefore 2n’=16+XX. In the female only one of the
U’s is present, the formula being 2m’=16+X. In the male meiotic
prophase all the chromosomes pair, giving nine bivalents, one of which
is larger than the others and curved; this is the X bivalent. At
metaphase I. this fails to dissociate, passing undivided to one pole, so
that the secondary spermatocytes contain either eight univalents or
eight univalents and the X bivalent. The latter dissociates in metaphase
II., so that two kinds of spermatids are produced with the chromosome
formulae 8 and 8+X respectively. Guyer gives reasons for believing
that the spermatids without the X chromosome degenerate without
metamorphosing into spermatozoa, though this could not be actually
demonstrated. The meiosis of the female was not worked out, but the

112 CYTOLOGY CHAP.

fact that she possesses an unpaired sex chromosome in her somatic cells
leads to the assumption that she produces two classes of eggs, of formulae
8 and 8+ xX. All the surviving spermatozoa being probably of the 8 + X
class, it follows that the female is heterozygous and the male homozygous
for sex, as the phenomena of sex-linked inheritance in birds demand.
The cytological evidence, however, is in need of confirmation from other —
species.

- In the Lepidoptera the cytological evidence of female heterozygosity
is stronger. The simplest case so far known is that of Talaeoporia tubulosa
(Seiler, 1917). In metaphase I. of the female meiosis there are thirty
chromosomes. In anaphase, one of these lags behind the rest, but
ultimately gets included in one or other of the telophase groups, 7...
in the nucleus either of oocyte II. or of the first polar body. This chromo-
some has evidently not divided, since the group which receives it has
30 chromosomes, while the other has 29. In metaphase II. all the
chromosomes appear to divide. Thus two classes of eggs are produced,
one with 29 and the other with 30, or 29+X, chromosomes. In the
male, 30 (bivalent) chromosomes appear in metaphase I., and all behave
alike, so that all the spermatozoa have 30 chromosomes. Two types
of embryos were also found, one with 59 chromosomes, presumably
females (58 + X), and the other with 60, or 58 + XX chromosomes, which
are presumably males.

It is interesting that in the anaphase of the first polar division the
unpaired X chromosome seems to go rather more frequently into the
polar body than into the oocyte nucleus, and that this corresponds with
the fact that females are more numerous than males in this species.

In Phragmatobia fuliginosa (Seiler, 1913) the heterozygosity of the
female is expressed in an unusual manner. The metaphase I. figures of
the two sexes are alike, containing twenty-eight bivalents, one of which
is very much larger than the others; this large one divides normally
in the male, but in the female the two chromosome groups formed in
anaphase I. differ from one another. One of them contains the expected
twenty-eight chromosomes, as in the secondary spermatocytes, but the
sister group contains twenty-nine chromosomes; moreover, in this
group the large chromosome, though still much larger than any of its
fellows, is not so large as its mate in the group of the twenty-eight
chromosomes at the other end of the spindle, and Seiler concludes that
the twenty-ninth chromosome has been produced by the breaking up
of the very large chromosome into a large and a normal-sized one. Thus
there is a physiological difference between the members constituting the
large bivalent in the female, for in anaphase I. one of them breaks up
into two and its homologue does not. This can clearly be compared

IV THE SEX CHROMOSOMES Ts

with the distinction between the X and Y pair in the males of many
other animals.

In Abraxas grossulariata Doncaster (1914 6) found that whereas 56
is the typical somatic number both for males and females, yet females
of certain strains have only 55 chromosomes and produce two classes of
eggs with 27 and 28 chromosomes respectively. It is not an unreasonable
hypothesis, therefore, that the females with 56 chromosomes have an X
chromosome paired by an inert Y, the latter having been lost in the
strain with 55 chromosomes.

(7) Some Special Life Histories

The apparently simple and obvious relation between the presence
or absence of the X chromosome in one of the gametes and the sex
of the resulting zygote in the examples already dealt with raises at
once some interesting questions regarding certain cases of reproduction
of a different type. What is the condition of the sex chromosomes, for
instance, in a case of alternation of bisexual and hermaphrodite genera-
tions, such as is found in Ascaris nigrovenosus ; or where a female
produces parthenogenetically both males and females, the sex therefore
being determined by something other than the spermatozoon (Cladocera,
Aphids, etc.) ; or again where all fertilized eggs develop into females
(Aphids, Apidae, etc.), males only developing from unfertilized eggs ?

The chromosome cycle in a number of these life histories has been
worked out.

(a) Ascaris nigrovenosus (Fig. 52).—The life history of this species
exhibits an alternation of hermaphrodite and bisexual generations, the
former being parasitic in the lung of the frog, while the latter is free-
living. The chromosome cycle in this species was worked out independ-
ently by Boveri (1911) and Schleip (1912), the two accounts agreeing
in all important points.

In the free-living bisexual generation the male has eleven chromo-
somes and the female twelve ; the male produces two kinds of spermatozoa,
one with five and one with six (5 +X) chromosomes, while all the eggs
have six (5+X). Now all the animals developing from the zygotes
formed by the union of these gametes are hermaphrodites, which are of
the female form of body and have: twelve chromosomes (10+ XX). It
is therefore to be supposed (though this matter was not actually deter-
mined by observation) that the spermatozoa without the X chromosomes
do not take part in fertilizing the eggs (cf. Aphis and Phylloxera,
below).

The hermaphrodites produce eggs and spermatozoa in the same
reproductive organ, and up to the onset of the meiotic phase the cells

I

114 fu Oe CYTOLOGY. CHAP.

(primary oocytes) which will give rise to eggs are indistinguishable from

FIG. 52.

Spermatogenesis in Ascaris migrovenosus. (After Schleip, 4.Z., 1912.) A, portion of gonad after the
primitive germ-cells have differentiated into oocytes and spermatocytes. In both, note the plasmosome, and in
the spermatocytes the single sex chromosome (cf. E). B, oocyte just before synizesis ; C, oocyte during growth
period. In both B and C note absence of compact sex chromosome. D, metaphase I., 9, six bivalents; E,
spermatocyte I.; one sex chromosome has condensed out ; F, later stage, both sex chromosomes condensed ; G,
late prophase I.; H, two secondary spermatocytes; I, metaphase II.; J, anaphase II., 5+X chromosomes
passing to each pole; K, late anaphase II., one X chromosome lagging behind; L, M, two pairs of spermatids.
In one of each the sex chromosome has been left out of the nucleus. N, first cleavage division of an egg
fertilized by aspermatozoon, without the X chromosome, and which will therefore develop into a male. The
groups of chromosomes from the g and 9 gametes still separate, showing the five chromosomes of the one and
the six of the other.

0, primary oocytes; s, primary spermatocytes; X, the sex chromosomes; the distinction between X
and Y made in the text is not shown here.

those (primary spermatocytes) which will give rise to spermatozoa. All

a a ee

IV THE SEX CHROMOSOMES II5

of course contain 10+ XX chromosomes. From now onwards, however,
the sex chromosomes of the two kinds of cells behave differently.

In the oogenesis there is nothing noteworthy, all the chromosomes
behaving alike, and all the mature eggs possessing six (5 +X) chromo-
somes.

In those cells which are going to give rise to spermatozoa, however,
and which may therefore now be called primary spermatocytes, one of
the X chromosomes undergoes a change which may perhaps legitimately
be expressed by saying that it turns into a Y chromosome. This
chromosome condenses out of the diffuse stage sooner than any of the
others (Fig. 52, E), its mate, the remaining X chromosome, following
soon after (Fig. 52, F). From now onwards the meiotic phase proceeds
in the typical manner for an animal with an XY pair, the second division
being the differential one so far as they are concerned.

Two classes of spermatids are formed, one with 5 +X, the other with
5+Y chromosomes. Of each pair of spermatids one (of the formula
5 +X) develops in the usual way into a spermatozoon containing six
chromosomes. In the other, however, the Y chromosome fails to enter
into the nucleus, but remains outside in the cytoplasm, to be ultimately
cast off with the excess cytoplasm (cytophore) when the ripe spermatozoon
is freed. Thus two classes of spermatozoa are formed, one with 5 and one
with 5 + X chromosomes.

It should be noted that Boveri found the process less regular than
this, but with the same end result—namely, the same two classes of
spermatozoa, in which five and six chromosomes can be counted
respectively.

The conjugation of these spermatozoa with the ova brings us back
to our starting-point—the bisexual generation, the males of which have
eleven and the females twelve chromosomes.

(6) Aphids and their Allies——The eggs laid in autumn are fertilized
and in the spring hatch into females, which reproduce parthenogenetically
(with one maturation division and no reduction of chromosomes). After
a lapse of one or more parthenogenetic generations, sexual forms (i.e.
males and sexual females) are produced ; copulation takes place, fertilized
eggs result, and the life-cycle is complete.

Here the double problem arises :

(t) How is it that all fertilized eggs produce females only ?

(2) What determines the sex of the individual developed from an
unfertilized egg, and which is sometimes male and sometimes female ?

The answer to the first problem is very clearly given by von Baehr
(1912) for Aphis salicett. In this species the diploid formula for the female
is 4+XX and for the male 4+X. Examination of spermatogenesis
(Fig. 53) shows that the X chromosome does not divide, but passes

116 CYTOLOGY CHAP.

intact to one pole, so that in anaphase I. there are three chromosomes

Fic. 53.

The chromosomes in the life-cycle of Aphis saliceti. (After von Baehr, L.C., 1912.) A, spermatogonial
prophase; B, primary spermatocyte, beginning of the meiotic phase; C,D,E,F,G, evolution of the
definitive chromosomes. Note.the two bivalents and the single X chromosome. H, metaphase I.; I, ana-
phase I.; J, K, telophase I. All the chromosomes now alike, all being univalent and split in preparation for
the second division. L, resting stage between the two divisions; M, prophase II.; N, metaphase II.; O,
telophase II. (N and O illustrate the case of the spermatocyte II., which contains the X chromosome.) P, cell
from a segmenting egg with five chromosomes (t.e. a ¢); Q, cell from an embryo with six chromosomes (1.e.
a Q).

p, plasmosome; X, the sex chromosome.

(2+X) at one pole and only two chromosomes at the other, in the
familiar manner. When cell division takes place, however, this is unequal,

IV THE SEX CHROMOSOMES Ti7

the cell (secondary spermatocyte) containing the X chromosome being
larger than the other one and, moreover, receiving the whole of the
chondriosomes. The larger cell proceeds to the second meiotic division
in the usual way, the X chromosome dividing this time so that both the
resulting spermatids have an X chromosome. On the other hand, the
smaller spermatocyte II. without the X chromosome proceeds as far
as prophase II. (Fig. 53, M), but degenerates without completing the
second division. In other words, only the one class of spermatozoon,
namely, the X-bearing or female-producing ones, are formed. Hence it
is clear why all fertilized eggs develop into females.

(2) The second problem is not completely cleared up by von Baehr’s
account for Aphis. The same individual gives rise parthenogenetically
to both males and females, so that presumably the mature egg is some-
times left with five chromosomes (male-producers) and sometimes with
six (female-producers) ; indeed, segmenting eggs and embryos are found
to have sometimes six and sometimes five chromosomes in their nuclei.
The mechanism by which one chromosome is eliminated in the formation —
of the male-producing egg could not, however, be determined in this
animal. The elimination of the second X chromosome during the
maturation of the male-producing eggs was, however, observed in the
following case.

Phylloxera (Morgan, 1909, 1915 a; Fig. 54). The life history of this
genus is the same in principle as that of Ap/is. All fertilized eggs give
rise to females (stem mothers) which hatch in the spring. These produce,
parthenogenetically, other females which in turn produce parthenogenetic-
ally males and sexual females. The eggs which will develop into males
are smaller than those which will develop into females. In Phylloxera
caryaecaulis all the daughters produced (parthenogenetically) from one
stem mother produce the same kind of egg—z.e. all are either male-
producing or female-producing. In the case of P. fallax both kinds of
daughters appear to be produced from the same stem mother, and
perhaps both kinds of sexual eggs from the same daughter.

Taking the case of P. fallax, which presents fewest complications
(conditions in P. caryaecaulis being the same in principle), the diploid
number is twelve in the female and ten in the male, the X chromosome
consisting of two components. The male may therefore be represented
by the formula 8 + X, and the female by 8+ X,X,. The two components
of the X chromosome act as a single compound chromosome as in
Syromastes, etc.

Spermatogenesis proceeds in the same way as in A. saliceti; in
anaphase I. the two X components pass intact into one secondary
spermatocyte, which is larger than its sister cell, and alone proceeds to
the second division ; thus all spermatozoa are female-producers.

118 GYTOLOGN CHAP.

v6 6@ @ -@

Said

Fic. 54.

The chromosomes in the life-cycle of Phylloxera fallax. (After Morgan, J.E.Z., 909, and 1915 a.)
A, B, polar body formation in a ?-producing parthenogenetic egg: all the chromosomes retained; C, D,
polar body formation in a ¢-producing parthenogenetic egg, showing elimination of the X chromosome:
E, metaphase I., sexual egg ; F, chromosome group from a 9 embryo, twelve chromosomes ; ‘G, chromosome
group froma ¢ embryo, ten chromosomes.

p.b. polar body; X, the (double) sex chromosome.

IV THE SEX CHROMOSOMES 11g

An analogous process is found to take place in the male-producing
eggs. It will be remembered that there are three kinds of eggs :

(1) The sexual eggs, which need fertilization and which all
develop into females. In them meiosis takes place in the usual
way, and the mature egg is left with 4+X, chromosomes. When
these are fertilized by spermatozoa, which, as we have just seen,
all have the chromosome formula of 4+ X,, all the resulting zygotes
are 8+ X,X,, 1.e. females.

(2) Parthenogenetically developing eggs which are going to
develop into females. These produce only one polar body without
reduction of chromosomes, and at the single maturation division all
the chromosomes divide as at a somatic mitosis ; the ripe egg (and
also the polar body) is therefore left with 8 +X,X, chromosomes.

(3) Parthenogenetically developing eggs which are going to
develop into males. Again only one polar body is produced, but at
the single maturation division one X, chromosome is left behind in
the anaphase and does not enter into the mature egg nucleus. This
is consequently left with ten chromosomes of composition 8 + X,.
This is shown in Fig. 54, C, D, where an X, chromosome (plainly
double in Fig. 54, C) is seen left behind when the groups of chromo-
somes separate at anaphase. The dark body in the cytoplasm which
is being nipped off with the polar body in Fig. 54, D, is presumably
this double X chromosome.

A very important point for the general theory of the sex chromo-
somes is the fact that the sex of the individual which is going to develop
from the parthenogenetic egg is in these cases determined before the
distribution of the sex chromosomes at polar body formation ; for the
male-producing and female-producing eggs are already differentiated
from one another by their relative sizes before this point is reached ;
the eggs which are going to eliminate an X, chromosome, and therefore
develop into males, are smaller than the female- producers. While
therefore we may probably still speak of the presence or absence of the
X, chromosome as determining the sex of the individual, we must realize
that in this case its presence or absence is not a matter of chance, but
that there is some earlier factor which determines whether it shall be
eliminated at maturation or not, and which consequently is a sex-deter-
mining factor earlier in the chain of causation.

In P. caryaecaulis this prior factor in sex determination must be sought
very far back, for not only are the male-producing and female-producing
eggs thus early differentiated from each other, but they are produced by
different females. Moreover, all the offspring of a single stem mother
are alike in respect to the type of eggs which they produce. Thus the
sex of the members of the sexual generation is determined by the con-

120 CYTOLOGY CHAP.

stitution of their grandmother or even earlier ancestor. Morgan (1909)
has, however, suggested a way in which even this determination may
be preceded and hence ‘‘ caused”’ by a differential partition of the
chromosomes.

(c) Some other Cases.—An entirely different method of sex regulation
is found in the gall-fly Neuroterus lenticularis (Doncaster, I9I0, IQIT).
This species produces two generations in a year. The generation
which hatches in the spring consists of females only, which reproduce
parthenogenetically. Their eggs hatch out into males or sexual females.
All offspring produced by one individual parthenogenetic female are
of the same sex; 1.e. her eggs are all either male-producing or female-
producing. It is also found that the parthenogenetic eggs fall into
two types: (I) in which there are the usual two meiotic divisions
resulting in the formation of two polar bodies ; (2) in which there are
no meiotic divisions or maturation processes. Embryos developing from
type (1) have haploid chromosome groups, and from (2) have diploid.
Moreover, all eggs laid by one individual are alike in this respect, being
either all of type 1 or all of type 2. Finally, the female of the sexual
generation has the usual diploid group in its pre-meiotic nuclei, but in
the male this is haploid.

Combining these observations, therefore, it becomes clear ‘iat the
parthenogenetic eggs with two polar bodies produce haploid embryos
which are males, while those with no polar body produce diploid
embryos which are females. The maleness of the haploid individuals is
in accordance with the general rule for facultative parthenogenesis
in the Hymenoptera (Chapter III.).

In the case of some groups of animals with alternation (though in most
cases irregular alternation) of sexual and parthenogenetic generations,
nothing is yet known of any accompanying changes in the chromosome
complex. In the Cladocera the common individual is the parthenogenetic
female, which produces females like itself for a variable number of genera-
tions. After a time, however, males and sexual females are produced.
The sexual eggs fertilized by the spermatozoa of these males invariably,
so far as is known, develop into females.

One great difficulty in the cytological investigation of this group is
that males and females are produced by the same parthenogenetic
female and it is not possible to determine into which sex an egg will
develop. So far as has been observed, all the parthenogenetic eggs produce
only one polar body, but whether this applies to the comparatively rare
parthenogenetic eggs which will develop into males, as well as to the
majority which will develop into females, is not known, though it is
probable from the fact that the male is diploid. The other problem,

IV DETERMINATION OF SEX I2I

why all fertilized eggs develop into females, is also quite unsolved.
Chambers (1913) described degeneration of large numbers of spermatozoa
in Simocephalus, and suggested that these, though not visibly different
from the others, were the male-producing spermatozoa. Taylor, however
(1915 0), found no evidence of degeneration of a whole class of spermatozoa
in the allied genus Daphnia.

(8) The Relation between the Sex Chromosomes and the Determination
of Sex

This question, having been fully discussed in a recent publication
(Doncaster, The Determination of Sex, 1914), will be treated very
summarily here to avoid unnecessary repetition.

In the cases where a dimorphism of the spermatozoa exists, it appears
plain that the sex of the zygote depends upon whether the egg was
fertilized by a spermatozoon with the X chromosome or without it ;
and similarly that where the spermatozoa are all alike and the eggs
dimorphic, it depends upon the nature of the egg which was fertilized.
It therefore seems legitimate to say that in these cases sex is determined,
or caused, by the sex chromosomes, but we must remember that the
sex chromosomes are only one of a number of causes. We have already
seen that in Phylloxera fallax a female gives rise parthenogenetically
through her descendants both to male-producing and to female-pro-
ducing eggs. It appears, therefore, that we must here look for an earlier
cause of sex than the presence or absence of the X chromosome—
namely, something which determines whether the X chromosome shall
or shall not be eliminated at maturation. Similar considerations apply
to the hermaphrodite Ascaris nigrovenosus.

In certain other cases it appears that the sex of the individual
can be determined by factors acting after fertilization, and at a time
therefore when we must suppose the chromosome equipment of the
embryo to be fixed. The experiments of King (1912) on toads, and
of R. Hertwig (1912) on frogs, make it probable that the sex of these
animals can be influenced by external factors acting on the egg either
before or after fertilization. An example from nature of the deter-
mination of sex by environment is afforded by the life history of the
marine worm Bonellia viridis (Baltzer, 1914). Here it appears to be
the environment of the larva which determines whether the adult shall
be male or female.

Finally, it has long been known that the secondary sexual characters
of one sex may appear in individuals of the opposite sex as the result of
castration or other causes.

Probably in every zygote, and indeed in every gamete, both sexes

122 CYTOLOGY CHAP, Iv

must be considered as potential, and which sex shall develop, or dominate
over the other, may depend upon a multitude of factors of which the —
sex chromosomes are only one. In most cases where sex chromosomes ~
are differentiated, however, the presence or absence of the second X
chromosome appears to be overwhelmingly the most important immediate
factor in sex determination, so that in the vast majority of such cases
when once the chromosomal constitution of the zygote has been fixed,
its sex is irrevocably determined. In certain rare cases, however, other
factors may be more powerful and thus be the immediate determiners
of sex.

As to the nature of the relation between the presence and absence
of the second X chromosome and of the sex of the zygote we have practi-
cally no conception. Any attempt to ascribe the influence merely to the
difference in the mass of chromatin is probably doomed to failure. It
is true that in the majority of cases the male has less chromatin than the -
female, owing to the absence of the second X, or to its representation
by the Y chromosome, which is usually smaller than its mate. In Acholla
multispinosa, however, the single Y chromosome is considerably larger
than the sum of the five X chromosomes (Payne). The chromatin content
of the moth Talaeoporia (p. 112) is also greater in the male than in the
female, since in this species it is the female which lacks the second X
chromosome.

Probably the problem of the determination of sex by the sex chromo-
some (on the occasions when this acts as sex determiner) is the same
as that of the dependence of any bodily characteristic upon hereditary
factors residing in the chromosomes (see Chapters V. and VI.).

CEA BAER TY.
THE CHROMOSOMES

OBSERVATION of the minute structure of the nucleus, together with the
evidence from experimental work on heredity, has led to the formulation
of a hypothesis which can be stated as follows :

The nucleus—and in particular that part of the nucleus constituting
the chromatin or, at least in most phases of the nucleus, indistinguishably
bound up with the chromatin—is the seat of the agency which initiates
and controls morphogenesis and function, and hence, since the chromo-
somes are carried on from one generation to another through the gametes,
it is also responsible for the phenomena of heredity. The chromatin
appears to act in this respect not as a homogeneous whole, but rather as
an aggregate of smaller bodies, each of which plays a different part
though the sum of them all is necessary to the general economy of the
organism (analogous to the parts played by the lungs, heart, liver and
other organs in the higher animals). These smaller bodies, which
constitute the lowest order of living units which need be considered for
the purpose of this hypothesis, are aggregated during mitosis in linear
series into bodies of a higher order, the chromosomes. The sum of the
chromosomes again forms the nucleus.

As the haploid number of chromosomes is sufficient to enable a normal
individual to develop (cf. facultative parthenogenesis and merogony),
each gamete must contain a complete set of all the units necessary for
the production of a normal individual of the species. Hence the diploid
zygote must contain a double set of these units, 7.e. two of each kind.

While it is impossible completely to separate the discussion of the
morphological and physiological aspects of this thesis (for their inter-
dependence is the chief evidence of the correctness of both) it is the
former aspect that will be the main consideration in this chapter, while
the physiological and more theoretical sides will be specially dealt with
in the following chapter.

123

124 CYTOLOGY CHAP,

A. THE CONTINUITY OF THE CHROMOSOMES
The first morphological hypothesis to be established is what has

come to be known as the tmdividuality, or better, the genetic continuity —

(Wilson) of the chromosomes. The meaning of this phrase is that the
material of the chromosomes is not resolved at telophase into a common
nuclear reticulum from which new chromosomes differentiate out in

the next prophase (as crystals might dissolve in a solvent and recrystallize ©

out again), but that the substance of each telophase chromosome is
concentrated again into a corresponding chromosome in prophase. Thus
each individual chromosome is the direct descendant of the corresponding
chromosome in the previous cell generation as described on p. 128.

This conclusion is at once suggested by the fact that the number of
chromosomes in any one species is constant (with certain, mostly well-
understood, exceptions) although it may vary greatly in nearly allied
species ; that the number is constant (with the same qualification) in ~
different tissues though the total amount of chromatin may vary greatly ©
from tissue to tissue ; and that the number of chromosomes is halved at
gametogenesis and subsequently restored at syngamy.

The truth of this hypothesis is indeed very generally accepted, being
supported by a great body of observations as well as by indirect evidence.
Indeed it would be exceedingly difficult to write a general treatise on
nuclear cytology without accepting the hypothesis as a basis, and the
reader will doubtless have noticed that it has frequently been implied
in this work. It will be necessary, however, to discuss briefly a few of
the problems which are raised thereby.

While the earlier cytologists were content with demonstrating the
constancy of the number of chromosomes in a given species it became
evident, with the extension of the study to a wider range of forms, that
in many species the chromosomes are not all alike, but differ from each
other in size, and especially in length ; as, except in the case of the meiotic
chromosomes, this is apparently the only dimension in which constant
differences occur, the thickness of all the chromosomes in a given nucleus
being approximately equal. Moreover, the length differences are constant,
so that in every nucleus (in mitosis) not only the same number of
chromosomes, but the same series, ranging from the largest to the smallest,
can be recognized. Again, it was found that there were in each diploid
nucleus two chromosomes of each size, so that if the chromosomes are
designated in order of size A, B, C, etc., the chromosome complex could
be designated thus :

A+A+B+B+C+C+ .

In the meiotic prophase the two chromosomes of each type, usually

Vv HOMOLOGOUS CHROMOSOMES 125

called homologous chromosomes, pair together to form the bivalents.
The nucleus of the primary oocyte or spermatocyte can therefore be
written :
AA+BB+CC +
At the reduction division the homologous chromosomes are separated,
as described in Chapter II., so that each gamete nucleus has the formula

A Bit C+
The diploid nucleus of the first formula is of course reconstituted at

syngamy.
Thus it follows that one member of each pair of homologous chromo-

9 > RIO ad
, ~
—_) 22 ~~ € *

74 ¢ @®-..
Qos Oss of opeg
rN tee = Od,”
f t 13 Ww f) @
a) ‘ea's 9
: ®@ @'@
+ 1 @
y 10
A
3
FIG. 55.
Illustrating the tendency of homologous chromosomes to lie near each other in somatic nuclei. (Miiller,

A.Z., 1912.) A-C, polar views of equatorial plates; D, prophase. A, Eucomis bicolor; B, Albuca fastigiata ;
C, Galtonia candicans ; D, Dahlia coronata. Some of the pairs are numbered,

somes in a diploid nucleus has been derived from the male and one from
the female parent.

This morphological fact, together with its theoretical consequences
for heredity (to be discussed in the next chapter) was first pointed out
clearly by Sutton in the case of the insect Brachystola magna.

The degree to which the chromosomes of a single nucleus differ from
one another in length varies greatly, and indeed in some species no
certain differences are detectable. Such species are of course of negative
value as evidence in this respect, the generalization being founded on
those numerous other forms in which the chromosomes exhibit marked
size differences. The seriation of the chromosomes according to size is
often facilitated by a tendency on the part of homologous chromosomes
to lie near or next to one another on the equatorial plate. This tendency
also varies in different species, in some indeed apparently not existing,
while in others (e.g., Yucca; Muller, 1912) it is pronounced (Fig. 55).

126 CYTOLOGY CHAP,

The paired arrangement of homologous chromosomes is most strik- -
ingly shown in the Diptera (Metz, 1916), in some species of which homo-
logous chromosomes are very closely approximated. This culminates
in Culex, where they are often so closely applied to one another as to
be distinguished only with difficulty. Fig. 56 shows the chromosome

H

Fic. 56.

The chromosomes of Culex as figured by different workers, showing the close approximation of homologous
chromosomes. (A, B, from Stevens, J.E.Z., 1910; C, from Taylor, 0.J.M.S., 1915; D, E, from Whiting,
J.M., 1917; F, G., from Hance, J.M., 1917; H, I, J, from Metz, J.E.Z., 1916.) A, spermatogonial pro-
phase; only one of the three chromatin threads is visibly double; B, spermatogonial metaphase, three pairs of
separate chromosomes; C, somatic prophase; D, spermatogonial prophase; E, spermatogonial anaphase ;
F, ovarian (diploid) prophase; G, spermatogonial metaphase; H, I, J, prophase, metaphase, and anaphase
of somatic mitoses.
complex of this mosquito (where 27 =6) as depicted by various workers.
As will be seen, the homologous chromosomes in prophase (Fig. 56,
A, C, D, F, H) are generally intimately applied to or twisted round one
another, quite as closely as are the daughter halves of split prophase

chromosomes in the somatic mitoses of many other forms (Figs. 3, 8).

Vv HOMOLOGOUS CHROMOSOMES 127

As they condense for metaphase the homologues become more distinct
from one another but still remain very closely paired (Fig. 56, B, G, I).
In some strains they remain indistinguishably fused even at this stage
(Taylor, 1915 a). In anaphase they again come into close application or
fusion (Fig. 56, E, J).

It is interesting to note that in Taylor’s material (1915 a, 1917),
although, in general, fusion of the homologous chromosomes was so
intimate that in somatic mitoses there appear to be only three chromo-
somes present, yet in the early cleavage divisions of the egg the six
chromosomes are as well separated from one another as in other animals.

The case of the Diptera, and especially of Culex, leads to the conclusion
that the fusion of the chromosomes in syndesis is only the climax of a
general mutual attraction between homologous chromosomes.

Further indirect evidence

of the continuity of the chromo-

somes is furnished by those

animals in which the bivalents

of the meiotic phase appear in A B Cc
various different shapes. In

these animals it is found that

the same shapes, and the same

number of each shape, reappear. }

in every meiotic nucleus (Figs.

1 Zz F
57, 65). : FE
The one difficulty in the way Fic. 57.
of the hypothesis of the con- The different forms of bivalents found in the meiotic

; ; : phase of the newt. (After Moore and Arnold, P.R.S., B 1906.)
tinuity of the chromosomes is_ Iwo of each type are found in all primary spermatocytes

Ree ae hae an the great (E1, E2, being alternative forms of the same type).
majority of cases they lose all visible signs of their identity in the
resting nucleus. This, however, is a piece of negative evidence which
cannot be allowed to outweigh the overwhelming indirect evidence from
their constancy in number, relative sizes, etc., which indicates that they
do actually maintain this continuity. Moreover, in many cases direct
evidence has been obtained that the chromosomes which enter into the
resting nucleus at telophase do not become diffused throughout the
whole nucleus and inextricably mingled up with one another, but retain
a definite localization in the nucleus though their boundaries may not
be visibly distinguishable. The classical piece of evidence on this
head is Boveri’s work on the cleavage nuclei of Ascaris megalocephala,
which on account of the small number of its chromosomes is plainly
a favourable object for such investigation.

Originally carried out on the eggs of Ascaris megalocephala bivalens
(Boveri, 1888), the work was repeated by Boveri in 190g on the wnivalens

128 CYTOLOGY CHAP.

form. He found that in anaphase the daughter chromosomes separate

in such a way that their arrangement in the two telophase groups is the —

same. Thus in Fig. 58, A, one chromosome is bent in a U-shape, so that
the two ends lie close together, while the other one is stretched out so
that one end lies with the two ends of the first chromosome, while the
opposite end is far removed. In D both chromosomes are U-shaped,

Fic. 58.

Telophase of the first cleavage mitosis to prophase of the following mitosis in Ascaris megalocephala univalens.
(Boveri, A.Z., 1909.) A, B,C, telophase, resting nucleus and following prophase of a nucleus with the chromo-
somes so grouped in telophase that one chromosome end projects by itself while the other three form a common
projection ; D, E, F, similar series in a nucleus in which each chromosome is bent upon itself, so that one projec-
tion contains the two ends of the one chromosome and another projection those of the other chromosome.

forming two separate groups of chromosome ends. When the telophase
becomes resolved into the resting nucleus it is found that the chromosome
ends form projections from the main mass of the nucleus, telophase
groups of type A resulting in resting nuclei such as shown in B, where
both sister nuclei have one thick and one thin projection (containing
three and one chromosome ends respectively). Telophase groups of

Vv CONTINUITY OF THE CHROMOSOMES 129

type D produce the nuclei shown in E, which have two equal projections,
each containing two chromosome ends. In the next prophases (C, F)
the chromosomes reappear in the same arrangement as they exhibited
in the previous telophases. It will be noticed that the orientation of the
nuclei towards each other in the two daughter cells has changed slightly
owing to the rotation of the nuclei within the cells. This however does
not affect their internal architecture.

It must be understood that what we have here described as a process
is, like all similar work in cytology, really pieced together from a series
of fixed stages. Thus it cannot actually be observed that the prophase
nuclei of types C and F are the outcome of telophase nuclei of types A
and D respectively, but this can be inferred without reasonable doubt
since (1) they are connected up by a close series of intermediate stages
of which B and E are examples; and (2) in the prophase, as in the

He

A B
Fic. 59.

Showing similar orientation of telophase (A) and prophase (B) in the epidermis of the salamander.
(Rabl, M.J., 1885.)

telophase, the arrangement of the chromosomes in the two sister nuclei
derived from the previous mitosis is the same.

An orientation of the chromosomes in telophase similar to that in the
succeeding prophase has been described by many cytologists from Rabl
(1885) onwards (Fig. 59), though the conditions are seldom so favourable
for observation as in Ascaris megalocephala.

Another fact directly supporting the hypothesis of the genetic
continuity of the chromosomes is that each chromosome may undergo
its telophase metamorphosis in a more or less separate vesicle within
the nucleus. This is especially characteristic of Orthopteran spermato-
genesis (Fig. 60). Sutton (1903) described the larger chromosomes of
the spermatogonial telophase of Brachystola magna as forming each its
own reticulum in a separate vesicle, which however is in communication
with the other vesicles at their polar ends, forming there a common
compartment from which the vesicles project like the fingers of a glove.
This general account has been confirmed by several cytologists. In
Phrynotettix (Wenrich, 1916) the telophase chromosomes first form closed

K

130 CYTOLOGY CHAP.

vesicles, then the walls of these vesicles break down to produce a common ~
nuclear cavity, in which, however, the regions of the vesicles can still be
recognized by the slightly denser core of chromatin occupying what was
formerly their axes. In prophase the chromosomes condense again in
the limits of these vesicles (Fig. 60, A-E).

In Locusta viridissima (Otte, 1907) the chromosomes of the spermato-

Fic. 60.

Formation of chromosome vesicles in the spermatogonia of Orthoptera. (A-E, Phrynotettix magnus, after
Wenrich, B.M.C.Z.H., 1916; F, Brachystola magna, after Sutton, B.B., 1903; G, H, I, Locusta viridissima,
after Otte, Z.J.A., 1907). A,B,C, successive stages in the formation of the resting nucleus out of the telo-
phase chromosomes; D, E, prophase; F, early prophase; G, telophase; H, resting ‘‘nucleus”’; I, prophase.
gonial telophases do not come into contact at all, but each one forms
a separate little nucleus, or karyomere, by itself (Fig. 60, G-I.) No
common nuclear membrane is formed to enclose them, but they remain
separate from one another, with cytoplasm extending in between them.
This account refers to the earlier spermatogonial divisions. In the last
one before the meiotic phase a compound nucleus is formed in the usual
way.

Karyomere formation—+.e. the formation of a separate little nucleus

v KARYOMERES 131

- by each chromosome—is a common occurrence in the cleavage divisions

of many animals. Instead of a single nucleus, we therefore find a mass
of small ones corresponding in number to the number of the chromosomes
(Fig. 61). Asa rule this condition is temporary, the karyomeres generally
fusing later into a single nucleus. Certain abnormal conditions, e.g. high
temperature (Tobias, 1914), accentuate the tendency to karyomere
formation.

Bic. 6F.

Formation of karyomeres in cleavage nuclei of various eggs. A. Chaetopterus pergamentaceus: 9 nucleus
has completed its maturation divisions, and, in the form of a group of chromosomic vesicles is moving inwards
to meet the ¢ nucleus (after Mead, J.M., 1895); B, C, resting and prophase nuclei from cleaving eggs of
Cyclops viridis, subjected to a high temperature (after Tobias, A.m.A., 1914); D, one nucleus of the 2-cell
stage of Polyphemus pediculus (after Kuhn, A.Z., 1908).

A difficulty which has been urged against the view of the continuity
of the chromosomes is the supposed power of the nucleus to form chromo-
somes after amitotic division. As this matter has already been discussed
(p. 24) it need not be dealt with again.

Very strong evidence in favour of the continuity of the chromosomes
has been obtained from the study of certain hybrids. Moenkhaus (1904)
crossed the Telostean fishes Fundulus heteroclitus and Menidia notata.

132 | CYTOLOGY CHAP.

The former has long (2-18 4), slender and generally straight chromosomes,
n being 18. The latter has about the same number of chromosomes,
which are short (I-00 «) and generally curved. The eggs of each species
are fertilizable by the sperm of the other, and the hybrids so formed
develop for a certain period apparently normally, though in later
embryonic stages abnormalities make their appearance, and the eggs
seem incapable of hatching (see Loeb, 1912). The chromosome conditions _
during the development of the hybrid embryo are especially interesting -
(Fig. 62). After fertilization the male and female gamete nuclei fuse
in the resting condition. When the chromosomes appear for the first

A Fic. 62.

A, anaphase of first cleavage mitosis in the egg of Fundulus heteroclitus; B, similar figure from Menidia
notata; C, D, anaphase of first (C) and later (D) cleavage mitosis of a hybrid (Menidia notata? x Fundulus
heteroclitus 8) showing the two distinct types of chromosomes, separately grouped in C and mingled in D
(Moenkhaus, Amer. Journ. Anat., 1904).
cleavage mitosis, however, it is found that the chromatin of the two
species has remained distinct, for the chromosomes appear in two groups,
one consisting of long chromosomes easily identifiable as derived from
the Fundulus parent, the other of short chromosomes derived from
Menidia. In telophase the two groups of chromosomes again become
indistinguishably merged into the resting nucleus, to reappear in the
same grouping at the next mitosis (2nd cleavage division). At the 3rd
cleavage the two types of chromosomes are still as sharply distinct
from one another, though they are no longer completely segregated into
two groups. By the 4th cleavage division the grouping is almost, and
in later cleavages quite, lost, the two types of chromosomes—still, how-
ever, perfectly distinct—being intermingled with each other.

Vv CONTINUITY OF THE CHROMOSOMES 133

J

The grouping of the chromosomes derived from the male and female
parents which is to be seen in the first few cleavage divisions is of course
an example of gonomery, such as occurs in Cyclops, etc. (Fig. 35), and,
as usual, disappears in later cleavages. The important fact is that the
two types of chromosomes introduced by the two parents, though mingled
together, are recognizable in all mitoses. From this we conclude that
their loss of identity in the resting nucleus is apparent only, and not real.

In the Lepidopteran cross Lycia hirtaria x Ithysia zonaria (Harrison

Fic. 63.

Fertilization of the egg of Ascaris megalocephala bivalens by spermatozoa of A. m. univalens. (After Herla,
Sona Ue eats ee appr eenone B, the chromosomes of the zygote nucleus; C, egg divided
and Doncaster, 1914) the large chromosomes of Lycia and the small
ones of Jthysia are distinguishable in the hybrid right up to the formation
of its gametes (Fig. 185). |

The persistence of an unusual number, though not of distinct types,
of chromosomes in a hybrid was observed by Herla in 1895. He found
five females of Ascaris megalocephala bivalens which had been fertilized
by the wnivalens variety. Consequently the nuclei of their hybrid embryos
had three chromosomes, two derived from the female and one from the

134 CYTOLOGY CHAP.

male parent (Fig. 63). This number could be counted (in the germ
track) up to at least the 12-cell stage, which was the latest stage examined.
This case must not be confused with the A. megalocephala with three
chromosomes described by Boveri (p. 145), though the two cases furnish
equally strong evidence for the continuity of the chromosomes.

B. THE COMPOSITION OF THE CHROMOSOMES
OF SMALLER UNITS

While the definitive chromosomes of the metaphase generally appear
homogeneous, they characteristically present a different appearance in
prophase, where they are often markedly moniliform, 7.e. consisting of
a row of bead-like swellings of chromatin, called chromomeres, joined
to each other by a thinner linin thread. This condition, which forms
one of the most characteristic sights met with by the cytologist, can
be illustrated by reference to almost any work dealing with mitosis,
whether in the soma, germ track or during meiosis. Many figures in
this book illustrate this point incidentally (eg. Figs. 20, 77). It is
equally characteristic of animals and plants (Fig. 64).

In the early prophase the chromomeres, if visible, are commonly
very small and numerous (when not visible it probably means that they
are so Closely distributed along the chromosome that their boundaries
are not distinct). As prophase proceeds they become larger and fewer,
obviously by fusion in groups. At about this stage they are often
extremely prominent, constituting comparatively large spheroidal
swellings joined to their neighbours by short stretches of very fine threads.
As the chromosomes contract, the now composite chromomeres become
more and more pressed together, the boundaries between them gradually
becoming obliterated till in the metaphase chromosome they are generally
no longer distinguishable from one another, and in consequence the
chromosomes appear homogeneous. Finally, in the greatly contracted
chromosomes of meiosis, or somatic telophase, the chromomeres appear
to lose their linear arrangement.’ We must however suppose that the
loss of the linear arrangement is only apparent, and that essentially it
is maintained so that the chromomeres appear in the same order in the
prophase chromosomes of successive mitoses.

In the early prophase the chromomeres are often the points of de-
parture for the linin threads which run out from the chromosomes into
the vanishing nuclear-reticulum (e.g. Figs. 3, 16). This suggests that the
substance forming the chromomere has travelled down the linin fibre to
the main trunk of the chromosome.

The correspondence, as regards number and sizes of the chromomeres,
between the daughter threads of the split somatic chromosome (Fig.

% CHROMOMERES 135

64, A, D) and between the conjugating chromosomes in the meiotic
prophase (Figs. 64, C, and 66) is very striking. The latter phenomenon
is specially significant, suggesting that syndesis does not concern the
chromosomes as wholes, but that it takes place between the separate
elements of which they are composed.

The thesis formulated at the beginning of this chapter requires that
the chromosomes should not merely be composed of smaller units, but
that these should be differentiated among themselves. It further follows

_ ie oS eas a MEE —.
: Pas

Fic. 64.
Chromomeres. A, epithelial cell of the salamander (Flemming, 1882); B, prophase of nucleus in root

tip of Najas marina (after Miiller, A.Z., 8, 1912) ; C, zygo-pachytene nucleus of oocyte I. of Enteroxenos (after
Bonnevie, J.Z., 1906) ; D, prophase chromosomes from alimentary canal of Culex (after Holt, J.M., 1917).

that if in syndesis corresponding elements of the homologous chromosomes
pair together, these elements must always be arranged in the same order
along the length of the chromosome. .

Evidence that the longitudinal differentiation of the chromosomes
is of a definite and relatively constant nature has been presented in the
case of Lepidosiven (Agar, 1913). In this animal the 38 somatic chromo-
somes are usually V-shaped, but in the shorter ones the limbs of the
V’s tend to diverge, till at length the chromosome, by straightening out,
becomes rod-shaped. The point of bending of the V can however be

136 CYTOLOGY CHAP.

traced through the gradually widening out V into the rod, where it
persists as a constriction, or an actual break in the chromatin, dividing
the chromosome into two portions connected by a linin bridge. This
transverse constriction is the same as that so characteristic of the meiotic
bivalents which causes them to appear in “ tetrad’”’ form as discussed
on p. 40. The significant fact is that the transverse break always
occurs in the same region in the same chromosome. It will be noticed
from Fig. 65 that one pair of chromosomes is much larger than any of
the others. The break in this chromosome—whether exhibited as the
angle of the V, or as a transverse constriction—is always found at about

C

Fic. 65.

B, the folic Bivalonts facut by the paling of the date eemueceres (ite pearls dei eee
anaphase I., each univalent split for the second division. Note that chromosomes 1 and 2 form a V with equal
limbs in A, and that each constituent of the corresponding bivalent is similarly constricted into equal portions.
The other three pairs of chromosomes have unequal limbs, both in A and in the bivalents.
the middle of the chromosome. The next two pairs of chromosomes are
of much the same size, but easily distinguishable both from the large
pair just described and from the next smaller pair. These two pairs
constantly have the break excentrically placed. Now if the break
were always in the middle of the chromosome, or varied in position in
the same chromosome, it weuld be without significance for the present
purpose, as it might then be due to purely accidental mechanical causes.
The fact that—however caused—it is constant in position in a given
chromosome, but differs in different ones, indicates that the chromosomes
possess a constant differentiation in a lengthwise direction.

Wenrich (1916) has found that in the prophase chromosomes of
the Orthopteran, Phrynotettix magna, the principal chromomeres are

q

;

v CHROMOMERES 137

constant in their arrangement in a given chromosome. Fig. 66 shows
one example of the corresponding chromosome from thirteen different
individuals. It is taken from the pachytene stage of spermatogenesis,
and is therefore bivalent, as indicated by frequent signs of duplicity.
The five principal chromomeres are numbered I-5 and it will be seen
how noticeably constant in arrangement they are. This regularity
extends also to the smaller chromomeres. For instance, in the segment
between Nos. 3 and 4 there are always two small granules of about the
same size, while there are never any prominent ones between Nos. 2
and 3.

Of course, the constancy is not perfect. A certain amount of variation
in the relative sizes of the principal chromomeres, and in the lengths of

i r :
: a ee
ea 2 8
Ne fA oy 4
ie ee
5 a =” ¢ ~~"
A
’
| hei |
14d kam
B
Fic. 66.

Various examples of the same chromosome in Phrynotettix.magnus. (After Wenrich, B.M.C.Z.H., 1916.)
A, the chromosome (bivalent) from the pachytene stage of thirteen different individuals. The principal
eran a enact - cites erat stages in the contraction of the pachytene chromosome to form
the segments separating them, can be observed, as well as in the number
and arrangement of the smaller granules in between. This variation
may be due to several causes, partly to errors of technique—for instance,
distortion by fixing agents, optical effects, etc.—partly to difference in
the extent to which fusion of smaller granules to form larger ones has
proceeded, but partly probably to real biological variation. The thesis
outlined at the beginning of this chapter requires that all genetic differ-
ences in organisms should be referred to preceding variation in the
idioplasmic elements, and hence it is no more surprising to find variation
in homologous chromosomes than in the somatic characteristics of
organisms.
Wenrich finds indeed, in the case of the particular chromosome under
consideration, that the chromomere numbered 5 is often absent. In

138 CYTOLOGY CHAP.

some individuals this chromomere is present in both members of the
pair, in some it is absent from both members, and in others it is present
in one chromosome but absent from its homologue. The same combina-
tion is of course constant for all the nuclei of a given individual. The
existence of the three possible combinations indicates promiscuous
syngamy between gametes which possess and those which do not possess
the chromomere in question.

Inequalities, or other visible differences, between the two members of
a homologous pair have also been described in Orthoptera by Carothers
(Brachystola, 1913; Trimerotropis and Circotettix, 1917) and by Robertson
(Tettigidea and Acridium, 1915).

In Trimerotropis (one of the grasshoppers) the metaphase chromosomes
are either rod-shaped, or bent into V’s (with equal or unequal arms).
These shapes are not transitory forms impressed on the chromosomes by
temporary forces acting in mitosis, but they mark the different methods
of attachment of the spindle fibres to the chromosomes, and are constant

in all the nuclei of an individual. In the

case of the rod-shaped chromosomes the

spindle fibre is attached to one end, and in

the case of the bent chromosome it is
A B C

attached to the angle of the V. Often homo-

logous chromosomes differ in this respect,

as shown by the shape of the bivalents in
diferent individuals, of Trimevairops. metaphase I. (Fig. 67). A bivalent may
Ce re nae be formed by two straight chromosomes
(Fig. 67, A) or by two bent chromosomes (B) or by one straight and
one bent chromosome (C), all these figures representing the same
bivalent as found in three different individuals.

A number of other cases of inequalities between homologous chromo-
somes have been recorded, of which further mention need only be made
of Gryllotalpa borealis (Payne, 1913 a) where the inequality is connected
with the sex chromosomes and may be of the same nature as found
in the unequal XY pair, which of course constitutes the most striking
example of unequal homologous chromosomes.!

Fic. 67.

C. VARIATION IN THE NUMBER OF CHROMOSOMES

Variations from the number of chromosomes typical for the species may
be due to the following causes :

(a) Transverse fracture of the chromosomes (fragmentation) leading
to increase, or end-to-end fusion (linkage) causing decrease, in number.

1 Boveri (1904) found a female A. megalocephala bivalens in which both the tetrads were
often formed of two rods of unequal length.

Vv NUMBER OF CHROMOSOMES 139

(>) Irregularities of mitosis, by which the chromosomes are unequally
distributed between the daughter nuclei, or certain of them are left out
of either nucleus.

(c) Ordinary longitudinal fission of the chromosomes without a
mitosis to separate the daughter chromosomes, thus leading to a doubling,
quadrupling, etc., of the number. Or longitudinal fusion of the chromo-
somes as in parasyndesis leading to an apparent halving of the number.

(1) Variation in Chromosome Number due to Fragmentation or Linkage

Since, as we have seen, the chromatin units are arranged in linear
series in the chromosomes, the total chromatin content of the nucleus
may be considered as ideally arranged in such a linear series along a
single long thread, which becomes divided into a number of segments
varying in number in different species.

Thus in the genus Cyclops (Braun, 1909) it may be segmented into
3(C. gracilis), 5 (C. vernalis), 6 (C. viridis), 7 (C. fuscus), 9 (C. bicuspidatus),
or I1 (C. stvenuus), these being the haploid numbers.

The older cytologists were indeed of opinion that this segmentation
of a single linear series actually occurred in the prophase of every mitosis,
the first stage in this process being the formation of the “ continuous
spireme,” which in later prophase gave place by transverse segmentation
to the “segmented spireme.”’ Though a continuous spireme probably
does not occur, at any rate as a regular stage, in prophase (see p. 9),
the process probably represents substantially the method by which in
evolution the varying chromosome numbers have been produced.

A special study of the phylogenetic derivation of chromosome numbers
has recently been made by American cytologists. The number of chromo-
somes in the grasshoppers (Orthoptera) is relatively constant. Thus in
ten species belonging to five genera of the family Tettigidae, Robertson
(1916) found in every case 2n=13 (male) and 14 (female). In over
forty genera of Acridiidae, 2 is 23 (male) and 24 (female) in all except
three, of which one (Chorthippus) has 17 and the other two from 20 to
24 chromosomes.

Robertson (loc. cit.) has shown how the smaller number in Chorthippus
(=Stenobothrus) has probably been derived from the type number for
the family. The Acridiid spermatogonial chromosome is typically rod-
shaped. In Chorthippus, however, only eleven of the chromosomes,
including the X chromosome, are of this shape, the remaining three
pairs being V-shaped. The angles of the V’s—that is to say, the points
of junction of the limbs—are marked by a constriction or non-staining
bridge between the limbs. Robertson makes the very reasonable sugges-
tion that the V’s have been formed by association or linkage of couples

140 . CYTOLOGY CHAP.

of the chromosomes of the type form, each limb corresponding to a
whole rod-shaped chromosome, thus accounting for the usual 23 chromo-
somes by I1 simple +6 double elements.

This association has of course not taken place between homologous
chromosomes, but between non-homologous ones. Thus, using the
notation on p. 124, linkage has occurred between each A and B, C and D,
E and F, etc., to form composite chromosomes of the formula AB, CD,
EF, etc. This interpretation is borne out by the facts that the six V’s
form three equal pairs, and also that the limbs of each V are not equal,
showing that the linkage has been between non-homologous chromosomes.

M‘Clung (1917) has come to similar conclusions regarding the variation
in the number of chromosomes in Hesperotettix viridis, one of the Acridiidae.
In this species the type number for the family (23 in the male) is found
in some individuals, but others exhibit fewer. An examination of the
bivalents of the primary spermatocytes shows that in the latter individuals
one or more of the chromosomes have a transverse constriction which
is not found in those individuals which possess the full 23 chromosomes,
probably indicating that the chromosomes in question are compound.

Thus, in their primary spermatocytes—

5 individuals had 12 separate chromosomes = 11 bipartite + the sex chromosomes.

7 i II BS =Io bipartite + 1 tripartite (the sex
chromosome attached to one
ordinary bivalent).

5 ” Io 3 = 8 bipartite + 1 quadripartite + 1 tri-
partite.

7 » 9 ” = 6 bipartite + 2 quadripartite + 1 tri-
partite. .

7 ” 10 53 = 7 bipartite + 2 quadripartite + the sex
chromosome.

6 ” II » = g bipartite +1 quadripartite + the sex
chromosome.

It will be seen that the number of chromatin segments in all cases
adds up to 23, the bipartite chromosomes being of course the ordinary
bivalents formed by the pairing of two simple homologous chromosomes,
the quadripartite ones being formed by a pair of composite homologous
chromosomes (each of which has been formed by the linkage of two
non-homologous chromosomes as in Chorthippus), and the tripartite
forms by the junction of the single sex chromosome with an ordinary
bivalent. The condition presented by the first example in the table is
the typical one for the family when there is no linkage of chromosomes.

Again, all the nuclei of a given individual have the same type of
chromosome complex. What happens in fertilization between different
classes of individuals is not known. M‘Clung suggests that the linkage is
resolved, and re-formed, at syngamy.

Woolsey (1915) found similar relations in the locustid genus Jamaicana

v NUMBER OF CHROMOSOMES I4I

(Fig. 68). In this genus the type number is 35 for the male; the
chromosomes are rod-shaped. In some individuals, however, the number
is reduced to 34 or 33 (spermatogonia). The 34 type has 33 rod-shaped
and one V chromosome, while the 33 type has 31 rods and two V’s.
The former case is in-
teresting as an example
of homologous chromo-
somes behaving dif-
ferently, for since there
is only one V instead of
a pair of them, linkage
must have taken place
between one member of
each of two pairs of
homologous chromo-
somes, the other mem-
ber remaining free.
That is to say, the
chromosome formula is
AB+A+B+C+C+...
What happens in this
case at syndesis? While
we do not know the
details of this process,
the result is clearly
shown by the structure
of the chromosomes of
the first meiotic division.
They consist, besides the
sex chromosome, of
fifteen ordinary biva-

lents and one tetrapar-
tite V of the type shown
in the figure. As the
spermatogonial divisions
show, the original linkage
was between two chromo-
somes of considerably

Fic. 68.

The chromosomes of Jamaicana subguttata (A-E) and J. unicolor

(F-G). (Woolsey, B.R., 1915.) A, spermatogonium of an individual
with the type number (35) of chromosomes. The chromosomes are
numbered in pairs from the smallest to the largest. No. 18 is the sex
chromosome. B-E, chromosomes of an individual in which one member
of two homologous pairs (14 and 16) have become associated ; B, sper-
matogonium ; C, late prophase I.; D, anaphase I.; E, another view of
the bivalent 14-16; F, G, isolated bivalent and spermatogonial group
from an individual in which both members of the two homologous pairs
are associated.

different sizes, so that a V with unequal limbs is formed. The tetrapartite

bivalent at meiosis is formed by the pairing of each limb of this V with its
A B
homologue, and may be represented thus: - a (Fig. 68, C). The

composite bivalent divides at the points of junction of the homologous

142 CYTOLOGY CHAP.

chromosomes composing it, so that the V (AB) goes to one pole, and the
two non-linked chromosomes (A and B) to the other (Fig. 68, D).

In the case of the individuals with 33 chromosomes—1.e. with two V’s,
or, in other words, in which linkage has occurred in both homologous
couples—we find instead of the open tetrapartite V in metaphase I. a
closed tetrapartite ring like the usual type of bivalent formed by syndesis
of two V’s (cf. Lepidosiren, Fig. 16).

Other similar cases could be cited, e.g., Notonecta (Browne, I9g13).
Here m= 13 or 14, the former number being produced by the linkage of
two chromosomes which are separate in the latter.

Fig. 69 illustrates five types of chromosome complexes found in
various species of the genus Drosophila, with their possible relationships

Vi
\\

}
ANA

Fic. 69.

Five types of chromosome complex found in the genus Drosophila, showing their probable relationships.
(After Metz, J.E.Z., 1914.)

(Metz, 1914). The remaining four types are all derivable from type I. by
(1) breaking across of one or both pairs of V’s, and (2) disappearance, or
attachment to another pair, of the pair of very small chromosomes. More
recently (1916) Metz has added several other types, all, however, simply
related to the above.

So far we have dealt with linkage or fragmentation of chromosomes
permanent for the individual or species. A fragmentation of the type
chromosomes, leading to a variation of chromosome number in different
tissues or cells of a given individual, has also often been observed or
inferred.

The classical case is that of Ascaris megalocephala, in which the
somatic chromosomes undergo fragmentation, so that somatic mitoses
exhibit far smaller.and more numerous chromosomes (about 60 in A. m.
untvalens) than do those in the germ track (see Chapter III.). It is note-
worthy that Payne (1913 }) found that subjecting eggs to radium emana-
tion causes the chromosomes in the germ track also to fragment.

Vv NUMBER OF CHROMOSOMES 143

In Ascaris canis (Walton, 1918) the chromosomes in the somatic
cells fragment into two, so that these mitoses have double the number
of chromosomes present in the germ track. This seems to be a not
uncommon phenomenon. Thus the bisexual form of Ascaris nigrovenosus
(Schleip, 1912) has in the germ track eleven ( ¢) or twelve (2 ) chromo-
somes, the somatic tissues having double that number of much smaller
ones. Doncaster (1910) found one male of the gall-fly Neuroterus
lenticularis in which the number of chromosomes in the somatic tissues
was double that in the spermatogonia. In the bee (Apis mellifica—
Meves, 1907; Nachtsheim, 1913) 2m is 32, judging from the meiotic
divisions, but the somatic cells often show 64. Armbruster (1913) found
also in the case of the solitary apid Osmia cornuta that the number of
chromosomes is much higher in the mitoses of the soma than in those
of the germ track.

Sometimes the process of fragmentation is less orderly, affecting
only certain cells, and these to varying degree, resulting in apparently
capricious variation of chromosome number. This has been studied by
Hance in the pig (1918 a) and in Oenothera (1918 5).

In the pig the spermatogonial chromosomes always number 40.
In the ninety-one somatic cells in which the chromosomes were counted,
the number varied from 40 to 57. It will be noticed that none had less
than the type number. Most of them had more, only four having exactly
40. In order to obtain evidence as to whether the increase is due to
fragmentation or multiplication, Hance measured the sum of the lengths
of all the metaphase chromosomes in the spermatogonia and in the
somatic cells. In the former the sum of the lengths of all the chromo-
somes varied from 118-6 to 177-6 units of measurement. The combined
lengths of all the chromosomes in the different somatic cells, in spite of
the variation in their number, fell within the same limits, with one
exception in which they totalled 117 units. This evidence, while not
conclusive in view of the variation of chromosome lengths in different
tissues, the possibility of regulations, and so on, is certainly greatly in
favour of the numerical increase having been caused by fragmentation
of the 40 “ type ’’ chromosomes.

Closely similar results were obtained from Oenothera scintillans. In
this evening primrose 2m =15, it being, like O. lata (p. 146) one of the
Oenothera mutants which possesses an extra chromosome. In the somatic
tissues, however, while the number 15 is the commonest, it varies from
15 to 21. By measuring the lengths of all the chromosomes and adding
them together, he found that the average total amount of chromatin
was approximately the same in nuclei with 15 chromosomes as in those
with 16, 17, 18, 19, 20 or 21. Hence he concludes that the higher numbers
have been derived from the lower by fragmentation of one or more of

144 CYTOLOGY CHAP.

the “type ’”’ chromosomes. It is significant that he found no fragmenta-
tion in the germinal tissues.+

It is plain that variation in chromosome number by linkage or
fragmentation is not by any means incompatible with the general thesis
laid down on p. 123. The primary object of the arrangement of the
units in linear series, which is to allow of their accurate distribution to
the daughter nuclei after fission in mitosis, is not in the least prejudiced
thereby. Syndesis of homologous chromosomes, or rather of the units
composing them, would, however, certainly be complicated if irregular
fragmentation were to occur in the germ track. It is precisely here, how-
ever, as the examples above quoted suffice to show, that the chromosome
number is particularly constant. Only a few cases of fragmentation or
linkage within the germ tracks of individuals have been described, and
these are mostly of a simple and orderly nature. The evolution of a
species with a chromosome number differing from that of the parent
species must indeed have been accompanied by a rearrangement of
chromatin units, or by a fragmentation or linkage of existing chromo-
somes. This case, however, goes far to furnish proof of the necessity for
the constancy of the linear arrangement, since there is strong reason to
believe that interspecific sterility arises through incompatibility of the
chromosomes of the two incipient species in syndesis (Chapter VI.).

The remaining two causes of variation in chromosome number do
not call into question the continuity of the chromosomes, since they
concern the multiplication or disappearance of whole chromosomes.

(2) Variation in Chromosome Number due to Irregularities of Mitosts

This is an abnormality which, if leading to an extensive loss of
chromosomes, must lead eventually to the death of the cell in which.
it has occurred. Since each diploid nucleus contains a double set of
chromosomes, and since one set contains all the units required for a
perfect organism, it follows that one member of each homologous pair
might be lost without much disturbance, but if both members of a pair
disappeared serious consequences must result.

Though mitotic irregularities usually lead to a loss of chromosomes
owing to one or more of these failing to get included in the daughter
nuclei, they sometimes have the effect of increasing the number. Probably
the earliest of these cases to be described was in Ascaris megalocephala
bivalens (Fig. 70). Boveri (for summary see 1904) found that sometimes
the spindle of the first meiotic division in the egg is placed tangentially

1 This perhaps is an indication of a differentiation between soma and gonad as displayed
in the germ tracks of so many animals.

Vv NUMBER OF CHROMOSOMES 145

FIG. 70.

Normal and abnormal polar body formation in Ascaris megalocephala univalens. (Boveri, Ergebnisse, 1904.)
A, normal series : B, abnormal series. In each, 1 is anaphase I,; 2, metaphase II. ; 3, polar body formation
completed ; 4, fertilization, the ¢ nucleus below in each figure. C, embryo derived from series B, showing
three chromosomes in the germ track, and only one polar body, with two chromosomes.

L

146 CYTOLOGY CHAP.

instead of radially. Hence the first polar body is not extruded, but
all the chromosomes—1t.e. two bivalents—remain in the egg. At the
second meiotic division all these chromosomes enter the spindle, with
the consequence that the egg and the single polar body are each left
with two univalents instead of one. When the egg is fertilized, the
zygote nucleus has therefore three chromosomes, two from the egg
and one from the spermatozoon. These three chromosomes appear
regularly in all the subsequent mitoses in the germ track. As the polar
bodies in Ascaris are conspicuous objects for a considerable time in
the early development of the embryo, the fact can be verified that
these rare embryos with three chromosomes have been produced
in this way, for they have only one polar body (containing two
univalents) instead of two (one with one bivalent and the other with
one univalent).

Some of the Oenothera mutants, e.g., O. lata, probably owe their origin
to an irregular distribution of chromosomes in meiosis, since they differ
from the parent forms in possessing an additional chromosome (2” =15
instead of 14). O.lata has been produced several times among the Oenothera
mutants, occurring in the frequency of about -4 per cent (Gates and
Thomas, 1914). It presumably owes its origin to the union of a normal
gamete with one bearing eight instead of seven chromosomes. In the
meiosis of this form the chromosomes are normally separated in groups
of seven and eight, though sometimes more irregularly, the anaphase
groups consisting of six and nine chromosomes respectively, etc. (Gates
and Thomas, 1914). Thus forms arise with still more varied chromosome
numbers, when gametes bearing these abnormal numbers of chromosomes
participate in syngamy.

In view of that dependence of somatic characters upon the chromatin
elements which is required by our thesis, it thus becomes of great interest
to know what are the characteristics of the offspring produced by O. lata.
For causes at present obscure, this plant produces practically no fertile
pollen grains. Consequently its behaviour in breeding can only be studied
in crosses in which other forms are the male parents. Crossed with O.
lamarckiana, in which 2n=14, it gives a mixture of lamarckiana and
lata offspring (de Vries, 1910). This of course is in accordance with
expectation, since all the microgametes have seven chromosomes, and
some of the macrogametes seven and others eight. As we have just
seen, however, the fifteen chromosomes of O. lata do not always separate
into seven and eight at meiosis, but sometimes into more unequal groups.
Lutz (1912) determined the number of chromosomes for fifty-two offspring
resulting from a cross between O. lata and O. gigas. In the latter 21 =28.
The numbers of chromosomes (2) found, together with the numbers of
plants exhibiting each number, were as follows :

a

v NUMBER OF CHROMOSOMES 147

Number of Chromosomes I5 21 22 23 29 += 30
Number of Plants . : 20156 GAO5 3 2 4

This variation is probably due to variation in the /ata gametes, since
O. gigas breeds true and therefore presumably always has the expected
fourteen chromosomes in its gametes. It will be noticed that the most
frequent numbers are 21 and 22, which corresponds with the above-
mentioned fact that the gametes of O. lata most commonly have seven
or eight chromosomes. These combining with the fourteen of the micro-
gamete produce of course these two diploid numbers. The six plants
with 29 and 30 chromosomes are probably examples of a phenomenon
to be described in the next section ; namely, they have presumably been
produced by fertilization of diploid egg cells.

(3) Variation in Chromosome Number due to Multiplication or F usion

Longitudinal fission of the chromosomes, as in ordinary prophase,
but without subsequent mitosis leading to separation of the daughter
halves, has frequently been described. By this means giant nuclei are
formed containing twice the normal number of chromosomes if the
process has taken place once, four times if it has happened twice, and
so on.

One of the most striking cases described is that of Culex pipiens
(Holt, 1917). Here 2n=6. During metamorphosis, while the alimentary
canal of the pupa is developing, the chromosomes in the cells of the old
larval alimentary canal undergo repeated fission, forming nuclei with a
large number of chromosomes, but always in multiples of three, and
generally in multiples of six. Nuclei were found, for example, with 9,
12, 18, 24, 36 and 72 chromosomes. Nuclei with this enormous increase
of chromosomes can proceed to apparently normal mitosis. In telophase
the chromosomes fuse into three masses (it will be remembered that in
the normal somatic mitoses of C. pipiens the pairing of the homologous
chromosomes is very pronounced, so that three double chromosomes
result, p. 126), from each of which in the next prophase one chromatin
filament is formed, which divides up by multiple longitudinal fission so
that three groups of chromosomes are produced. The chromosomes
within each group are of closely similar lengths, though the length type
is different in the different groups.

These cells, being in the larval alimentary canal which is being re-
placed by that of the pupa, are destined to perish. Nuclei with a multiple
supply of chromosomes seem often to be produced in this way, however,
and the process as such does not seem to have a necessarily deleterious
effect.

148 CYTOLOGY cua

Multiplication of chromosomes has been obtained by experimental
methods by Némec (1904, 1910). If the root tips of plants (Vicia, ©
Pisum, Allium) are subjected to the narcotizing action of chloral hydrate,
any cell divisions that are in progress at the time are inhibited to this
extent, that the telophase reconstruction of the nuclei takes place, but
cell division is not completed. Thus binucleate cells are formed. If
the root tips are allowed to recover from their narcotization, the
two nuclei, which lie close together, may fuse into a single giant
nucleus. This may proceed to mitosis, in which case it exhibits 4n
chromosomes.

If the root tips are repeatedly narcotized, being allowed to recover
after each narcotization, the above process may also be repeated, resulting
in giant nuclei with 8” or probably even 16” chromosomes.

From the fact that such polyploid nuclei become fewer in proportion
to the length of time that has elapsed since the plants recovered from
the narcotization, Némec concludes that a reduction to the normal
number of chromosomes ultimately takes place, and indeed found certain
mitoses which he believed to be the reduction divisions ; this interpreta-
tion is, however, necessarily uncertain.

There are many cases known where of two nearly allied species one
has double the number of chromosomes of the other, and the possibility
naturally suggests itself that the chromosome number may have been
doubled in this way, namely, by a fission of the chromosomes unaccom-
panied by nuclear division. Forms thus derived would obviously have
four sets of chromosomes; that is to say, they would be éetraploid
instead of diploid in their somata, and diploid instead of haploid in their
gametes. It must not, however, be assumed without special evidence
that any species is tetraploid, since in the great majority of cases where
one species has twice as many chromosomes as nearly related species,
the larger number has almost certainly been derived from the smaller
either by the aggregation of the chromomeres into double the number
of chromosomes or by the transverse fragmentation of all the chromo-
somes into two—as, for instance, happens in the somatic cells of Ascaris
canis and the other examples mentioned on p. 143. If transverse con-
strictions in chromosomes are signs of a weak point where fragmentation
is liable to take place, there is no difficulty in understanding how the
number of chromosomes comes to be exactly doubled, for it may happen
that every chromosome exhibits this constriction (e.g. Lepidosiren, p. 40),
and there are very few references in literature to a chromosome exhibiting
more than one such transverse joint.

Out of the numerous cases of numerical doubling of chromosomes
known, the following few striking examples may be quoted :

v TETRAPLOIDY 149

Species, Number of Chromosomes
F (Diploid).
Ascaris megalocephala univalens
bivalens : 2 *
Cypris veptans, parthenogenetic . ‘ : : et
| fuscata, 24 f Schleip, 1909.

a, Many authors.

Ophryotrocha Bae some sri 4\ Korschelt 1895,
Oenciera lamarckiana . ; : ; . . 14 Various authors.

35 gigas : , ; . 28 Gates, etc., Ig09 a.
Musa sapientum (the banana), one variety . : 16

3 3 another variety . 32 | Tischler, 1908.

” ” ” - 48,

In a species in which distinct size differences exist between the
different chromosomes, the question of tetraploidy should be capable of
easy solution, since in this case the chromosomes should be grouped
according to sizes in fours instead of in pairs. Practically nothing seems
to be known about this matter in supposed tetraploid forms. Montgomery
(1909), however, has described the chromosomes of the bivalens variety
of A. megalocephala as consisting of a longer and a shorter pair. The
difference between the pairs is indeed slight, but if established it
would make it probable that this form is not tetraploid. This case is
especially instructive, as here if anywhere one might have expected
the four chromosomes to have been derived by doubling of the two
chromosomes of wnivalens, since the total volume of the four chromosomes
in bivalens is even more than double that of the two in wnivalens (Brauer,
1893).

Two genera of the Oligochaete family Enchytraeidae present similar
relations (Vejdovsky, 1907). In Fredericia hegemon 2n=32, and in
Enchytraeus humicultor 2n=64. Nevertheless, the chromosomes in the
latter genus are much longer and thicker than in the former.

In certain cases, however, especially those in which the forms with
doubled chromosome number have arisen under experimental conditions,
the comparison of the volumes of the chromosomes is of value in deciding
how the doubling has occurred. Two such cases are found in the plant
genera Oenothera and Primula.

Oenothera gigas is one of the well-known “ mutants ” of O. lamarckiana.
It first appeared as a single individual in de Vries’ cultures of 1898, and
in the next twenty years the appearance of six more individuals was
recorded from the cultures of de Vries and others, so that its origin from
the parent form was observed altogether seven times in that period.
Once arisen, it breeds pure to its peculiar characteristics (except for a
tendency to give off exceptional mutants of the same order as those
produced by the parent species). These characteristics are the possession
of 28 chromosomes in place of 14, and the greater size of nearly all its
parts, such as stalk, leaves, petals, etc.

Gates (1909 a) has compared the relative sizes of the cells in O. gigas

{

150 CYTOLOGY CHAP,
;

and O. lamarckiana. Those of O. gigas are 1:5 to 3:8 times as large as

those of O. lamarckiana, averaging about 2-5 times as large (cells of —

anther epidermis). Since in general the size of the cell is proportional
to the surface of the nucleus, which again is proportional to the volume
of chromatin (Boveri, 1904, 1905), we may assume that O. gigas has
twice as much chromatin as O. lamarckiana, and therefore that its 28
chromosomes comprise a double set of those present in the parent form.
O. gigas is probably therefore a true tetraploid species.

The origin of such a form as this can be conceived as being due either
to (1) syngamy of two diploid gametes, i.e. gametes in whose formation
the meiotic division has been omitted ; or (2) a doubling of the chromo-
somes in the zygote nucleus owing to fission of the chromosomes not
followed by mitosis.

It is at present impossible to decide between these alternatives. As
we have seen, a doubling of the chromosomes may take place under
certain experimental conditions (p. 148), and there appears to be no
reason to deny that this might happen in nature under abnormal
conditions. If the doubling occurred in the undivided zygote cell, it
might result in the whole individual being tetraploid. On the other
hand, diploid egg cells are normally formed in obligatory parthenogenesis
and may possibly occur, and be capable of fertilization, in sexual repro-
duction. Diploid microgametes are, however, not known to occur, though
the giant spermatozoa occasionally found in many animals are sometimes
supposed to be due to the omission of the meiotic division.

If diploid male and female gametes do occur, they are certainly very
rare, and it must be still more rare that two such gametes should meet.
In the enormous majority of cases a diploid gamete must meet a normal
haploid gamete. This, if syngamy took place, would result in a triploid
zygote. Triploid Oenotheras, with 21 chromosomes, have indeed frequently
been described (Lutz, 1912; Stomps, 1912). They have been observed
as ‘‘ mutants ” both in pure cultures of O. lamarckiana and from crosses
between the various Oenothera mutants. Their characteristics vary to a
certain extent according to their origin, but in their general growth they
appear to stand mid-way between O. gigas and the ordinary diploid
Oenotheras (Stomps, Ig12).

Stomps estimates that three triploid (‘““Hero’’) plants are found
among about 1000 plants in certain Oenothera cultures, and that O. gigas
appears at most in the proportion of 1: 10,000, though the total
number of O. gigas mutants known is too small for the percentage to
be reliably estimated. So far as the figures go, however, the greater
rarity of tetraploid forms compared with the triploid varieties favours
the view that they have arisen through syngamy of diploid gametes,
rather than from a multiplication of the chromosomes in the zygote.

Vv NUMBER OF CHROMOSOMES I5I

While O. gigas is probably a true tetraploid form, a case of chromo-
some doubling has been described by Farmer and Digby (1914), in which
it appears probable that the doubling was produced by fragmentation.
Primula floribunda crossed by P. verticillata gave a hybrid of a distinct
type known as P. kewensis. In both parents 2n=18, and this was also
the number in the hybrid. The hybrid plant produced only “ thrum ”’
flowers, and was therefore self-sterile. It was reproduced vegetatively
by cuttings, and eventually a single “ pin” flower appeared, which
allowed of fertilization by one of the thrum flowers, and from this a
fertile race of P. Rewensis was obtained. This race was found, however,
to have double the number of chromosomes present in the original P.
kewensis, 2n being 36. The chromosomes are also smaller in the fertile
race, measurements giving the following results :

Mean volume of single Total volume of all
Chromosomes. Chromosomes.
Race with 18 chromosomes “S141 14°65
Race with 36 chromosomes -4088 TA71

These measurements clearly suggest that in this case the doubling
of the chromosome number has been brought about by transverse
fragmentation, and not by chromosome fission unfollowed by nuclear
division. A true tetraploid Primula has, however, been found in another
case, as described in the next chapter (p. 170).

The last cause of variation in chromosome number which we have
to consider is fusion of chromosomes. An example of this has already
been given in the case of Culex pipiens (p. 126). It is obvious that here
we are dealing with an apparent variation only, since all the chromosomes
are present, though they may be indistinguishably fused in pairs.

A Sycon sponge (Jorgensen, 1910 a) probably presents a case similar
to Culex. In this sponge the number of chromosomes in the gamete is
eight, and in accordance with this sixteen chromosomes are found in the
metaphase of the first cleavage division. In all other cells examined,
however (mesoderm cells, oogonia, oocytes), the number is always eight,
except in the early prophases, when it is more, though the number could
not be exactly counted. Jorgensen brings forward evidence to show that
a fusion of pairs of homologous chromosomes takes place in the telophase
of the first cleavage division.

A particular class of cases of the halving of the chromosome number
falls to be mentioned here. A union of the chromosomes—now in the
haploid number—in pairs sometimes takes place in the anaphase of the
first meiotic division or between the two divisions. This has been de-
scribed in Birds (Numidia and Gallus—Guyer, 1909, 1916; Columba—
Smith, 1913), Mammals (Man—Guyer, 1910; Didelphys-—Jordan, 1912) ;

152 CYTOLOGY. CHAP. V

Lepidoptera (Phragmatobia—Seiler, 1913); Hymenoptera (Osmuia cor-
nuta—Armbruster, 1913).
The chromosome numbers in prophase are as follows in these cases :

: Spermatogonia. Spermatocyte I. Spermatocyte II.
Man . : ‘ ‘ ; ‘ 22m 12 5 Or 7
Didelphys virginiana . ; : 7a 9 4 0F 5
Numidia meleagus : 3 : 17 9 4 OT 5
Gallus gallus ; : : : 184 9 4 0F 5
Columba. : , : 3 ; 16 8 4
Phragmatobia fuliginosa : : [56]® 28 14
Osmia cornuia . ; a 16® 16 8

The only cases in which we have any further information as to the
behaviour of these secondarily united chromosomes are Phragmatobia
and Gallus. In these the secondary spermatocyte pairs are resolved into
their elements (though only exceptionally in Gallus) before the second
meiotic division takes place. It is to be presumed that in the other
cases mentioned they also separate before syngamy.

Another instance of pairing between chromosomes in a haploid
nucleus is found in the spermatogonia of the bee (see p. g1, footnote).
In the female bee, according to Nachtsheim (1913) the 32 chromosomes
unite into 16 bivalents in the oogonia. In oocyte I. they pair a second
time to form 8 tetravalents. The mature egg has therefore 8 bivalents,
which, however, fall apart into univalents about the time of fertilization.

At present the above cases cannot be said to fit without forcing into
any general scheme of chromosome behaviour. Probably, however,
fusion of the chromosomes should be regarded, not as a pairing strictly
speaking, but as a general tendency to fusion which usually stops short
at fusion in couples. Cutler (1918) found that in the pheasant, where
2n is probably 20-22, the number of chromosomes in the first meiotic
division is 10-11, while in the secondary spermatocytes 1-8 masses of
chromatin appear, indicating a tendency to fusion carried to various
degrees. Even in Gallus, though the fusion in couples is usually complete,
giving 4 chromosome masses in secondary spermatocytes which lack the
X-chromosome, and 5 chromosome masses in those which contain it,
yet metaphases IT. with six or seven such masses are not unusual.

1 Two are sex chromosomes. Other workers, however, have given the number of chromo-
somes in man, in whom they are very difficult to count, in varying figures up to 40.

2 One is a sex chromosome.

3 One is a sex chromosome. The exact number of chromosomes was, however, impossible
to determine beyond doubt. In view of Guyer’s more recent conclusions as regards Gallus
it seems probable that there are two sex chromosomes, with a total of 18 chromosomes.

4 Two are sex chromosomes.

5 Spermatogonial number inferred from the number of bivalents in meiosis.
6 This is the haploid number, the case being parallel with that of the bee (p. 91).

CHAPTER. VI
HEREDITY AND MORPHOGENESIS

In this chapter we have to consider the thesis that the nucleus and
not the cytoplasm is the substratum by which hereditary qualities are
transmitted from parent to offspring, and the correlative hypothesis
that it is the nucleus which is the initiator and controller of the activities
of the cell and especially of morphogenesis. It is obvious that these two
theses are mutually interdependent, and if either were established the
other would follow as a corollary without the necessity of further proof.
In the meantime, any evidence obtainable for or against the one is in
equal degree evidence for or against the other, and therefore the two
may be considered together.

Both these theses are capable of expansion far beyond the limits
of a volume of this scope, and have, moreover, so often been made the
subject of special treatises that they can only be dealt with briefly here.

The student must also realize that in this chapter the amount of
theory bulks larger in proportion to the amount of fact than in the
preceding chapters, and therefore that it is all the more necessary for
him to keep in mind the proper scientific spirit which is always ready
to modify its ideas when the discovery of new facts makes this necessary.

The principal threads of evidence under this head may be classified
as follows :

(a) The equality of inheritance from male and female parents.

(0) The process of mitosis and its implications.

(c) The process of meiosis.

(d) The parallel which exists between chromosome behaviour and

the results of breeding experiments.
(e) The case of sterile and partially sterile hybrids.
(f) Morphogenesis and mode of action of the nucleus.
(g) Chromidia and chondriosomes.
We will consider these points in order.
153

154 CYTOLOGY, CHAP.

A. THE EQUALITY OF INHERITANCE FROM MALE AND
FEMALE PARENTS

The fact that, on the average, offSpring inherit with approximately
equal intensity from both parents, whereas the macrogamete as a whole
is nearly always enormously larger (often a million or even more than
a billion times larger) than the microgamete, immediately suggests that
the hereditary substratum is not the substance of the gametes as a whole,
but some special portion of them which is of more approximately equal
mass in the two cells. This consideration led Nageli in 1884 to postulate
two substances in the gametes, one of which is present in equal amount in
the micro- and macro-gamete. This is the bearer of hereditary qualities—
the idioplasm. The other has mainly a nutritive function and is present
in far greater amount in the egg and is indeed responsible for its larger
size. Knowledge of the processes of fertilization naturally led to the
idioplasm being identified with the nucleus (independently by O. Hertwig
and Strasburger in 1884), since the nuclear substance appears to be the
only one that is contributed in approximately equal amounts by the
two gametes. Mere study of the anatomy of the gametes therefore at
once leads us to suspect the all-importance of the nucleus and the
essential passivity of the cytoplasm in the transmission of hereditary
qualities.

It is of course not necessary to assume that equal quantities of living
matter produce equally powerful effects, nor that inequal masses cannot
produce equal effects. It is certain also that the amount of chromatin
in the nucleus cannot always be taken as denoting the amount of
idioplasm therein. Nevertheless, in corresponding stages of the life-
cycle such as the ripe male and female gametes, one would expect to
find approximate mass equality of the idioplasm, at any rate not the
enormous disproportion that exists between the cytoplasm of the egg
and that of the spermatozoon.

The statement just made, that the amount of chromatin in the nucleus
cannot always be taken as an indication of the quantity of idioplasm is
supported by many considerations. Thus the amount of chromatin in
the nucleus varies greatly in more or less closely allied forms (Fig. 71).
We cannot suppose that the amount of idioplasm in the nucleus of
Lepidosiven is much greater than in that of the salamander, and enor-
mously greater than in the nucleus of a rabbit. Moreover, the amount
of chromatin varies in different tissues of the same organism, or even
in different periods of the life-cycle of the same nucleus. Riickert pointed
out long ago that the amount of chromatin in the oocyte nucleus in the
growth period (Selachians) is very much greater than the combined
bulk of the chromosomes on the spindle of the first maturation division,

|

j

vI CHROMATIN AND IDIOPLASM 155

though here a complete complement of hereditary substance is bound
to be present. This is probably a universal rule in oogenesis (cf. Fig. 22,
etc.). Thus Gardiner (1899) calculated that in Polychaerus not more
than so part of the chromatin of the germinal vesicle at its most
chromatic stage is used up in the formation of the chromosomes of the
meiotic divisions.

In the sea-urchin Strongylocentrotus, Erdmann (1gog) finds that the
volume of the chromosomes in the pluteus is only 9's of their volume in
the two-cell stage.

Such considerations have led to the hypothesis of two kinds’ of
chromatin—idiochromatin, the essential hereditary substance, or idio-
plasma proper, and ‘vophochromatin (see later under chromidia). Prob-

C

FIG. 72.

Equatorial plates of spermatogonia of three Vertebrates. A, Lepidosiren; B, salamander; C, rabbit.
The three figures are drawn to the same scale to show the relative amounts of chromatin present in each.

ably, however, the idioplasm cannot, strictly speaking, be identified with
any subs'ance. The hereditary factors should probably be considered as
elementary organisms, consisting mainly indeed of chromatin but possessing
an organic structure on which their activities depend.

The argument therefore from equality or otherwise of mass, though
weighty, is not conclusive in deciding the claims of the various constituents
of the gametes to be considered as the idioplasm. There is no doubt that
in animals, at any rate, a certain very small amount of cytoplasm is
introduced by the male gamete along with the nucleus (see also under
chondriosomes, below). This has, however, been denied in the case of
some of the higher plants where it has been held that no cytoplasm
enters the egg cell with the nucleus (Strasburger, 1908), but a negative is
proverbially hard to prove.

156 . CYTOLOGY a

The statement made above, that inheritance from male and female
parents is on the average of approximately equal intensity, besides being
a matter of common knowledge, is established beyond question experi-
mentally. Contrary opinions have, however, been expressed by workers
in one special field of biology—namely, the study of hybrids (especially
of Echinoderms) in embryonic or early larval stages, where it has been
held that the influence of the female parent is predominant. From this
the conclusion has been drawn that the cytoplasm also contains
idioplasm.

The Echinodermata have shown themselves to be very adaptable to
these experiments, crosses being comparatively easily effected between
species, genera or even classes, and an efficient technique has been
evolved for dealing with this work, which has attracted a large number
of investigators. Many of these have found that the hybrid embryos
and larvae from certain crosses exhibit purely maternal characters.
Thus Vernon (1898), making reciprocal crosses between seven different
genera of sea-urchins, found that, as a rule, the hybrid larvae (plutei)
were of the maternal type. The results varied considerably in this respect,
however, in different seasons. Thus the cross Sphaerechinus 9 x Strongylo-
centrotus ¢ gave in May-July mostly maternal larvae, while in December-
January the larvae from the same cross were all paternalin type. Shearer,
de Morgan and Fuchs have also shown (1913) how the prepotency of
one or other species may vary at different times. Their crosses were
made between Echinus esculentus or acutus and muliaris. The pluteus
larvae of the former two species exhibit, in the older stages, posterior
ciliated epaulettes but no masses of green pigment, while the larvae of
miliaris at the same stage have masses of green pigment but no posterior
epaulettes. In Igro-II crosses between esculentus or acutus and miliaris,
in whichever direction they were made, produced larvae resembling the
species used as female parent (in regard to these two features). In 1912,
however, similar crosses gave a different result, miliaris appearing
practically incapable of transmitting its characters at all, so that when
this species was used as the female parent the larvae resembled the
male parent species. This result appeared to be due to something un-
favourable to miliaris in the environment, since pure cultures of this
species, which in 1910-11 were easy to rear, proved very difficult to bring
up in IQI2.

Godlewski (1906) succeeded in fertilizing Echinus eggs by the sperm
of Antedon, which belongs to a very different class of the Echinodermata.
Union between male and female gamete nuclei took place, and the two
sets of chromosomes, though of such different origins, appeared to har-
monize on the spindle and to co-operate at telophase to form the
resting nucleus as in normal fertilization and cleavage. The hybrids

VI ECHINODERM HYBRIDS £57

developed as far as gastrulation and in a few cases even reached the
pluteus stage. They exhibited solely the characters of the female parent
(Echinus). Godlewski even managed to fertilize eggs of Echinus from
which the nuclei had been removed (by shaking) with Antedon sperm.
These all developed to embryos of pure Echinus type. None of them,
however, passed the gastrula stage. From these experiments Godlewski
argues that the egg cytoplasm and not the nucleus is the determining
factor in early development.

Boveri's classical experiments on the cross Sphaerechinus 2 x Echinus 3
led to exactly the opposite conclusion (1896). These larvae are normally
intermediate between the two parents. By violently shaking the eggs
before fertilization, Boveri succeeded in breaking many of them into
two or more fragments, of which of course only one contains a nucleus.
On adding Echinus sperm to a mass of these broken-up Sphaerechinus
eggs a culture was obtained which consisted of a mixture of—(rz)
normal larvae, intermediate between the two parents; (2) dwarfs, also
intermediate ; and (3) dwarfs, resembling the male parent exclusively.
Having previously, by direct observation, ascertained the power of
non-nucleated egg fragments to be fertilized and to develop normally,
Boveri interpreted these three classes as having originated as follows:
(1) from unfragmented eggs, (2) from egg fragments containing a nucleus,
(3) from egg fragments containing no nucleus—all of course fertilized
by Echinus sperm. From this result Boveri draws the conclusion that
the nucleus is the sole bearer of hereditary factors. Taking, however, a
general view of the results of Echinoderm crosses, it undoubtedly is the
case that the hybrids in the larval stages do tend to resemble the female
parent more than the male, though the tendency is by no means a
universal rule, and though the prepotency of one or other parent is
strongly influenced in some way or other by factors of the external
environment.

One cause of the frequent prepotency of the female parent in
Echinoderm crosses has been discovered by Baltzer (1910) in a work
which is of the greatest importance in interpreting the results of crosses
between distantly related forms. To take the case of two Echinoderm
genera, Strongylocentrotus and Sphaerechinus, the cross Sphaerechinus
x Strongylocentrotus g'gives embryos which develop regularly and with-
out pathological phenomena into plutei, which as regards the skeleton
are intermediate between the two parent species. Cultures from the
reciprocal cross, however, Strongylocentrotus 2 x Sphaerechinus $ run a
different course. When the hybrid embryos reach the blastula stage,
instead of turning into a hollow sphere, the cavity (blastocoele) becomes
filled with masses of degenerating cells and nuclei, and the larvae, which
should be transparent, become in consequence opaque and the great

158 CYTOLOGY CHAP.

majority die. Those that reach the pluteus stage are found to exhibit
exclusively the characters of the female parent (Strongylocentrotus).
Cytological examination of these hybrids (Stvongylocentrotus 2 x
wae Sphaerechinus $) pro-
Pe Zea? vides a satisfactory ex-
Z planation both of the
pathological course of
development and of the
purely maternal char-
acteristics of the plutei.
In the anaphase of the
first cleavage division a
number of chromosomes
fail to travel up to the
poles with the other
chromosomes, but remain
lying in between the two
daughter groups (Fig. 72).
They fail to enter into
the daughter nucleus, re-

ene |

— ~ . .
ret ! Ques a = <> maining as extra-nuclear
y / I ere ? Ty s 4 2
hee rey ANSP 79 WW masses of chromatin in

B the blastomeres. A
further number of chro-
mosomes get left out in
the second cleavage
mitosis. The nuclei from
now onwards have only
about twenty or twenty-
one chromosomes (vary-
ing in different larvae
from nineteen to twenty-
four) instead of the
expected thirty-six (m in
both parent species being

eighteen). Thus about
The chromosomes of the hybrid Strongylocentrotus Q x Sphaer-

echinus g. (Baltzer, A.Z., 1910.) A, B, anaphase and telophase of fifteen chromosomes have

the first cleavage division. A number of chromosomes fail to enter coe

either daughter nucleus. C, 4-cell stage. The eliminated chromosomes been eliminated. These

form irregular masses of chromatin between the daughter nuclei. 3

rejected chromosomesare,
in all probability, those brought in by the male gamete. For instance,
two pairs of the Sphaerechinus and one pair of the Strongylocentrotus
chromosomes are distinguishable by their shapes and sizes. After the
elimination we never find these two Sphaerechinus chromosomes, but the

BIGsE 72:

VI ECHINODERM HYBRIDS 159

Strongylocentrotus chromosome is still present. In regard to these three
chromosomes, therefore, there is no doubt that it is the male chromosomes
that are eliminated. There are other reasons, which cannot be gone into
here, for supposing that this is so in regard to all the fifteen chromosomes
which are got rid of. Thus the overwhelming influence of the female
parent in the formation of the pluteus skeleton is easily intelligible on
the hypothesis that the idioplasm is contained in the nucleus, since the
larval nuclei contain eighteen chromosomes from the female parent but
only about three from the male.

Furthermore, the pathological phenomena in the blastulae owe their
origin to the same process. The eliminated paternal chromosomes do
not lie altogether passive in the cytoplasm, but undergo repeated
fission, forming great masses of chromatin. At the formation of the
blastula these chromatin masses with surrounding cytoplasm are extruded
into the blastocoele, where they form the masses mentioned above.
In the cross Strongylocentrotus 2 x Arbacia g, which also results in plutei
with almost purely maternal characters, the paternal chromatin also
appears to be eliminated, but by an entirely different method. The
development of these larvae runs a somewhat similar course to that of
the Strongylocentrotus 9 x Sphaerechinus ¢ hybrids, only the patho-
logical phenomena in the blastulae make their appearance slightly later.

An examination of the cleavage mitoses shows that there is here
no elimination of the chromosomes, all the expected 38 being present
at all stages up to the blastula (in Strongylocentrotus, n=18; in Arbacia
n=20). In correspondence with this we find that the size of the nuclei
(which is proportional to the number of chromosomes) is about the
same in the hybrid larvae as in those of the pure parent species
(Strongylocentrotus). The nucleiin the pluteus stage, however, have many
fewer chromosomes (about 18, judging from direct counts), and corre-
spondingly their nuclei are smaller than those of pure bred nuclei, in the
proportion of about 21:36. Hence it appears that somewhere between
the beginning of the blastula stage and the pluteus an elimination of
about 20 chromosomes has taken place. Sections of the blastulae at the
time of the critical period of their development show numbers of nuclei
obviously pathological, others apparently in the process of extruding
chromatin into the cytoplasm, while still others have lying beside them
in the cytoplasm an extra-nuclear mass of chromatin. The conclusion
to be drawn from these observations is that the Arbacia chromatin is
eliminated from the hybrid nuclei at this stage. As in the case of the
hybrids described above, the blastocoele is filled up with these masses
of degenerating chromatin and their surrounding cytoplasm.

While this explanation of the female prepotency in this cross is not
quite conclusive (for exceptions occur in the form of plutei, also purely

160 CYTOLOGY CHAP.

maternal in character, but with apparently the full complement of |
chromosomes), it may be accepted as reasonably satisfactory.

Elimination of chromosomes in cleavage of other Echinoderm crosses
has also been described by Tennant (1912) and by Doncaster and Gray
(1913). The results of Baltzer’s crosses can be tabulated as follows :

(r) Development is normal. There is no elimination of chromosomes
or chromatin. Plutei are intermediate between the two parents,
(Echinus 9 x Strongylocentrotus 3, Strongylocentrotus 9 x Echinus ¢,
Sphaerechinus 9 x Echinus ¢, Sphaerechinus 2 x Strongylocentrotus ¢).

(2) Development is pathological. Most of the parental chromosomes
are eliminated in the first two cleavages. Plutei are of maternal type
(Echinus 9 x Sphaerechinus ¢, Strongylocentrotus 9 x Sphaerechinus ¢,
Arbacia 2 x Echinus 6, Arbacia 2 x Strongylocenirotus 4).

(3) Development is pathological. The chromatin is eliminated in
the blastula stage. The plutei are of maternal type (Echinus 2 x
Arbacia ¢ , Strongylocentrotus 9 x Arbacia 3).

(4) Development is pathological. No chromatin is eliminated. The
plutei are maternal, either predominantly (Sphaerechinus 2 x Arbactia ¢)
or completely (Echinus 9 x Antedon 3, Strongylocentrotus 9 x Antedon ¢).

It will be noticed that the first three of the above results accord well
with the view that the idioplasm is exclusively contained in the nucleus,
classes 2 and 3, if finally established, even constituting strong evidence
in favour of that hypothesis. As regards the 4th class, the following
considerations suggest themselves.

(a) All these three crosses, it will be noticed, are between distantly
related genera—in the case of the two last indeed, between different
classes. Now, granting that the idioplasm is contained in the nucleus,
it can only exert its morphogenetic function through, and on, the
cytoplasm. When idioplasm finds itself in such a strange environment
as cytoplasm belonging to a distantly related genus, or even class, it is
not unlikely that it may be unable to exert any of its normal functions,
though able to nourish and maintain itself, as is shown by the fact that
the parental chromosomes multiply in mitosis with the maternal.

Extreme examples of the behaviour of chromatin in foreign cytoplasm
are furnished by the crosses made by Kupelweiser (1909). In these
the male chromosomes were unable even to maintain themselves and
multiply. He succeeded in fertilizing eggs of Echinus with sperm of
Molluscs and Annelids (Fig. 73). The sperm nucleus did not fuse with
the egg nucleus, but remained practically unchanged in the egg cytoplasm,
obviously inert. The egg indeed developed, but the rédle of the sperm
in this case was plainly merely to supply the stimulus to development.
These cross fertilizations may indeed be considered as another of the
methods of bringing about artificial parthenogenesis. Under these

v1 ECHINODERM HYBRIDS 161

circumstances it is not surprising that the embryos were of the purely
maternal type.

(0) We have already just emphasized that the nucleus can only
exert its morphogenetic function through the cytoplasm. Now the
earlier stages of development proceed with little or no increase of cyto-
plasm. They consist indeed largely in the remodelling of the substance
of the egg into that of the embryo. Hence in these very early stages
the nucleus of the zygote has little opportunity to exert its morphogenetic
function. This can only get full scope after the embryo has begun to
form new cytoplasm by assimilation either of food substances supplied
from without, or of reserve food material stored within it. Thus the

Fic. 73.
Diagram of the fertilization of the egg of Echinus microtuberculatus by the sperm of Myzilus galloprovin-
cialis. (Kupelweiser, A. E-M., 1909.) A, B, ¢ nucleus, preceded by aster, approaching 9°; C, D, first cleavage

nucleus formed entirely from the ? nucleus, ¢ nucleus unchanged; E, 2-cell stage. The ¢ nucleus, still
unchanged, lies inert in that one of the blastomeres to which cell division has chanced to relegate it.

very early form of the embryo must be determined to a large extent
by the physical constitution of the egg cytoplasm.

The resemblance to the maternal species during cleavage, gastrulation,
etc., which is brought about by the purely mechanical factors of size
and number of yolk granules, viscosity of cytoplasm, etc., could only
by a confusion of ideas be brought into the same category as the
resemblance to parents due to the presence of the living, self-repro-
ducing idioplasm. To apply the word ‘‘ Heredity ”’ to both these cases
would be to confuse the meanings of the term as used biologically and
socially, as is done when a child who has been infected im utero with
Spirochaeta pallida is said to have inherited the disease of syphilis.

Finally, it must be mentioned that the only Echinoderm hybrids
which have been examined in the adult condition, namely, the offspring
of the crosses Echinus miliaris 2 x Echinus acutus g (Shearer, de Morgan

M

162 CYTOLOGY CHAP.

and Fuchs, 1914) are found, like all other hybrids, to exhibit certain
characters of one parent and certain of the other. MacBride (1911)
examined larvae from the cross Echinocardium 9 x Echinus ¢ at a much
later stage of development than has usually been employed by workers
on Echinoderm hybrids, and also found them to exhibit characteristics
of both parents.

Summing up, the predominance of maternal characters sometimes
found in the young stages of hybrids, mostly between distantly related
forms, is reconciled with the equality of inheritance from male and
female parents which is the general rule in those vastly more numerous
hybrids which have been studied in the adult condition, by (1) the
observation of elimination of paternal chromatin from the hybrid nuclei ;
(2) the hypothesis of the impotence of idioplasm in a foreign cytoplasm ;
(3) Gn the case of very early embryos) by the fact that the first
stages of development consist in the remodelling of the substance of
the egg into the embryo, without the formation of any new substance
under the influence of the zygote nucleus.!

B. THE PROCESS OF MITOSIS AND ITS IMPLICATIONS... THE
FUNCTIONAL DIFFERENTIATION OF THE CHROMOSOMES

The complicated processes of mitosis—the rearrangement of the
apparently irregularly distributed chromatin of the resting nucleus into
linear series forming long chromatin threads, the longitudinal fission of
these involving the division of each element of the series, and the
separation of the daughter threads in anaphase, one of each pair going
to the one daughter cell and the other to the other—are obviously adapted
to ensure the accurate division and distribution of a mass consisting of
a number of differentiated elements, such as we must suppose the ~
hereditary substance to be. On the other hand, nothing of the kind is
recognizable in the division of the cytoplasm of a mother cell into two
daughter cells.

This lack of a distributing mechanism for the cytoplasm at cell
division argues an essential homogeneity of the cytoplasm. This does
not imply that it may not contain a mixture of different substances
nor possess a definite structure, but that it is not composed of localized
elements of differentiated function essential for the life of the organism,
and incapable of being regenerated if lost. We must qualify this thesis
by an apparent exception, of a parallel nature to that already made
to the thesis of equality of inheritance from the two parents, which we

1 See also the discussion on ‘‘ organ-forming substances,” p. 188.

VI DISPERMIC EGGS 163

have just discussed, for experimental embryology has established the fact
of the local differentiation of the cytoplasm of the egg (the so-called
‘‘ organ-forming ’’ substances). This matter is discussed on pp. 188, etc.

We have already found morphological evidence of the differentiation
of the chromatin in the genetic continuity of the chromosomes, the
constant size differences often visible between them, their composition
out of chromomeres of different but constant sizes, etc., and it is a
necessary corollary of the hypothesis of the dependence of Mendelian
phenomena upon the chromosomes (see below under (D)). Direct experi-
mental evidence of the functional differentiation of the chromosomes
has been obtained by Boveri (1907) from his remarkable experiments
on the development of polyspermic Echinoderm eggs.

Among normally fertilized Echinoid eggs it occasionally happens

.

Se

C4

FIG. 74.

Triaster and tetraster mitotic figures from a sea-urchin, Strongylocentrotus lividus.
(After Baltzer, Verh. Phys. Med. Gesells, Wurzburg, 1908.)

that two spermatozoa enter the egg instead of one. By increasing the
concentration of sperm, the percentage of such dispermic (or poly-
spermic) eggs can be enormously increased. Thus in two parallel experi-
ments, eggs placed in water with only a few spermatozoa resulted in a
hundred normal monospermic and no di- or polyspermic fertilizations.
On the other hand, eggs placed in very concentrated sperm gave only
eleven monospermic and eighty-nine di- or polyspermic fertilizations.

When an Echinoid egg is fertilized by two spermatozoa both sperm
nuclei (typically) fuse with the egg nucleus, and the centrosome intro-
duced by each spermatozoon divides as if it were the only one—hence
we get a zygote nucleus with 3” (=54) chromosomes and four centro-
somes. A four-pole spindle figure is then produced (tetraster), and at the
first division the egg divides simultaneously into four blastomeres instead
of into two.

In the most usual type of tetraster (and the only type which we will

164 CYTOLOGY CHAP.

here consider) the two cleavage planes separating these first four blasto-
meres are identical in position with the two planes formed by the first
two cleavages of the normal monospermic egg. Hence the four simul-
taneously formed blastomeres of the tetraster are identical as regards
cytoplasmic contents with the first four blastomeres of the monospermic
egg.
By shaking the eggs immediately after fertilization it may happen
that the sperm centrosome fails to divide, and in the case of dispermic
eggs it often chances to be brought about that one centrosome divides
and the other does not. In this case the 3m zygote nucleus is provided
with three centrosomes and a ¢riastey spindle results, the egg dividing
into three blastomeres at the first cleavage.

Now, it is obvious that in the tetraster the 32 chromosomes—or rather
the products of their division—that is to say, 6” chromosomes, have to
be distributed amongst four nuclei; and as the arrangement of the
chromosomes on the various parts of the four-pole spindle seems to be
by chance, the number and combination of chromosomes received by
each nucleus are various. As an illustration, one of the very many
possible arrangements in the spindle, and the resulting number of chromo-
somes in the daughter nuclei, is given in Fig. 75.

Even when a blastomere receives more than (=18) chromosomes
there is still a strong chance that it may be lacking in one or more
members of the series. Among the four nuclei, the daughters of three
members of each series are to be distributed—that is to say, there are
two (33A+3B+3C+ ... 3R) to be distributed among the four nuclei.
Hence in the vast majority of cases one or more’ of the blastomeres will
lack a representative of one or more members of the complete series of
chromosomes. A pictorial example of one out of the many possibilities,
showing only four of the eighteen chromosomes, is given in Fig. 75, D, E.

Once the first four blastomere nuclei have been constituted, of course
the number and series of chromosomes contained in them will be per-
petuated in all the nuclei descended from them.

Now, it has been established by many workers, including Boveri
himself, that if the first four blastomeres of the normal Echinoid egg are
separated (which can most conveniently be effected by placing the
egg in sea water from which the calcium salts are lacking—as discovered
by Herbst) each will develop into a dwarf pluteus of one quarter the
normal size. The plutei generally indeed have minor defects, and the
four plutei derived from a single egg may exhibit differences from each
other, but these are comparatively slight. Hence the great abnormalities
among the embryos developing from isolated blastomeres of tetraster
eggs, which will be described immediately, cannot be ascribed to their
cytoplasm, since this does not differ from that in the first four blastomeres

VI DISPERMIC EGGS 165

of monospermic eggs, which, as we have just seen, develop practically
normally. The same applies in principle to the triaster egg.

Boveri found, however, that if the blastomeres of tetraster eggs are
separated, some or all of them develop abnormalities which lead to
their early death, while very rarely, or never, do they all grow into normal
plutei. Moreover, and this is very important, the abnormalities appearing
in the four embryos which are derived from the four separated blastomeres
of a single egg are often of quite different types. Thus, to take one

Fic. 75.

Diagram of distribution of chromosomes in the first cleavage division of dispermic sea-urchin eggs. (Boveri,
Zellen-Studien, 1907.) A, B, C, showing one of the many possible arrangements of the 54 chromo-
somes on the tetraster spindle figure, and the consequent distribution of the 108 daughter chromosomes between
the four blastomeres; D, a possible arrangement of the three sets of chromosomes belonging to the three gametic
nuclei (only 4—designated a, b, c, d—out of the 18 chromosomes of each gamete are shown); E, the four
blastomeres resulting from D, Only the cell in the bottom left-hand corner has a representative of each of the
four types of chromosomes, and therefore it is the only cell that can develop normally.

example from the twenty-one given by Boveri, the four blastomeres of
one dispermic egg gave:

One good gastrula.

One very thick-walled stereoblastula (i.e. blastula ywith blasto-

coele filled with cell masses).

One compact clump of cells.

One heap of isolated cells.
Similar differences were found amongst the larvae developed from isolated
blastomeres of triaster eggs, only here a far greater proportion of them
developed normally. This, it is to be noted, is in agreement with the

166 CY LOLEOGH CHAP.

fact that the first three nuclei of a triaster have a far greater chance of
each getting a representative of all the chromosomes of the series than
have the four nuclei of the tetraster.

Fic. 76.

Development of dispermic Echinoderm eggs. (After Boveri, Zellen-Studien, 1907.) A, blastula of the
tetraster type (Echinus). About one quarter of the wall of the blastula is*falling into separate cells; B, C,
D, plutei from triaster eggs (Strongylocentrotus). B, with a normal skeleton on the left side, rudimentary on
the right ; C, with skeleton present on left side only; D, a perfect pluteus. The dotted lines indicate the
boundaries of the regions derived from the three primary blastomeres. Note the difference in the size of the
nuclei in the different regions.

Boveri also reared tetraster and triaster dispermic eggs as whole
embryos (t.e. without separating the first four or three blastomeres

ee ee ee ee eee eee eee

VI DISPERMIC EGGS 167

respectively) and found that these larvae frequently or generally showed
various and different abnormalities in one or more quarters (or thirds) of
their bodies. That is to say, the regions of the larval bodies derived from
the first four (or three) blastomeres were different from each other (Fig. 76).

Finally, Boveri found that the quarters or thirds derived from different
blastomeres often had nuclei of different sizes. Now, as Boveri has shown,
in Echinoid larvae the surface of the nucleus is proportional to the
number of its chromosomes. Hence the different sizes of the nuclei in
various regions of the body give additional evidence that the blastomeres
from which they were derived differed as to the number of their chromo-
somes. To give specific examples: he calculated the number of chromo-
somes present in the nuclei of four larvae derived from whole triaster
embryos and found that the numbers in the three thirds derived from
the three primary blastomeres were as follows in the different larvae :
(8 28730,54 (2) 218,45; 45 3. (3)'29, 43,30 5 (4) 28,40,,40. Pig. 76, D;
shows the first of these larvae.

Now, as we have seen, cytoplasmic differences between the first four
or three blastomeres of tetraster and triaster eggs cannot account for
the varying manner in which the primary blastomeres develop. It
follows therefore that we must look to the nuclei for the cause of this
variation. A moment’s consideration shows us that it is not the nwmber of
the chromosomes that is of primary importance, since we have abundant
proof that, at any rate, ”, 2, 3m or 4n chromosomes are perfectly
compatible with a normal organism (see Chapter V., etc.). It is only very
rarely that even one, and still more rarely that two, of the primary
blastomeres of a tetraster or of a triaster will have less than m chromo-
somes. Moreover, the four triaster larvae, the number of whose chromo-
somes are given in the preceding paragraph, were normal in all their
parts, although the number of chromosomes was so varied. We are
left with the conclusion that the cause of the abnormalities is the lack
of certain members of the chromosome series in certain of the primary
blastomere nuclei (cf. Fig. 75, E). The fact that the abnormalities which
arise in development are not identical in all the blastomeres of a single
egg, is due to the fact that different members of the series are lacking
in the different blastomeres. This, then, is direct experimental evidence
of the functional differentiation of the chromosomes.

C. ‘FHE: PROCESS OF MEIOSIS

If we take it that the hereditary substance is composed of smaller
differentiated elements, each with its own particular function, it is
plain that some arrangement must be sought by which a doubling of
the number of these elements in each successive act of syngamy is

168 CYTOLOGY CHAP.

avoided. Moreover, since the elements are differentiated, a mere mass
reduction—as, for instance, by diminished growth of the chromatin
between mitoses—would not meet the case. Weismann indeed long ago
postulated from theoretical considerations the occurrence of a reduction
such as takes place in gametogenesis. The process of meiosis has now
been universally recognized as peculiarly adapted to bring about the
qualitative reduction required. This is especially the case since the dis-
covery that, of the two chromosomes which unite to form each bivalent,
the one is derived from the male and the other from the female parent.
The consequences of this for the processes of heredity are discussed under
the next section (D). The existence of meiosis, and the entire absence of
any discoverable analogous process for the cytoplasm, is such an obvious
argument in favour of the view that the whole of the idioplasm is con-
tained in the nucleus, that it is unnecessary to enlarge further upon it.

D. THE PARALLEL WHICH EXISTS BETWEEN CHROMOSOME
BEHAVIOUR AND THE RESULTS OF BREEDING EX-
PERIMENTS.

The results of Mendelian work find a ready-interpretation in the dis-
coveries of cytologists, and this forms one of the most fascinating chapters
of Biology, which, however, can only be treated in brief fashion here.

The fundamental step in establishing the parallel—to call it for the
moment by no more committal name—between breeding experiments
and cytological observations was taken by Sutton (1903), when he
pointed out that the nucleus contains a double series of differentiated
chromosomes, one series contributed from each parent, and that in
syndesis the chromosomes from the one parent pair with the homo-
logous chromosomes of the other.

If we consider the simple Mendelian case, involving one character
only—say the feather colour in certain fowls (A =black, a=‘‘ splashed
white ’’; see Bateson, Saunders and Punnett on the Andalusian fowl)—
the results of crossing the two forms may be represented in the usual way:

Zygotes AA tx ae P
black white
Hess Oa
Gametes a ee | Fie I
Zygote Aa F,
grey

| Sale

Zygotes 1 AA black + 2 Aagrey + 1 aa white F,

vi MENDEL’S LAW 169

The F, and F, represent what is actually found in the breeding-
pen. To bring the notation into conformity with cytological observation
we must suppose that the colour of the feather is dependent on a
“ factor ’’ or gene residing in a chromosome, and we may call this chromo-
some A when the factor is present in the black-producing form, and a
when it is present in the white-producing condition.

In the case of the original pure (homozygous) black parent, both of
the homologous chromosomes are present in the A form, and in the white
parent in the a form. Hence the gametes of the two birds contain one
A or one @ chromosome respectively, and the zygote contains both A
and a, 1.e. it is a hybrid or heterozygote. When this hybrid comes to
form its gametes, A and a being homologous chromosomes, pair together
in syndesis, A going to one spermatocyte II. (or oocyte II.) and a going
to the other (or Ist polar body). Thus the segregation of the hereditary
factors, and hence of the characteristics which they represent, is brought
about. In the second division of the meiotic phase each of the chromo-
somes of course divides longitudinally, and therefore gametogenesis
has proceeded (having regard to this pair of homologous chromosomes
only) according to the following scheme :

A+a — somatic cell or germ track.
Aa — _ syndesis.
foe
a a — spermatocyte II., oocyte II., and polar body I.
Ans 7 t d polar bodi
vin aa __ spermatozoon, or mature egg and polar bodies
I. and II.

In the male, therefore, we have obviously an equal number of
spermatozoa carrying the A and the a chromosomes respectively, and
as in the female it is a matter of chance which chromosome stays in the
egg and which goes into the polar body, we have, on an average,
approximate equality of the two kinds of female gametes also. If now
two of these hybrids are mated together, we get. the following
possible combinations of .gametes, all of which will occur, on the
average, in equal numbers :

A @ may be fertilized by A g¢ =zygote AA —black.

A ? ” ”? a ref = ” aA |

a9 h ' Ae, ie 2 greys.
a : ” ” a 5 = 23 aa —white.

If two or more independently inheritable characters are under
consideration we can again describe, by an interchangeable notation,

170 CYTOLOGY CHAP.

the results of breeding and the distribution of the factors in gameto-
genesis, on the assumption that these are located in different chromosomes.
Thus supposing we are dealing with two characters—say colour (green
or yellow) and surface (smooth or wrinkled) of the pea cotyledon—we
may call the chromosome in which resides the colour factor by the
first letter of the alphabet A or a in accordance with whether the factor
is present in its yellow (A) or green (a) producing form, and similarly
we may call the chromosome which contains the surface factor B
(smooth) or 6 (wrinkled). Regarding these two chromosomes only, the
pure parent plants, say the one green and smooth, the other yellow
and wrinkled, possess chromosomes aaBB and AAbb respectively, thcir
gametes being of course aB and Ad, and the hybrid offspring AaBb.

Now in the meiosis of this hybrid, chromosomes A-a-pair together in
syndesis and likewise B-b, and at the reduction division A and a go
one to each spermatocyte II. and B and b behave similarly. As now
it is a matter of chance whether B goes into the same secondary
spermatocyte as A or as a, there are formed four different classes of
spermatocytes II. and therefore of gametes, namely, AB, Ab, aB and ab.
These gametes, uniting at random when the hybrids are bred together,
combine to give the same classes of zygotes, and in the same pro-
portion as the various classes of individuals found in F, in the actual
breeding experiment.

Since this is a treatise on cytology and not on heredity, it would be
out of place to give any account of the immense number of character-
istics, both in animals and in plants, which have now been found to
be inherited in accordance with Mendel’s Law. One or two interesting
side lines of evidence as to the correspondence between the distribution
of characteristics in heredity and of chromosomes in gametogenesis,
may, however, be mentioned.

(1) The Genetics of a Tetraploid Plant

Gregory (1914) obtained two tetraploid individuals of Primula
simensts (of independent origin), and as they proved to be heterozygous
for certain characteristics, he was able to carry out breeding experiments
with them. As we have just seen, if we fix our attention on a single
characteristic, represented in the idioplasm by a factor which we may
call A, or a, according to the form in which it is present, the formula
for a pure homozygous individual is AA or aa, and for the heterozygote
Aa. Ina tetraploid individual, however, if its quadruple set of chromo-
somes means a quadruple set of factors, the formulae for the pure forms
will be AAAA and aaaa, while there are three kinds of heterozygotes
possible, namely, AA Aa, AAaa and Aaaa.

VI CHROMOSOMES AND SEGREGATION 171

The zygote AAAa produces gametes AA and Aa, and therefore if
bred with its like will produce among its offspring no pure aaaa.

The zygote AAaa produces four kinds of gametes, AA, Aa, Aa and
aa, and therefore will produce one pure aaaa among every sixteen off-
® spring, or I: 15. 7

The zygote Aaaa produces gametes Aa and aa, and therefore we will
find one aaaa among every four offspring, or I : 3.

Similarly, if crossed with a pure aaaa plant (producing gametes aa)
the first kind of heterozygote (AAAa) will produce no aaaa among its
offspring, the second kind (A Aaa) will produce one to every three others,
and the third (Aaaa) equal numbers of aaaa and Aaaa.

The last two of the three types of heterozygotes described above can

be identified among the tetraploid Primulas. Thus, taking the charac-
teristics green style (dominant) and red style (recessive), one heterozygous
plant, self-fertilized, gave forty-four green and two red, i.e. approxi-
mately 15:1. It was therefore an AAaa plant. Ten others gave ninety-
nine green and thirty-four red, i.e. 3:1. They were therefore Aaaa
plants.
Again, taking the characteristics short style (dominant) and long
style (recessive), one heterozygous short styled plant crossed with a
long styled plant (7.e. pure recessive, aaaa) gave thirty-seven short and
fifteen long, or approximately 3:1; i.e. this heterozygote was A Aaa.
Another plant gave, with the same cross, forty-nine short and forty-seven
long—approximate equality, so that this plant was Aaaa.

Thus the quadruple set of chromosomes is shown to be associated with
* a quadruple set of hereditary factors.

(2) Segregation and Parthenogenesis

If the segregation of characteristics which is found in inheritance is
due to the separation of homologous chromosomes in gametogenesis,
it is clear that there should be no such segregation in obligatory
parthenogenetic reproduction, since all the offspring contain the same
chromosomes as their parent. Many species of the plant genus Hieracium
produce some egg cells in which meiosis takes place (and therefore,
having the reduced number of chromosomes, are capable of fertilization),
and also others in which reduction does not take place and which develop
parthenogenetically. In many species the latter egg cells are much more
numerous and hence reproduction is mainly parthenogenetic; the
occurrence, however, of a certain number of haploid egg cells permits the
possibility of sexual reproduction and hence allows of crossing between
different species. It is found that the hybrids so formed are constant
under parthenogenetic reproduction—that is to say, the parental char-

172 CYTOLOGY CHAP.

acteristics do not segregate among their offspring, these all resembling
their hybrid parent (Ostenfeld, 1904, and Rosenberg, 1907). Indirect
evidence of the absence of segregation in parthenogenesis has also been
obtained in the case of the Cladoceran Simocephalus (Agar, 1914). A
“ population ’”’ of females hatched from fertilized eggs was allowed to
reproduce itself parthenogenetically. The females included representa-
tives of numerous size-types, and having been collected from the same
locality must have included many heterozygotes between the various
types. Now, if segregation were taking place, the resemblance between
parent and offspring, as measured by the correlation co-efficient, must
get more and more perfect generation after generation in parthenogenesis,
since heterozygotes could split into homozygotes, but, in the absence
of syngamy, these could not recombine into heterozygotes. Thus the
original mixed population must get more and more nearly homozygous,
and the correlation between parent and offspring must consequently
rise from generation to generation. This correlation co-efficient was
measured for the first five parthenogenetic generations from the fertilized
eggs and was found to remain practically constant—at any rate it showed
no sign of increasing. Hence we conclude that no segregation was taking
place.

(3) Segregation and Bud-V ariation

A few cases have been described where segregation appears to take
place in vegetative (somatic) growth in plants. The most famous case
is the laburnum Cytisus adami, which is a hybrid between the purple
C. purpureus and the yellow C. laburnum. C. adami has dingy red flowers
which are sterile. It occasionally, however, gives rise to pure, or almost
pure, branches or single flowers of C. laburnum and C. purpureus. The
flowers on these branches are fertile, and give rise to C. laburnum and
C. purpureus plants respectively (accounts differ as to whether these
plants are quite pure or show traces of the other species).

Many cases have been described where a dominant plant hybrid
has produced, by bud-variation, branches or flowers with the character-
istics of the recessive parent. Before these can be put down as examples
of vegetative segregation, however, two possibilities have to be taken
into account. Firstly, do the recessive flowers breed true to the recessive
character ? Otherwise the appearance of the recessive character in one
part of the plant may be due to some somatic condition preventing the
dominance of the normally dominant characteristic—and many cases
are known where dominance of one or other characteristic in a hybrid
is affected by somatic conditions (for examples, see Cramer, 1907).
Secondly, even if the recessive bud-variations do breed true to the
recessive character, showing that they no longer contain the dominant

vI SEGREGATION IN ASEXUAL REPRODUCTION 573

factor, we must always reckon with the possibility that mutation, and
not segregation, has taken place, as in the case of Mirabilis (p. 182).
Suppose a plant with blue (dominant) flowers is crossed with a white
variety (recessive) of the same species, the hybrid then contains, according
to hypothesis, a pair of homologous chromosomes, one containing the
colour factor in its blue condition, which we may call A, the other one
the same factor in its white state (a). Now in many instances we know
that a was originally derived by mutation from A, and there is no reason
to suppose that this may not happen again, leaving the heterozygote
with two a chromosomes, ?7.e. pure white.

While many cases of supposed vegetative segregation may probably
be explained in this way, there can be little doubt that true vegetative
segregation does take place as a very rare occurrence.

A case which can hardly be explained otherwise has been described
by Bateson and Pellew (1915). Many varieties of peas (Pisum) produce
a small percentage of “ rogues,’’ or plants with a somewhat vetch-like
habit. The genetic behaviour of heterozygotes between rogues and
typical plants is remarkable. As young plants they usually differ very
little from the type form, but as they grow older the rogue characters
appear in their upper parts, and as adults they are always pure rogues.
Moreover, though of heterozygous origin, they produce, when self-
fertilized, exclusively rogue offspring. These exhibit the rogue characters
even as young plants.

Thus, as the above-mentioned authors point out, the normal and
rogue characteristics of the heterozygote seem to separate during the
growth of the plant, the normal characteristics being left behind in
the older or lower parts of the plant, leaving purely rogue characteristics
in the upper parts, and therefore also in the gametes.

What the underlying cytological conditions of vegetative segregation
may be we do not know, but it may be fairly confidently conjectured
that something analogous to the separation of homologous chromosomes
in meiosis is concerned in it.

Summing up, we see that, except as extremely rare exceptions of
which nothing is known as to their cytology, segregation in heterozygotes
does not take place unless meiosis occurs, and that when meiosis does
occur segregation does take place, thus adding direct experimental evidence
to the other considerations which lead us to suppose that the separation
of homologous chromosomes in meiosis is the cause of segregation.

It is not to be expected that the distinction between the members of
a pair of homologous chromosomes which differ in regard to one or even
more of their factors should be visible in all cases under the microscope
—though we have seen some examples of visible differences between

174 | CYTOLOGY CHAP.

homologous chromosomes in the last chapter, and it is a plausible
hypothesis that these differences are due to their different factorial
composition.

(4) The Interchange of Hereditary Factors between Homologous —
Chromosomes

The simple proposition that the characteristics of organisms are
represented by factors in the chromosomes and that their distribution,
according to the classical Mendelian scheme, depends upon the movements
of these chromosomes in meiosis, requires some elaboration, however.
For if each independently inheritable character resided in a separate
chromosome, it is clear that there could only be as many such characters
as there are chromosomes in the gamete. By separately inheritable
characters we mean those which can be separated from each other and
made to enter into fresh combinations by crossing. To put it in another
way, all the characteristics of an organism should be capable of correla-
tion into the same number of groups as there are chromosomes in the
gamete, each group of characters behaving in heredity as a unit.

Now organisms present far more characters which are separately
inheritable than they have chromosomes. This has been shown definitely
for some organisms, and there can be little doubt that it is the general
rule. Thus in the fruit-fly Drosophila ampelophila, which has been the
subject of such exhaustive study in America, more than a hundred
separately inheritable characters are known, though the number of
chromosomes (haploid) is only four.

The hypothesis that hereditary factors are located in the chromosomes
has therefore to be supplemented by the supposition that there are
many factors in each chromosome, each located in a definite part of the
chromosome (t.e. represented by a definite unit in the structure of the
chromosome) and that exchange of corresponding factors may take
place between homologous chromosomes. It is natural to suggest the
chromomeres as the seat of the separate factors, and syndesis as the
moment at which exchange takes place. By chromomeres, in this case,
must be understood the numerous small bead-like bodies often observable
in syndesis, not the few, much larger swellings on the chromosomes,
sometimes found in late prophase. According to this view, a chromo-
some is to be considered as containing a linear series of factors ABCDE

. and two chromosomes in syndesis can be represented thus :

ABCDE. = 2s.
abcde

1 Although even in syndesis the chromomeres are probably often already compounded
of several smaller units.

v1 FUNCTION OF SYNDESIS

During their apposition exchange of corresponding factors takes ;
and the chromosomes after separation may be constituted in varic
ways, ¢.g., AbCdE and aBcDe ; or abCDe and ABcdE, etc., etc.

By this means the Mendelian inheritance of any number of separate
characters can be accounted for.

The necessity of assuming the interchangeability between homologous
chromosomes of the chromosome components makes it highly desirable
to determine whether parasyndesis is or is not of general occurrence.
Parasyndesis obviously
offers a favourable op-
portunity for the mutual
exchange between con-
jugating chromosomes
of their elements which,
as we have seen, are
arranged in linear series
—and indeed at this
stage the corresponding
chromomeres of the two
chromosomes are often
most regularly and con-
spicuously in close ap-
position (Fig. 77). The
evidence for parasyn-
desis was discussed in
Chapter IE, and” its
general occurrence

provisionally accepted. Fic. 77.
We have, moreover, Examples of the correspondence between the chromomeres in homo-
logous chromosomes during syndesis. A, Spinax niger & ; B, Myxine

pe aoe ee ein caren ee ee
ing that the function of

syndesis is not merely that of bringing the members of homologous pairs
into apposition so as to effect their sorting out into different daughter
nuclei at meiosis. For syndesis often takes place months or years
(mammalian oocytes) before the reduction division, and is frequently
followed by complete separation of the ex-syndetic chromosomes (many
oogeneses, spermatogenesis of Lepidosiren, etc.). Between syndesis and
metaphase I. the chromosomes may even undergo metamorphoses
as great as those undergone in the resting nucleus (most cases
of oogenesis). The separated homologous chromosomes then pair
again in prophase I. immediately before the metaphase (oogenesis,
Lepidosiven spermatogenesis). This second pairing, which is not of the

CYTOLOGY CHAP.

ate nature of the syndesis proper, has obviously the function of
aging the homologous chromosomes in pairs on to the meiotic spindle.
this being effectively achieved by this late prophase pairing, we are
compelled to look for another function for syndesis in these cases, and
this function, as indicated above, we believe to be the exchange of
hereditary factors.

In what way may we conceive that this hypothetical interchange of
factors is effected ? Two possibilities have been suggested—the first
is that which naturally presents itself, namely, that while the chromo-
somes are longitudinally apposed to one another in parasyndesis, an
exchange of chromatin units takes place analogous to the exchange of
nuclei between two conjugating Infusoria. The other suggestion was first
made by Janssens (1909) (chiasma-
typie) on purely cytological grounds.
Since the homologous chromosomes
separating after syndesis are often
spirally twisted round one another
(strepsitene stage), he suggested that
fusion may occur at the points of
junction, and that when these break
apart at final separation the chromo-
somes may be composed of alternate
iA B C segments of the original chromosomes
(Fig. 78).
: ° ; A view similar to Janssens’ has
Diagram illustrating the hypothesis of “ chias-

ciniinmomes belige ayunese,) <0) dhigiess Coro SUPPorted, Dy Morgan) (ctey 2

(= strepsitene) stage ; C, the chromosomes fully forming a cytological foundation for

separated after syndesis. _ . i
certain breeding results with Dyroso-

phila and other forms. It is known as the “‘ crossing over ’’ hypothesis,

and is supported by the following experimental evidence.

A number of cases are now known in which characters which can
be separated in crossing, nevertheless show a preference to remain in
the combinations in which they were present in the parents, rather than
to become rearranged in different combinations. Thus if two forms,
differing as regards two characters, are crossed, say AB x ab, the F, hybrid
will of course form four classes of gametes AB, Ab, aB, and ab. In most
cases these classes are formed in equal numbers, but in some cases the
gametes representing the parental combinations are found in excess of
the others. In the cross AB x ab we have an excess of the gametes AB
and ab, and a deficiency of the Ab and aB classes. On the other hand,
the cross Ab x aB gives an excess of Ab and aB and a deficiency of AB
and ab. It must not be supposed that this is a mere exception to the

Fic. 78.

vt CROSSING OVER 177 -

general Mendelian scheme and the theory of its cytological basis, and
therefore invalidates their generality. On the contrary, the departure
from the ordinary equality of different classes of gametes is quite orderly
and, as we shall see, inte'ligible. Thus if 4% is the total number of gametes
produced we find that they occur in the following proportions :

In the first case (AB x ab) the gametes are:
(x+y) AB+(x-y) Ab+(x-y) aB+(x% +y) ab.

And in the second case (Ab x aB):
(x-y) AB+(x+y) Ab+(x+y) aB+(x—-y) ab.

This will be apparent from the following experiment with the sweet
pea (Lathyrus) described by Bateson and Punnett (1911) and by Punnett
(1913) 1: the characters concerned are the colour of the flower, blue (B)
and red (4), and the shape of the pollen grain, long (L) or round (J).
Blue is dominant over red and long pollen is dominant over round.
Now the F from the cross BL x b/ is different from the F, from the cross
Bl x bL, as the following parallel experiments show :

Generations :
Blue, long x red, round P Blue, round x red, long
|
Blue, long FB, Blue, long
| | :
| | | eee | | |

3915. | 312 294 1079 226 95 97 I
Blue, blue, red, red, Blue, blue, red, red,
long round long round long round long round

The F, is of course the same in both cases (blue, long), since blue is
dominant over red, and long over round. The number to be expected
in F,, if it were a simple Mendelian case with no connection between
colour and pollen shape, would be the same for both experiments, and
would be in the proportion g blue, long : 3 blue, round : 3 red, long :
1 red, round. These proportions are what would be obtained supposing
the four kinds of gametes BL, Bl, bL and 01 were produced in equal
numbers. The actual F,’s obtained? show that this cannot have been
the case in this instance. Supposing, however, that the gametes had been
produced in the first experiment (blue, long x red, round) in the pro-

portion
Bie bbe Tl, 4 7308,

1 The hypothesis of reduplication advanced by these authors in explanation is funda-

mentally different from Morgan’s view of ‘‘ crossing over,’’ which is here adopted. :
* Included in F, are a number of F, families descended from heterozygous I, plants.

N

178 CYTOLOGY CHAP.

this would give a proportion in F, of
177 blue, long ; 15 blue, round ; 15 red, long ; and 49 red, round ;
or, out of the total of 5600 in the above F,, the numbers :
3871 blue, long ; 328 blue, round ; 328 red, long ; and 1073 red, round.

This we see is a very close approximation to the numbers obtained in
the experiment, and it is to be noted that the gametes produced in excess
are those which exhibited the same combinations of characters as the
original parents (BL and 0/). Similarly, if the gametes in the second
experiment were formed in the proportions

EBL a Bl 3 FOL an OF
the numbers which would be given in F, are
210 blue, long ; 103 blue, round ; 103 red, long ; and 1:5 red, round ;

again a sufficiently close approximation to the numbers actually obtained.

Thus, we conclude that the hybrid produces about seven times as
many gametes with the parental combinations of characters as with the
reciprocal combinations.

The explanation of this, on the “ crossing over” hypothesis, is that
the factors for colour of flower and shape of pollen grain lie in the same
chromosome.! To take the first case (BL x bl) : during syndesis in the
hybrid, the BL and the 6/ chromosomes come together, and separate
again in the diplotene stage, during which they become twisted round
one another and liable to exchange segments as described above. There
is, however, a greater chance of the BL and the b/ factors remaining
together than of the combination having been broken up, and conse-
quently of B and / being found in one of the separating chromosomes
and 6 and L in the other; for (1) the chromosomes may have separated
without any crossing over of the particular segments containing the
flower colour and pollen grain shape factors respectively ; or (2) if crossing
over has taken place in this region, a length of chromosome sufficient
to include both factors may have crossed over. It is plainly only when
one of the factors has crossed with its mate, and not when both or neither
have done so, that a recombination of the B and 6 with the L and / will
have taken place. It is also plain that the nearer to each other the two
factors are located in the chromosome, the more likely they are to be
included in a segment behaving as a unit in crossing over—or, in other
words, the less likely is a cross over and rupture of the chromosome to
take place between them. Acting on these considerations Morgan and
his colleagues (1915 b) have mapped out the arrangement of a large number
of factors in the chromosomes of Dyosophila, calculating their relative

‘

1 Such factors are said to be “linked.”

vi CROSSING OVER 179

distances from one another by the relative frequency of crossings over
between them.

In Drosophila, for some unknown reason, crossing over takes place
in the female only, never in the male. This would be explicable if only
the sex-linked characters were concerned (see below), since the factors
for these are presumably carried in the X chromosome, of which the male
has only one (its mate, the Y, being apparently inert). The rule applies
equally, however, to characters which are not sex-linked and of which
the factors must therefore be carried in other chromosomes. Thus the
characters for body colour (grey, dominant over black) and shape of
wing (long, dominant over vestigial) behave in the male as an inseparable
couple, all the gametes of a male hybrid between a grey, long and a black,
vestigial, being either grey, long or black, vestigial. The correspond-
ing female hybrid, however, gives indeed a majority of grey, long and
black, vestigial gametes, but also a small percentage of grey, vestigial,
and black, long. Conversely, a male hybrid between a grey, vestigial,
and a black, long, gives only gametes grey, vestigial, and black, long,
while the female hybrid gives a majority of these, with, however, a small
percentage of grey, long and black, vestigial. It appears therefore that
in Drosophila exchange of substance between homologous chromosomes
occurs in syndesis of the female but not in that of the male; a fact
which seems to indicate that the full explanation is not given by the
hypothesis of crossing over in its simplest form.

Tanaka (1915) has described a case among silkworms where the
reverse condition obtains, crossing over occurring in the gametogenesis
of the male hybrid but not in that of the female.

The above brief summary of the points of contact between the
observations of cytologists on one hand, and the results of the Mendelian
method of studying heredity on the other, must suffice, for to follow it
up by multiplication of detail and instances would lead us beyond the
subject-matter of this book. Moreover, it is not necessary, as the subject
has quite recently been treated in special publications—e.g. Doncaster,
The Determination of Sex, 1914a; and Morgan, Sturtevant, Muller and
Bridges, The Mechanism of Mendelian Heredity, 1915—to which the
reader desirous of further information is referred. These works also
deal with the problem of sex-linked (or sex-limited) inheritance (the
connection between the sex chromosomes and the determination of
sex has already been dealt with in Chapter IV.). This term covers those
cases in which certain characters are transmitted only by gametes bearing
the sex chromosome ; e.g. in Drosophila certain characters such as red
eye can be transmitted by any egg, but only by the female-producing
spermatozoa. Similar cases are known in the cat and in man (e.g. colour

180 CYTOLOGY ; CHAP.

blindness and haemophilia). It is an obvious hypothesis that the reason
for this is that the factors of the characters in question are located in
the X chromosome. As the female has two X chromosomes, and as conse-
quently all her egg cells have an X chromosome, therefore any of them
are in a position to transmit this character. Since, however, half the
spermatozoa lack the X chromosome altogether, or possess in its place
the Y chromosome (which we must suppose to be inert) only half of
them, that is to say the X-bearing (female-producing) spermatozoa, can
transmit the character. Thus in Dvosophila a red-eyed female can
transmit this character to any of her offspring, but a red-eyed male only
to his daughters, 7.e. through the X-bearing spermatozoa.

In other cases (Lepidoptera, Birds) it is the male which transmits
certain characters to all his offspring (7.e. through all gametes), and the
female only to her sons (7.e. through male-producing gametes only), thus
indicating that the chromosome formula for male and female is reversed,
male being XX and female XY (or X—). This again corresponds with
the somewhat scanty cytological observations on these two groups.

For further information on the subject of sex-linked inheritance,
which furnishes one of the strongest pieces of evidence in favour of the
chromosomes (chromomeres) being the seat of the Mendelian factors,
the reader is referred to the above-mentioned works, where he will also
find reference to certain exceptions and the supplementary hypotheses
which have been put forward to account for them.

(5) The Cytological Basis of Mutation

If morphogenesis and heredity have their physical basis in the nucleus,
heritable variation must be due to changes in one or more of the chromo-
somes, or, rather, of their constituents. This of course is, in the present
state of our knowledge, impossible to prove directly, but certain relative
evidence can be obtained from the study of the origin of variation, or
mutation.

If a hereditary factor undergoes a mutational change, whether in
gametogenesis or at some other time, the first individual to possess this
altered factor in its diploid nuclei will presumably most often possess
one chromosome containing the factor in its new state, and its homologue
possessing it in its old state. A moment’s consideration will make this
clear. If the variation has taken place in gametogenesis, the mutated
gamete will almost necessarily have to fertilize a non-mutated gamete
from another organism, for its chances of meeting another gamete which
has undergone a similar mutation will in most cases be negligible, and
hence the first individual to possess the new factor will be heterozygous
between it and the old form. If on the other hand the mutation takes

vI MUTATION 181

place in the fertilized (diploid) egg, the individual will again be in the
heterozygous condition unless both members of the homologous pair
concerned have undergone the same mutation.

A number of cases of mutants appearing in the first instance in the
heterozygous form are on record.

Nilsson-Ehle (1911) described such a case in oats. He found in
pedigree cultures of this cereal a small proportion (one in ten to twelve
thousand) of plants exhibiting certain atavistic features in respect to
the awns and the hairiness of the flower base. When these atavists
were self-fertilized they gave offspring of three classes, viz. normals,
atavists like themselves, and more pronounced atavists, in the
proportion of 1:2:1. Obviously therefore the original semi-atavists,
as we may call them, were heterozygotes between the normal and
fully atavistic forms—that is to say, they were produced by syngamy
of a mutated and a non-mutated gamete.

Gates (1914) has shown that in the case of Oenothera rubricalyx, which
arose as a mutation of an O. rubrinervis, the first rubricalyx individual
was a heterozygote between rubricalyx and rubrinervis, the new char-
acter rubricalyx being in this case dominant. Its heterozygous nature
was therefore not obvious from its external characters, but was only
disclosed by breeding.

There is one great obstacle in the way of discovering the mode of
origin of mutations, and that is that a large number, probably the great
majority of them, are partially or completely recessive to the type condi-
tion, and therefore the heterozygotes are indistinguishable from the type
form. In these cases, since it is only the homozygous recessives which
exhibit the new character, this will make its first external appearance
in the homozygous condition. As moreover the new factor may have
been in existence a considerable time, and may have become widely
distributed, but invisible, owing to being always concealed in individuals
possessing also the dominant type character, these recessive mutants
are liable to appear suddenly in relatively large numbers, when sufficient
heterozygotes have accumulated in the population to allow of the meeting
between two mutated gametes to take place fairly frequently.

Also it must be remembered that mutation, striking enough to be
recognized and investigated in the first animal or plant exhibiting it,
is rare.

It must not be supposed, however, that mutation can only take place
in the germ-cells, nor is there any theoretical reason for supposing that
it should, like segregation, be connected with meiosis, except in that
limited class of mutations due to irregular distribution of the chromo-
somes or their constituents in the reduction division.

An example of a mutation which almost certainly did not originate

182 CYTOLOGY CHAP.

during gametogenesis is afforded by the origin of the peculiarly malformed
“cretin’’? sweet pea, described by Bateson and Punnet (1911) and
Punnet (1919). The malformation concerned is recessive to the normal
condition, and therefore only manifests itself in homozygous individuals.
The “cretin’’ arose in a pedigree culture, and was the only one of its
kind among a large number of direct and collateral ascendants and
descendants (excluding, of course, its own offspring). Had the mutation
occurred during gametogenesis two possibilities are open: (I) it might
have occurred during the gametogenesis of the grandparent, so that the
immediate parent of the cretin was heterozygous, though normal in
external appearance. In this case, however, it should have produced
one cretin among every four of its offspring, whereas it actually produced
only the one cretin and 51 normals. (2) The mutation may have occurred
during the gametogenesis of the immediate parent. If this were the
case, more than one gamete must have been similarly affected, since
the cretin itself, being homozygous, must have been produced by syngamy
of two such affected gametes. As, however, the parent plant produced
only one cretin among a large number of normals (the latter again pro-
ducing only normal offspring) it is plain that the number of mutated
gametes produced must in any case have been very small, and the chances
against two of them having united to form the cretin very great. The
evidence seems to indicate therefore that the mutation occurred in the
zygote cell, and affected both members of the homologous pair of chromo-
somes concerned.

A well-known class of mutations occurring in somatic cells constitutes
the phenomenon of bud-variation in plants. Innumerable examples of
this could be quoted, but very few of them have been thoroughly investi-
gated. As an example of one which has been more fully worked out,
we may take a case described by Correns (1910) in Mirabilis. It was
found that plants with variegated foliage occasionally gave rise to
branches with pure green leaves. Seeds from flowers on these green-
leaved branches yielded 25 per cent variegated plants and 75 per cent
green. On further breeding, twenty-five of these 75 per cent of green
plants gave only green plants, and the remaining fifty gave again 25 per
cent variegated and 75 per cent green offspring. Thus it is plain that
the green branches appearing as bud-variations on the variegated plants
were heterozygous between variegated and green, green being dominant,
and produced the usual proportion of offspring for this type of hetero-
zygote, namely, 3 dominant : I recessive.

In terms of cytology, we must suppose that the factor for chlorophyll
distribution exists in two forms—one to produce a uniform distribution of
chlorophyll, giving green plants (G), and the other giving a patchwork
distribution, resulting in variegated plants (g). In the variegated plants

vl HYBRIDS 183

both of the homologous chromosomes bearing this factor have it in the
gform. Sometimes, however, one of these, by mutation, changes into the
G condition, and since G is dominant over g, all future somatic descendants
of this cell will be green; thus we find green branches occasionally
appearing on the variegated plants. These branches are now in exactly
the same condition, cytologically, as if they belonged to a hybrid between
a green and a variegated plant, with the result that the ordinary Mendelian
proportions which are to be expected among their offspring are realized.

EH. oLERILE, AND PAR TIAEBY STERILE; HYBRIDS

We can provisionally distinguish two types of these hybrids: (1)
where the disturbance in meiosis seems to be mainly of a mechanical
nature, depending upon a numerical, rather than a physiological dis-
crepancy in the chromosomes of the two parents; (2) where the
disturbance is mainly physiological We must remember that this
distinction, even if justifiable, is by no means sharp, the two types
overlapping and grading into each other.

As an example of the first of these two types of crosses, we may
take the cross between the two species of sun-dew Drosera longifolia
and D. rotundifolia (Rosenberg, 1909). This cross results in a hybrid
intermediate in character between the parent species, and known as
D. obovata. In D. rotundifolia 2n is 20 and in D. longifolia it is 40, the
latter species being probably a tetraploid form. In D. obovata the somatic
number is 30.

In syndesis of the hybrid, Rosenberg found ten bivalents and ten
univalents—that is to say, syndesis had taken place between the ten
votundifolla chromosomes and one of the two sets of their homologues
from the tetraploid longifolia, leaving the other set of ten unpaired.
In anaphase the constituents of the bivalents were regularly, but the
univalents irregularly, distributed to the daughter nuclei, which therefore
sometimes received very unequal numbers of chromosomes ; for example,
18 and 12. Fertile pollen grains were seldom or never formed, but fertile
ova fairly frequently.

Probably a closely analogous case is afforded by the crosses between
Oenothera gigas and other Oenotheras, which may be considered here
though they do not necessarily result in any notable degree of sterility.

We have already (p. 149) seen reason to believe that O. gigas is, like
D. longifolia, tetraploid ; its somatic chromosome number is 28. It
therefore becomes of interest to see how this form behaves in crosses
with the diploid forms (2n=14). The cross most studied is O. lata! x O.

1 The fact that this Oenothera has £5 instead of 14 somatic chromosomes (p- 146) need not
concern us here.

184 CYTOLOGY CHAP.

gigas. In the male meiosis of this hybrid, which has 21 chromosomes
(14+7), according to Geerts (I9II), we get 7 bivalents and 7 univalents ;
i.e. of the triple set of chromosomes, two sets of homologues have paired,
leaving the other set free, as is probably also the case in Drosera. Gates,
however (1909 6), thinks it probable that there is no syndesis, and finds
that there are 21 univalents in metaphase I. (in the Oenotheras, the associa-

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FIG. 79.

Side and polar views of the equatorial plate in the meiosis of certain Lepidoptera and their hybrids. (After
Federley, Z.A.V., 1913.) A, B, Pygaera anachoreta, 30 bivalents; C, D, P. curtula, 29 bivalents; E, F, P.
anachoreta x P. curtula, hybrid; in E, 3 chromosomes are bivalent, the rest univalent; in F, 56 or 57 chromo-
somes can be seen, 7.e. two or three are bivalent, the rest univalent. G, H, secondary hybrid obtained by
crossing the first hybrid back with P. anachoreta 9; in G a mixture of univalents and bivalents; in H, 56
chromosomes can be counted ; namely, about 30 large bivalents (the anachoreta chromosomes), and about

26 small univalents (the curtula chromosomes). ;
tion between the constituents of the bivalents in the meiotic division
is characteristically very loose). According to the latter authority these
21 chromosomes are separated at anaphase into groups of ro and 11,
rarely 9 and 12. According to Geerts the constituents of the bivalents
separate normally, sending 7 to each pole, while the remaining 7 uni-
valents suffer various fates—some being irregularly distributed to the

VI HYBRIDS 185

daughter nuclei and some being left behind and failing to enter either
nucleus.

The nature of the progeny obtained by breeding from this hybrid
has already been described (p. 146).

Somewhat intermediate between the two types which we have
provisionally distinguished as mechanical and physiological, stand
probably the Lepidopteran crosses examined by Federley (1913) and by
Harrison and Doncaster (1914).

Federley’s crosses (Fig. -79) were between Pygaera curtula (2n =58)
and P. anachoreta (2n=60). The hybrid has about 29 + 30 =59 chromo-
somes. In meiosis of the male hybrid very little syndesis takes place,
only two or three chromosomes being paired, so that there results a few
bivalents while the remainder are univalent. The former separate into
their constituents in the usual way, while the others divide as in a
somatic mitosis. Thus each gamete has the diploid number of chromo-
somes, except for those few which paired at syndesis, in respect of which
it is haploid.

This hybrid was crossed back with anachoreta 9. The number of
chromosomes to be expected in this secondary hybrid is 59 +30 =809 ; i.e.
a double set of anachoreta and a single set of curtula chromosomes—or
nearly so, as the hybrid gametes have not quite the full 59 chromosomes.
This of course is a very high number to count satisfactorily, but in
several nuclei over 70 were counted. In meiosis of this secondary hybrid
we find a mixture of bivalents and univalents, leading to the presumption
that the anachoreta chromosomes introduced by the mother have paired
with those of the same species introduced by the hybrid gamete, while
the curiula chromosomes of the hybrid are left univalent.

Harrison and Doncaster’s cross was between Lycia hirtaria and
Ithysia zonaria, and gave results closely comparable to those of Federley
(Fig. 80). This cross exhibits an advantage over the last one described,
in that the chromosomes of the two parent species are distinguishable
from one another in the hybrid by their relative sizes.

The chromosomes of L. hivtaria (2n =28) consist of 11 pairs of large,
I pair of small, and 2 pairs of very small ones. Those of I. zonaria
(2% =112) are all very small, the largest being no bigger than the smallest
of L. hirtaria.

In the hybrid diploid cells (spermatogonia) the two types of chromo-
somes are easily recognizable, the total number being of course 14 +56 =70.
In the first meiotic division it is found that there can be counted rather
less than 70, but always many more than 35 (varying from about 53 to 63).
It is therefore to be presumed that about a dozen pairs of chromosomes
have entered into syndesis, and that the rest remain unpaired.

These hybrids are completely sterile.

186 CYTOLOGY CHAP.

In the following three cases the meiotic disturbance must be ascribed
to physiological causes, since the degenerative changes that take place
in the nucleus are more profound, even though in some cases the number
of chromosomes in the parent species is the same.

Matings between the magpie pigeon ( ¢) and dove (?) (Smith, 1913)
result in male offspring only. These are found to exhibit a meiosis
differing from the normal in that there is an irregular metaphase I.
and, doubtless in consequence of this, the second meiotic division appears
to be omitted. In any case it was never found, though spermatogenesis
proceeds without it, resulting in the formation of spermatozoa, 77 per cent
of which are about twice the size of those of-either parent. The

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The chromosomes ot certain Lepidoptera and their hybrids. (After Harrison and Doncaster, J.G., 1914.)
All are polar views of equatorial plates. A, B, Lycia hirtaria. A, oogonium showing 28 chromosomes; B,
spermatocyte I., 13 bivalents (one compound). C, D, Ithysiazonaria. C, spermatogonium, about 112 chromo-
somes : D, spermatocyte I., 56 bivalents. E, F, the hybrid, I. zonaria x L. hirtaria. E, spermatogonium, r4 large
(hirtaria), and about 56 small (zonaria) chromosomes; F, spermatocyte I., 63 chromosomes. G, spermato-
cyte I. of the reciprocal hybrid, about 60 chromosomes, showing the two parental types.

experiments indicate that the spermatozoa are not functional, for two
of the hybrid males were paired with female pigeons, which laid and
incubated eggs which, however, proved unfertile.

In the hybrids between different species of pheasant (Smith and
Thomas, 1913), and pheasant and domestic fowl (Cutler, 1g18), no normal
stages can be observed after synizesis. These hybrids are of course
sterile. When they are females the resulting anatomical abnormality
of the ovaries is very marked, since oogenesis ceases at synizesis and
therefore there is an entire lack of yolked oocytes. Thus the ovaries
remain minute and sometimes invisible.

The best known sterile hybrid is of course the mule. The male
meiotic phase of this animal has been worked out by Wodsedalek (1916),

vi HYBRIDS 187

who also examined the spermatogenesis of the horse. That of the ass
has, however, not yet been described. The number of chromosomes
(diploid) in the mule is 51, one being a large unpaired X chromosome.
As 2n in the horse is 36+ X, the ass may be presumed to possess 64 +X
chromosomes. Evidences of physiological disturbance appear early in
the meiotic prophase of the mule, the diplotene stage found in the horse
being replaced by a reticular stage showing only occasional and irregular
duplicity of threads. The majority of cells perish in this stage; in
those that reach the late prophase the number of chromosomes varies
between 34 and 49, the commonest numbers being 40 to 45, i.e. there are
about 5 to ro bivalents, the remainder being univalents. The few cells
that reach metaphase I., or even anaphase I., are marked by numerous
abnormalities, especially multipolar mitoses and the failure of many of
the chromosomes to gain attachment to the spindle fibres. No secondary
spermatocytes or any later stages were ever observed.

F. THE NUCLEUS IN MORPHOGENESIS

All arguments in favour of the nucleus being the bearer of the
hereditary factors are of course equally arguments for its control of
morphogenesis. As to its mode of action in this respect, however, we
are almost wholly ignorant. One thing which does seem certain is that
all the nuclei of the body—at any rate up to a late stage of development
—are identical in their potentialities, 7.e. contain a complete (double)
set of hereditary factors. There are no differential nuclear divisions
in the embryo by which the endoderm factors are sorted out into the
nuclei of the cells which are to form endoderm, mesoderm factors into the
mesoderm nuclei, etc., as originally supposed by Weismann and Roux.

This has been abundantly proved by the pressure experiments of
Driesch (1893), etc., as well as by many other well-established facts of
embryogeny, regeneration, etc. The particular experiments referred to
consisted in making sea-urchin (Echinus) eggs undergo their early
development under pressure between two glass plates. Under such
conditions the eggs may continue to develop as far as the 4th cleavage
(16 cells). Instead of forming a spherical blastula, however, the cells are
all arranged in one plane in a flat plate. On removing the pressure,
the embryo gradually recovers its proper shape and proceeds to normal
development, in spite of the fact that, as can be easily verified, the cells
have a quite different mutual arrangement and contain different parts
of the cytoplasm from those which they have in the normal larva. Thus
a cell which in the normal larva would have given rise to ectoderm, now
gives rise to endoderm, etc.

What is it then that causes the cell differentiation which leads to the

188 CYTOLOGY: CHAP.

formation of the Various tissues and structures of the body ? A discussion
of this question would again lead us far beyond the scope of this book,
and would indeed involve nearly the whole subject of experimental
embryology. Here we can only allude to the question of the so-called
“ organ-forming substances,” referred to on p. 163. Lack of space and
the uselessness of repeating what has already been made the subject
of recent text-books must restrict us to a very brief, and therefore
necessarily dogmatic, summary. The reader is referred for further
information to Korschelt and Heider’s Lehrbuch (1902), or to the smaller
text-book of Jenkinson (1909), where all the essential facts are given, and
where references to the more important original works may be found.

It is found that a single blastomere isolated from an embryo of the
16-cell stage (termed a j'; blastomere) of a sea-urchin will live and develop,
at least as far as gastrulation, as if it were a whole miniature egg. It
is only blastomeres from the lower or vegetative pole of the cleaving
egg, however (t.e. that part of the egg from which gastrulation starts in
a normal, whole embryo), which will gastrulate. Cells from other parts
develop irregularly and do not gastrulate; 4} blastomeres, however,
whichever part of the egg they are taken from, will develop into embryos
which gastrulate. If the egg is divided into its blastomeres at the two-
cell stage, both develop into a perfect normal, though dwarf, pluteus.

This is expressed by saying that the blastomeres of an Echinoid egg
are equipotential as far as the eight-cell stage, then gradually become
inequipotential. The eggs of a large number of animals can be arranged
in a series, according as to how long they retain their totipotentiality,
down to the forms where even the undivided egg is inequipotential in
its various parts.

Thus in the radially symmetrical Ctenophora, if a segment of the
cytoplasm of the undivided egg is removed, the resulting larva lacks the
organs on the radius represented by the removed egg cytoplasm.

The eggs of several Molluscs and Chaetopods exhibit a swelling of
the cytoplasm (after fertilization but before cleavage has begun) termed
the yolk lobe. If this is cut off, the egg will nevertheless develop to a
certain stage, but the resulting larva, though it may become free-
swimming, does not develop any mesoderm.

These and many other experiments have led to the hypothesis of
the presence of ‘‘ organ-forming substances ”’ in the egg cytoplasm exhibit-
ing a stratified arrangement, generally according to their specific gravities.
For instance, ectoderm-forming substance is most concentrated towards
the upper pole of the egg and least concentrated towards the lower
pole, and endoderm-forming substance has the reverse arrangement.
In the cleaving Echinoderm egg, up to and including the eight-cell stage,
there is sufficient of all organ-forming substances in all the blastomeres

VI ORGAN-FORMING SUBSTANCES 189

to allow of them all developing as complete organisms. By the time
that the 16-cell stage is reached, however, successive cleavages have
resulted in the cells in the upper pole having so little endoderm-forming
in proportion to ectoderm-forming substance, that they are now no
longer able to gastrulate, though isolated cells from the lower pole
(which contain the endoderm-forming substance) can still do so—as can
even a z's blastomere, if taken from this pole.

The action of the cytoplasm in determining which cells shall develop
into soma and which into gonad in Ascaris (p. 85) is clearly that of an
“ organ-forming substance.”

It must be granted that the egg cytoplasm contains substances that
are necessary to the formation of various tissues and organs. There is,
however, no reason to suppose that these substances play an active
formative part, or that they are anything other than the conditioning
environment or the releasing stimulus through which the nucleus exerts
its activities. The external environment of the developing egg contains
elements which act in quite as specific a manner as the so-called organ-
forming substances in the egg cytoplasm. Thus if Echinoderm eggs
are made to undergo their development in water identical with that of
sea water, except that it lacks the SO, radicle, the gut of the larva is
not properly formed; if the calcium normally present is absent, the
blastomeres fail to cohere into a blastula, but fall apart, swim away
by means of their cilia, and eventually die without undergoing any
differentiation. Eggs of the Teleostean fish Fundulus heteroclitus,
developing in sea water to which MgCl, has been added, produce
embryos with a single median ‘‘ Cyclopean”’ eye, instead of a pair of
lateral ones (Stockard, Ig0g). It would clearly be possible to speak
of the SO, radicle as a gut-forming substance, of calcium as a blastula-
forming substance, and of MgCl, as a monocularity-producing body,
with as much justification as we call the substances in the egg cyto-
plasm which we have just discussed, organ-forming (rather than organ-
conditioning) substances.

The fact appears to be that all these substances, whether within the
cytoplasm or without, including yolk and vitelline membrane (the
degree of the permeability of which is of vital importance to the embryo),
or the relation of the embryo to the mother in viviparous forms, are all
alike components of the environment of the morphogenetic factors
residing in the nucleus. The fact that certain of them, such as the
vitelline membrane, the fine and coarse grains of the yolk, and the
“ organ-forming substances,’’ are not uniformly distributed, but are
more or less localized in various parts of the cytoplasm, does not
seem to raise a problem different from that raised by any other adaptive
arrangement.

Igo CYTOLOGY CHAP,

Finally, we may point out that the local differentiation, or anisotropy,
of the egg cytoplasm belongs to the class of exceptions which prove
the rule. Not only is there no mechanism for an equal partition of these
substances among the daughter cells at cell division, but their mode of
action depends upon the fact that they are not so distributed. It is
therefore very improbable that they can retain their continuity through
the numerous cell divisions leading from the unfertilized egg through
the cleavage divisions and all the divisions in the female germ track
till the cycle is complete with the formation of the next generation of
oocytes. It would appear therefore that they must be formed anew
in these cells in each generation, and all the arguments in favour of
the general morphogenetic activity of the nucleus in moulding the
cytoplasm apply equally in favour of the view that these substances also
are formed by the agency of the nucleus. Whatever view therefore is
taken of the ‘“ organ-forming substances’’ of the egg cytoplasm, their
presence does not affect the question of the monopoly by the nucleus
of the hereditary substance, which stands or falls on other grounds.

G. CHROMIDIA AND CHONDRIOSOMES

We now come to a very difficult chapter of cytology, in which state-
ments of fact and theory are so contradictory that it is at present
scarcely possible to do more than give an abstract of the work done
and the interpretations put upon it ; the questions involved are, however,
too important to pass over, in spite of the necessity of reserving judge-
ment on the issues.

(1) Chromidia

As we have already pointed out, we are in almost complete ignorance
as to the way in which the nucleus exerts its regulative and morpho-
genetic functions, but these appear to be usually exerted on the cyto-
plasm through the nuclear membrane. Occasionally, however, the
chromatin comes to lie naked in the cytoplasm instead of forming part
of a nucleus with a definite architecture enclosed by a membrane. This
is very commonly the case in Protista, where granules of chromatin
called chromidia often lie in the cytoplasm. These may be in addition
to the formed nucleus, or may take the place of this at definite stages
of the life history, or again may constitute the whole chromatic garni-
ture of the animal, a formed nucleus being absent. Sometimes these
chromidia are destined to take part in the reproduction of the organism
(generative chromidia) ; in other cases they are finally absorbed by the
cytoplasm without playing the part in reproduction usually allotted to

vI CHROMIDIA 191

the nuclear material. They are then known as vegetative chromidia,
and have been compared to the macronucleus of Infusoria, which is
absorbed at the time of conjugation without playing any part in that
process. Often, however, the extrusion of vegetative chromidia from
the nucleus seems to be merely a means by which a nucleus which has
through unfavourable conditions become hypertrophied, gets rid of its
excess chromatin. The classical example of this is Actinosphaerium
(R. Hertwig, 1904). Under certain unfavourable conditions (either
starvation or over-feeding) the nuclei become greatly enlarged and
hyperchromatic ; the excess of chromatin may then be got rid of by
the emission of large quantities of it into the cytoplasm, where it
degenerates into brown pigment.

The formation of vegetative chromidia in the Metazoan oocyte I.
has been described by many writers, and some have described it in other
Metazoan cells also.

In oocyte I. it is said to take place at the beginning of the growth
period, characteristically at the bouquet stage, when there is to be
observed in many animals a deeply staining mass in the cytoplasm
just outside the polar surface of the nucleus (Fig. 81), e.g., Proteus
(Jorgensen, 1910 6), Paludina (Popoff, 1907), Gryllus (Buchner, 1909).

This mass consists of granules or filaments which stain like chromatin,
and are often so closely applied to the nuclear membrane that they
appear to be continuous through this with the intranuclear chromatin.
This fact has led many cytologists to conclude that the mass above
referred to consists of chromatin extruded through the nuclear membrane
into the cytoplasm, that is to say, of chromidia.

Often, however, the extrusion of chromidia occurs after the bouquet
orientation has been lost ; in this case, as in the forms where there is
no orientated bouquet stage, the emission takes place diffusely through
the nuclear membrane instead of only at the polar surface, e.g., Aricia
(Schaxel, 1912; Fig. 81). .

Often when emission is diffuse, and sometimes even when it takes
place from the polar surface only, in the bouquet stage (various
Orthoptera—Wassilieff, 1907; Buchner, 1909), the nucleolus has been
described as acting as an intermediary in the process, the chromatin
which is to be extruded being first collected into it, and thence emitted
into the cytoplasm.

A precisely similar process has also been described in spermato-
cyte I.; ¢.g., Blatta (Wassilieff, 1907 ; see Fig. 81).

While descriptions of chromidia formation in the Metazoa have
mainly been restricted to oocytes and spermatocytes, they have recently
been extended to somatic cells. Here—doubtless in correlation with
the absence of a well-marked bouquet stage—the emission is diffuse,

192 CYTOLOGY CHAP,

taking place apparently from any part of the nuclear surface
(Fig. 81, G).
As to the way in which chromidia get out of the nucleus, opinions

BIGH ier.

Illustrating the supposed emission of chromidia from various Metazoan cells. A, B, C, oocyte of ‘Aricta
foetida (aiter Schaxel, Z.J.A., 1912). A, young oocyte, chromosomes still filamentar, cytoplasm destitute of
chromidia ; B, older oocyte, still no chromidia in the cytoplasm; C, still older oocyte, showing emission of
chromidia from the nucleus into the cytoplasm. D, oocyte of Antedon bifida (after Chubb, Phil. Trans., 1906).
Discharge of comparatively large masses of chromatin from the nucleolus. E, spermatocyte I. of Blatta ger-
manica (after Wassilieff, A.m.A., 1907). _F, oocyte of Proteus anguineus (after Jorgensen, F.H., 1910). G, H,
somatic cells of Musca (after Popoff, F.H., 1910). G, emission stage: H, the chromidia congregated into a
band round the nucleus.

are divided as to whether they pass as formed bodies through deficiencies
in the nuclear membrane (Buchner, 1910), or whether they are passed

VI CHROMIDIA 193

through in a state of solution. Various views are held regarding the
meaning of the chromidial formation. Following Hertwig, it has been
supposed (Popoff, 1907) that it is a means by which the mass relations
between nucleus and cytoplasm are restored, if for any reason the
quantity of chromatin relative to the cytoplasm has become too great.
Chromidia have also been supposed to give rise by degenerative trans-
formation to reserve food material such as yolk (Popoff, 1907 ; Paludina
oocyte: Moroff, 1909; Copepod oocyte) or fat (Popoff, tg10 ; Musca
fat cells).

Others again have ascribed to them a much more important role,
supposing that they have a formative function, being in fact the inter-
mediaries through which the nucleus produces the necessary changes
in the cytoplasm to bring about the differentiation of indifferent
embryonic cells into the specialized cells—muscles, nerves, etc.—of the
soma. According to Goldschmidt, the originator of this view, chromidia
may give rise to cell structures by direct transformation into them.
Thus in oocyte I. they are transformed into yolk granules, in embryonic
cells into muscle fibrillae, zymogen granules of gland cells, ‘etc.

This view was founded partly on observation of muscle and gland
cells in Ascaris (Goldschmidt, 1905, 1910), which have not been supported
by subsequent observers on the same material, and partly on analogy
with certain Protista; for example, Trypanosomes, where a darkly
staining body (‘ kinetonucleus ’’) which is in close anatomical relation
to the flagellum and therefore apparently concerned with the function
of locomotion, is supposed by many to have been derived from the nucleus
and to consist of chromatin. At present, however, we cannot be said
to possess reliable evidence of the direct transformation of chromidia
into functional cell structures, though it seems not unlikely that they
may by fatty degeneration be transformed into yolk, fat, etc.

Goldschmidt, however, also allows of a different mode of action of
the chromidia, considering that besides giving rise to functional cell
structures by direct transformation into them, they are in other cases
the formative bodies under whose agency the cytoplasm is moulded
into its various forms; in other words, the morphogenetic activity of
the nucleus, to which we have so often alluded, is not exerted directly
by the nucleus as a whole, but by portions emitted through the nuclear
membrane, as chromidia, into the cytoplasm which it is to mould.
Goldschmidt is thus led to distinguish between two kinds of nuclear
material, propagative and somatic, or to select one of the various terms
that have been proposed by different workers—idtochromatin and
trophochromatin. Idiochromatin is the idioplasm, which we have
sufficiently characterized already. Trophochromatin is derived from
the idioplasm, and is the intermediary by which the latter, the master

O

194 CYTOLOGY. CHAP.

substance of the body, reacts upon the cytoplasm on which, in the last
instance, the forms and functions of the cells, and therefore of the
organism, directly depend.

This view is again largely founded upon the analogy of the Infusoria,
where the nucleus is divided into two portions, the micronucleus, which
alone takes part in conjugation, and the macronucleus, which is supposed
to be concerned with the physiological activities of the cell, and which
disappears before conjugation, a new one being derived from the micro-
nucleus after syngamy. The ordinary Metazoan nucleus contains both
kinds of nuclear substances, the trophochromatin being separated from
it from time to time as required in the form of chromidia, which therefore
correspond to the Infusorian macronucleus. The only nuclei in the
Metazoa which exactly correspond with the Infusorian micronucleus,
consisting entirely of idiochromatin, are the gamete nuclei, from which
all the trophochromatin is supposed to have been eliminated during the
growth period of the oocyte or spermatocyte (Goldschmidt, 1905, rgIo).

While probably no useful purpose is to be served by labouring the
distinction between the two kinds of nuclear substances (since in any
case the one is directly derived from the other), if it were established
that the nucleus exerts its morphogenetic action through the agency
of chromatin extruded naked into the cytoplasm, a step, though a small
one, would undoubtedly have been taken towards the understanding
of this dark problem. Practically our only direct evidence in favour
of this view is to be found in a series of papers by Schaxel.

His results in a large number of forms are remarkably uniform, and
may be illustrated by the development of the Polychaete Aricia foetida
(1912) and Strongylocentrotus (IgII a).

There is no emission of chromidia during the process of cleavage
(cell multiplication), but before cell differentiation begins, e.g. before
the endoderm cells take on the specific character of the cells of the
alimentary canal, or mesoderm cells develop into muscle cells, an emission -
of chromatin takes place.

To take a specific example: the conversion of an undifferentiated
mesenchyme cell of Stvongylocentrotus into a skeletal cell is preceded,
according to Schaxel, by an emission of chromidia from the nucleus
into the cytoplasm. These chromidia congregate into a mass, in the
middle of which a globule of the skeletal secretion soon appears. This
increases in size at the expense of the chromidia—that is to say, the
chromidia are destroyed or used up by their own formative action,
though it is not suggested that they are actually transformed into the
secretion. As the Echinoderm skeleton is extracellular, this secretion
has to be extruded from the cell to take part in the formation of the
skeletal spicule. At present the work of Schaxel stands in need of con-

VI CHROMIDIA 195

firmation. Indeed, even the occurrence of chromidial extrusion is not
undisputed. Thus Duesberg (1911 a) denies the nuclear origin of the
‘‘chromidia’’ described by Wassilieff in Blatta (Fig. 81, E) and holds them
to be chondriosomes (see below), and therefore of purely cytoplasmic
origin. Meves also denies the derivation from the nucleus of certain
bodies, which have been described by the upholders of the chromidia
theory as having been so derived. Beckwith (1914) has subjected the
egg of Hydractinia to a very careful examination, and comes to the
conclusion that the staining particles there present, which must un-
doubtedly be of the same nature as the similar bodies described by
Schaxel in so many oocytes (including those of several Hydrozoa), are
of cytoplasmic, and not of nuclear, origin. She bases this conclusion
on the fact that they make their first appearance scattered through the
cytoplasm and not concentrated round the nucleus, and also on the
fact that though they react similarly to chromatin to many stains,
they show striking differences in their reaction to others. The doubts
thus thrown on the nuclear origin of the ‘‘ chromidia’’ in the Metazoa lead
us on to a consideration of the chondriosomes, which, as we shall see,
may or may not be identical with chromidia.

(2) Chondriosomes }

These are granular or filamentar bodies present in the cytoplasm,
about the nature of which there has been much controversy during the
last few years. Some cytologists have ascribed to them a role in
morphogenesis and heredity equal to that of the chromosomes. This
theory has been especially developed by Meves (1908, 1g11, etc.) and
Duesberg (1911 0, etc.), following on the work of Benda. An exhaustive
review of the literature on the subject up to the year IgII is given by
Duesberg (1911 0).

An account of the chondriosomes in the spermatogenesis of Blatta has
already been given (Chapter III.). They may take the form of granules,
chains or filaments. They stain strongly with many stains, including
the commoner chromatin stains, though towards others they react
differently from chromatin, which fact is an important argument against
their being the same as chromidia. By certain fixatives, especially

1 This subject has suffered, like most other branches of science, from changes of nomen-
clature accompanying extension of knowledge or change of view. The following summary
will be of use to the student who wishes to follow up this subject in the original literature :

General term =-Chondriosome (Meves) = Plastosome (Meves).
Chondriosomes in form of granules = Mitochondria (Benda) = Plastochondria (Meves).
Chondriosomes in form of rods or filaments =Chondrioconts (Meves) = Plastoconts (Meves).
Chondriosomes in form of chains of granules=Chondriomites (Benda).

The change in the prefix in Meves’ latest set of terms signifies his view of their histo-
genetic function (1911). Paraplastic bodies (Meves) are bodies developing from Plastosomes.

196 % CYTOLGGY CHAP.

acetic acid, their non-chromatic nature is made especially evident, since
in their usual condition chondriosomes are dissolved, or at any rate
caused to disappear, by this reagent, which on the contrary forms one
of the commonest and most useful of chromatin fixatives. Largely for
this reason, doubtless, chondriosomes do not often appear in cytological
figures, which are mostly taken from material which has been fixed
with regard to the preservation of the nuclear structures. Chondriosomes
are readily seen, and their movements followed in detail, in living cells
(see, for example, Lewis and Robertson, 1916).

Meves (1910) identifies the chondriosomes with the filaments of
Flemming (except that the latter included certain structures under this
term, such as the fibres of the achromatic figure, which appear to be
of a different nature) and with the granules of Altmann.

That the chondriosomes possess a peculiar significance in morpho-
genesis and heredity is based on the following claims :

(1) They are permanent cell structures, persisting from one cell
generation to another, reproducing themselves by fission.

(2) There are indications that in some cases arrangements exist for at
any rate an approximately equal partition of the chondriosomes between
two daughter cells at cell division.

(3) They are carried into the egg by the spermatozoon at fertilization.

(4) The chondriosomes found in the fertilized egg can be traced in
the tissues of the developing embryo, and have been described as actually
giving rise to permanent cell structures of the adult—for example,
neurofibrillae, muscle fibrillae of striated muscle fibres (Meves, 1908 ;
Duesberg, 1g10) and to various organs of plant cells (see Meves, Ig18).

It must suffice to comment very briefly on these four points.

(r) Since the chondriosomes lie in the cytoplasm, and since the
cytoplasm of the mother cell is divided among the two daughter cells,
it follows that the chondriosomes also must be passed on from cell to
cell; the spermatogenesis of Blatta illustrates this (Fig. 29). There is
no evidence, however, to show that they may not disappear and re-form
in the cytoplasm from time to time ; their origin de novo in the cytoplasm
has indeed been described in certain cases (Schaxel, 1912; Beckwith,
1914), while evidence of their regular multiplication by fission is practi-
cally non-existent.

(2) So far very little evidence exists for this, though at cell division
the chondriosomes are often doubtless distributed together with the
cytoplasm into two approximately equal masses. On the other hand,
certain cases have been described where the division is unequal. This
is manifestly the case in the polar body formation, where practically
the entire mass of the chondriosomes of the oocyte I. are left in the ripe
egg, exhibiting nothing corresponding to the reduction of chromosomes.

VI CHONDRIOSOMES 197

In the division of the spermatocytes the chondriosomes are presumably
generally more or less equally divided, along with the cytoplasm, between
the daughter cells, but definitely unequal distribution of chondriosomes
among spermatids has been described in Myxine (A. and K. E. Schreiner,
1908) and Euschistus (Montgomery, 1911). An unequal distribution of
the chondriosomes among the daughter cells also takes place in the
developing embryo of Ascidians (Duesberg, 1915). Thus precise quantita-
tive and qualitative distribution of the chondriosomes between the
daughter cells at cell division is not at any rate of general occurrence,
nor is there any reduction in their mass at oogenesis. These facts weigh
heavily against the theory that the chondriosomes are the seat of
morphogenetic factors.

(3, 4) The chondriosome apparatus always! plays a part, though
a variable one, in the structure of the adult spermatozoon. In what
may be called the ‘“‘ typical” spermatozoon it occupies the middle piece of
the tail, forming a sheath for some distance round the axial fibre. In
the tailless forms of spermatozoon, such as are found in Nematodes,
Arthropods, and a few other animals, it assumes various forms and
positions.

It is quite possible that chondriosome apparatus, or part of it, always
enters the egg in fertilization. In several cases, it is true, the tail of
the spermatozoon fails to enter the egg, but in many spermatozoa only
the extreme base of it would be necessary, or in others (Ciona ; Duesberg,
1915) none of it, the chondriosome apparatus being alongside the nucleus
in the head. If the chondriosomes are to be accepted as hereditary
substance, however, it is necessary to show that having entered, they
mingle with the egg chondriosomes, and are distributed with them to
the cells of the developing embryo.

The first form in which the behaviour of the chondriosomes at
fertilization was worked out was Ascaris megalocephala (Meves, 1911),
and this appeared to give brilliant support to the theory that the
chondriosomes are hereditary material. The chondriosomes in the
spermatozoon are in the form of a number of comparatively large
granules, those in the egg being much smaller, and scattered throughout
the egg cytoplasm. After the spermatozoon has entered the egg, the
egg chondriosomes concentrate round it; the male chondriosomes
leave the spermatozoon and break up into numerous small granules of
the same size as those of the egg. The two sets of chondriosomes now
become indistinguishably mingled.

It soon appeared that the case of Ascaris, in the spermatozoon of
which the chondriosomes are unusually bulky, is not typical. Meves

1 Montgomery (1912), however, describes it as being thrown off by the developing
spermatozoon of Pertpatus.

198 CYTOLOGY CHAP.

(1912) himself found that in sea-urchins the chondriosome apparatus
introduced by the spermatozoon undergoes no metamorphosis, but is
relegated unchanged to one of the first two blastomeres. An, exactly
similar process has been described for various mammals by van der
Stricht (1910) in the bat, and by Lams (1913) in the guinea-pig (Fig. 82).
In order to save the theory of chondriosome inheritance, it was suggested
that the blastomere lacking the chondriosomes takes no part in the
formation of the adult, but that in the mammals (van der Stricht) the
trophoblast develops from it, and in Echinoderms (Meves) that part
of the pluteus larva which is cast off at metamorphosis. This conjecture
must be considered very improbable, for the pluteus is an organism
quite as specific as the adult sea-urchin and quite as much in need of

Fie. 82:

The chondriosomes in the fertilization and cleavage of the guinea-pig’s ovum. (After Lams; A.B., 1913.)
A, entry of the spermatozoon; B, 2-cell stage, the tail (including the chondriosome apparatus) of the
spermatozoon lying unchanged in one blastomere.

p.b. I., first polar body; p.b. I1., second polar body ; ¢, tail of spermatozoon ; II., metaphase IT.

a complete hereditary outfit. It has, however, been rendered still more
improbable—if not indeed definitely disproved—by Meves himself,
who later (1914) traced the chondriosome mass of the male gamete
in Parechinus up to the 32-cell embryo. At this stage it is still compact
and unchanged, and is therefore of course only to be found in one cell.
All the other 31 cells therefore contain no part of the male chondriosome
apparatus, so that it is established that this is not essential to the develop-
ment of the Echinoderm.

Whilst thus the behaviour of the male chondriosome mass in
fertilization is alone enough to destroy all claim to the idioplasmic
nature of chondriosomes, unless and until new knowledge of an unfore-
seen nature is forthcoming, there are several other almost equally con-
vincing items of evidence. It is not necessary to labour the point of the
colossal excess of the chondriosomes in the egg over those brought in

ae en ee ee ee

=a

vi CHONDRIOSOMES 199

by the spermatozoon. More serious perhaps is the fact that these egg
chondriosomes may be very unevenly distributed amongst the blastomeres
in cleavage (Duesberg, 1915). An unequal distribution of the chondrio-
somes may be brought about experimentally in Hydractinia (Beckwith,
1914) by centrifuging the egg. This does not prejudice the normal
development of the larvae.

The persistence of the chondriosomes which are undoubtedly present

Fic. 83.

The chondriosomes in the fertilization and cleavage of the egg of Parechinus miliaris. (After Meves, A.m.A.,
1g12 and 1914.) A, spermatozoon; B, shortly after entry of the spermatozoon into the egg; C, 2-cell
stage; D, 32-cell stage.

c, chondriosome apparatus ; , head (nucleus) of spermatozoon.

in the fertilized egg and embryo, and their development into certain
specific cell structures of the adult, especially into neurofibrillae and
muscle fibrillae, have been described by Meves (1908) and Duesberg
(1910) for the chick, and also for some mammals. According to Arnold
(1912) they give rise to the zymogen granules in the pancreas.

200 CYTOLOGY CHAP. VI

(3) The Relation between Chromidia and Chondriosomes

This last supposed characteristic of the chondriosomes obviously
raises at once the question whether these bodies are not the same as
chromidia, and here we are once again face to face with a controversy |
which must at present be left undecided. There can be no doubt that
in certain specific cases the same thing has been described by one worker
as chondriosomes and by another worker as chromidia—we may compare
Fig. 29, D, and Fig. 81, E, both representing the primary spermatocyte
of Blatta germanica. We have indeed all possible views of the relation
between the two structures supported by different workers.

(1) Chondriosomes have no relation with chromidia, though in some
cases they have been erroneously described as such (Meves, Duesberg).

(2) Chondriosome is simply a name given to chromidia by those who
have failed to recognize their true origin from the nucleus (Goldschmidt,
Popoff, Buchner).

(3) Chondriosomes and chromidia are independent bodies, found side
by side in the cytoplasm, the one of cytoplasmic, the other of nuclear
origin (Schaxel, Jorgensen).

Finally, it should be mentioned that both chondriosomes and
chromidia have been interpreted in some cases as metamorphosed parts
of the achromatic figure. This applies to certain of the very diverse
bodies known under the comprehensive terms of “‘ Nebenkern”’ and
yolk nucleus, but it appears certain that most of the structures described
as chondriosomes and chromidia cannot be so explained.

CHARTER: Vit
THE NUCLEUS OF THE PROTISTA AND PLANTS

Ae BROTISTA

THE nucleus of the Protista is not constructed on such a uniform plan
as that of the Metazoa or Metaphyta. Certain bacteria indeed are said
to exhibit no differentiation into nucleus and cytoplasm, being alterna-
tively interpreted as consisting wholly of the one or the other. Dobell
(1911), however, who has made a special study of the larger forms of
bacteria (on which alone reliable cytological observations of this kind
are possible) finds a differentiation between nucleus—or, at least, chromatin
—and cytoplasm in all the forms which he examined.

In many other Protista there is no organized nucleus, the chromatin,
etc., being scattered through the cell in the form of chromidia. This
may possibly be the permanent condition of the nucleus in some of the
more lowly organized forms, but much more often it is a temporary
phase, a compact nucleus being formed at other phases of the life cycle,
as in certain bacteria (Dobell, Joc. cit.) and many Protozoa, of which
examples are given below.

Even when an organized nucleus is present in the Protista, its form
is more varied than it is in the higher organisms.

Two chief types of organized nucleus are commonly distinguished in
the Protista, namely, the vesicular and the granular. In the commonest
type of the former, which appears to be the less specialised form of
nucleus, the greater part or possibly sometimes all the chromatin is
aggregated into a single central mass or karyosome, which lies in a
vacuole containing fluid, probably of the same nature as the Metazoan
karyolymph. The chromatin in the karyosome is probably in the form
of granules bound together by linin or plastin (as in the amphinucleolus
of Metazoa). In certain Protista the karyosome also contains a body
which has been identified as the centrosome, which is therefore intra-
nuclear in these forms.

In the granular type of nucleus the chromatin is distributed in the
form of granules or small masses over a linin framework. This type

201

202 CYTOLOGY ‘CHAP.

of nucleus approaches therefore the commoner type of Metazoan nucleus.
The vesicular and granular types of nuclei grade into one another, however.

Many Protista have two nuclei differing greatly in structure, and
presumably in function. Two principal types of this bimuclearity are
found.

(r) In the Infusoria, one of the two nuclei (macronucleus) is much
larger than the other (micronucleus). The former divides amitotically
during reproduction by fission, while the latter exhibits a form of mitosis.
Moreover, the macronucleus disintegrates before conjugation, and takes
no part in that process. In the exconjugant it is formed anew from the
zygote micronucleus. It is thus clear that the macronucleus is a nucleus
physiologically of a lower order than the micronucleus, and it is supposed
to be concerned exclusively with the somatic functions of the cell.

(2) In the second type of binuclearity, found in the Trypanosomes
and their allies, the two nuclei are called respectively the trophonucleus
and the kinetonucleus. (It must be mentioned, however, that the nuclear
nature or origin of the “‘ kinetonucleus’”’ has been questioned in many
cases.) The former is the larger, and the one most nearly corresponding
to the nucleus of uninucleate Protista. The kinetonucleus is in close
anatomical relation to the flagellum, and is therefore supposed to be
specially concerned with the movements of the animal.

A general review of binuclearity in the Protista is given by Dobell

(1909).

Compared with the Metazoa and Metaphyta, the modes of nuclear
multiplication in the Protista are of bewildering variety. This refers
both to the behaviour of the chromatin and to the development of the
achromatic figure. The latter we will not consider specially, except to
point out that the centrosomes when present in the Protista are not
uncommonly intranuclear, as indeed is the whole achromatic figure (in
cases where this is identifiable). In such cases the whole mitosis may
take place without rupture of the nuclear membrane. Centrosomes
are, however, sometimes absent ; e.g. in certain Amoebae.

As regards the chromatin, we may classify the modes of nuclear
multiplication into three principal categories—amitosis, mitosis, and
through the intermediation of chromidia formation.

Before discussing mitosis and amitosis in the Protista we must make
quite clear the precise meaning which we attach to each of these terms.
The word mitosis was coined to describe nuclear division in the Metazoa
and Metaphyta, and correctly emphasizes the most significant feature
of the process—namely, the linear arrangement of the chromatin elements
into the threadlike chromosomes. The function of this is universally
assumed to be to facilitate the exact partition among the daughter nuclei

——_— ~ ret alten LE tem me

~~ a

vu AMITOSIS IN PROTISTA 203

of the products of division of differentiated chromatin particles. In the
Protista, however, we must extend the word to cover all cases of nuclear
division accompanied or preceded by rearrangements of the chromatin
which can be interpreted as having this function of bringing about a
qualitative rather than a purely quantitative division of the chromatin,
even though they do not result in the formation of regular chromosomes.

The term amitosis is confined, as in the case of the Metazoa and
Metaphyta, to a purely mass division of the nucleus without any attempt
at a qualitative equality among the daughter nuclei.

Amitosis, in the sense just defined, has been described repeatedly
in the Protista, both in the vesicular and granular types of nucleus.
Undoubtedly, however, the trend of modern research in Protistology
is to discover in more and more supposed cases of true amitosis a pre-
liminary internal readjustment of the chromatin, which suggests that it
undergoes a qualitative rather than a purely quantitative partition.
Very often these readjustments do not go so far as the formation of
the regular chromosomes which we find universally among the higher
organisms, but they in all probability represent a primitive form of
mitosis. It would seem as well therefore to suspend judgement for
the present as to whether purely quantitative mass division of simple
nuclei ever does take place. The term “ simple ”’ is a necessary qualifica-
tion, for the Protozoan nucleus sometimes has a very different composition
from that of the higher organisms, and in these cases the problem of
‘““amitosis ’’ bears a different complexion.

Pure mass division of the macronucleus of Infusoria and Acinetaria
does indeed appear to be demonstrated. Especially in the latter group
is it difficult to see how the division can be qualitative during the process
of bud formation in such a form as Ephelota. Here a process of the
macronucleus grows out into the developing bud and then becomes
nipped off to form the macronucleus of the bud. The macronucleus.in
these groups, however, appears to be composed of trophochromatin only.
It is at any rate destined only to last during the asexual portion of the
organism’s life cycle; before conjugation the macronucleus breaks up
and disappears, only the micronucleus taking part in syngamy. In both
groups the latter nucleus, in contrast to the macronucleus, divides by
mitosis throughout the whole life cycle, thereby retaining its qualitative
composition.

The process of mitosis in the Protista ranges in complexity from the
merest indication of a sorting out of the chromatin elements before
division of the nucleus as a whole, to a mitosis as perfectly developed
as any found in the Metazoa, with fully formed chromosomes, an equatorial
plate, centrosomes and complete achromatic figures.

As examples of primitive mitosis we may take the nuclear division at

204 CYTOLOGY CHAP.

two different phases of the life cycle of Coccidium schubergi (Schaudinn,
1900). In the schizont (the asexual cycle), before nuclear division the
chromatin granules become massed together in little clumps and
irregular threads, in which, however, no definite longitudinal splitting
can be made out, and they do not get collected into an equatorial plate.
They sort themselves out in some way or other into two groups which
appear to be pushed apart by the elongation of the karyosome, which
contains, or takes the place of, the centrosome and achromatic figure.
The nuclear divisions in the oocyst of the same species, which cover

Fic. 84.

Nuclear division in the asexual cycle of Cocctdium schubergi. (After ScHaudinn, Z.J.A., 1900.)
A-E, the schizont; F-L, the oocyst.

the first few divisions of the zygote nucleus after syngamy, are instructive
as showing how nuclei which appear to divide in the most purely amitotic
fashion may have undergone a previous reorganization which is presum-
ably connected with the accurate partition of differentiated chromatin
elements between the daughter nuclei (Fig. 84, F-L).

There is a prophase closely resembling that. of a Metazoan mitosis
resulting finally in the formation of a relatively very thick and short
spireme. This, however, breaks up into irregular fragments which become
united to form a reticular nucleus again, and in this condition the nucleus
divides. Although, therefore, the actual division appears to be amitotic
it is difficult to avoid the conclusion that the previous arrangement of

—"

a

VII MITOSIS IN PROTISTA 205

the chromatin granules in linear series had the same function as postu-
lated for the chromosomes of a Metazoan mitosis, namely, to effect their
accurate division and partition among the daughter nuclei.

In some Protista very well-developed mitosis, closely resembling
that in the Metazoa and Metaphyta, is found (Fig. 85).

In the Coccidian, Aggregata ebertht (Dobell and Jameson, 1915) the
nuclei of the primary gametocytes (¢ and $?) show in mitosis six
chromosomes of very different sizes (labelled, from largest to smallest,
a-f in Fig. 86). The macrogametocyte is transformed into the macro-
gamete without any reduction of
chromosomes, the macrogamete
having theretore the same series
of six chromosomes. The _ pri-
mary microgametocyte nucleus
undergoes repeated division to
form the microgamete nuclei, the
same series of six chromosomes
appearing throughout, though
becoming greatly reduced in size.
Both gametes have therefore, like
the gametocytes, six chromo-
somes. Syngamy results in a
zygote with twelve chromosomes
which can be sorted out into
pairs as in a typical Metazoan
diploid nucleus.

In the metaphase of the first Pisa.

Mitosis of the gametocyte nucleus of Monocystis.
division of the zygote nucleus the ee Pie dae Cea a Ue ge e3)) Wa oa
homologous chromosomes become
united into bivalents, the constituents separating at anaphase. This
division therefore is a reduction division and the daughter nuclei
have only six chromosomes. This number is retained throughout
all the subsequent nuclear divisions of the life cycle, which include
spore formation, the asexual multiplication of the schizont, and the
gametocyte divisions again., Thus in this animal the relation be-
tween the duration of the haploid and diploid phases is the reverse of
what obtains in the Metazoa, the nuclei being haploid throughout all
the life cycle except in one cell generation—the zygote cell—while
meiosis takes place, not at gametogenesis, but at the first division of
the zygote nucleus, which is comparable to the first cleavage mitosis
of the Metazoan egg. Many plants also exhibit a much longer duration
of the haploid generation relatively to that of the diploid part of the life

Fic. 85.

206 CYTOLOGY CHAP.

cycle than is found in the Metazoa, where with the exception of certain
cases of parthenogenesis the haploid stage is represented by at most two
cell generations (p. 214).

A closely similar account of the nuclear cycle of the Gregarine Diplo-
cystis schneideri is given by the same authors (loc. cit.).

The third principal mode of nuclear multiplication in the Protista—
by the intermediation

hee’ of chromidia formation
doer —is supposed to be of
i B frequent occurrence.
; The three forms, Mas-
. oy % tigella, Coccidium and

See Arcella will serve as
examples.

During the asexual
multiplication of Mas-
tigella vitrea (Gold-
schmidt, 1907) the nuc-
leus divides by mitosis
with well - developed
chromosome formation.
In gametogenesis, how-
ever, the gamete nuclei
are produced from the
nucleus of the gameto-
cyte by a very different
process (Fig.87). There
‘s is a copious emission

The chromosomes of Aggregata eberthi. They are designated ce of chromidia from the
from the largest (a) to the smallest (f). (Dobell and Jameson, P.R.S nucleus into the cyto-

1915.) A. microgametocyte nucleus in prophase, with six chromosomes ;

B, equatorial plate, and C, anaphase of same ; D, zygote nucleus in pro- plasm. In the mass of
phase for its first division: E, equatorial plate of same division—homo- ft

logous chromosomes united into bivalents; F, anaphase of same Chromidia numerous
(reduction division).

Fic. 86.

nuclei (up to two or
three hundred) are formed by aggregation of numbers of chromidia into
clumps. The nuclei thus formed then (in the case of the macrogamete)
divide by mitosis (with chromosome formation) at least once. Gold-
schmidt interprets this as a reduction division, as it results in only one
of the two daughter groups of chromosomes forming a functional nucleus,
the other degenerating into a body strikingly reminiscent of the Metazoan
polar body.
The micro- and macrogametes unite in syngamy, the zygote nucleus
dividing by mitosis to introduce the asexual cycle with which we began.

vu CHROMIDIA IN PROTISTA 207

Another example of multiplication of nuclei by chromidia formation
alternating with multiplication by mitotic division, though attended by
more complications, is afforded by Arcella (Fig. 88), the life history of

Fic. 87.

Stages in the formation of macrogametes in Mastigella vitrea. (After Goldschmidt, A.P.K., 1907.) A,
macrogametocyte nucleus surrounded by extruded chromidia, B, a later stage. As the chromidia get
further away from the nucleus they are formed into the macrogamete nuclei. C, gametocyte full of macro-
gametes. Some are undergoing the (? reduction) division. D, four of the macrogametes from C on a larger
scale. Three are in mitosis, and the fourth has completed the mitosis and contains the mature gamete nucleus
and the polar body-like ‘‘ reduction body.”

ch, chromidia ; m, macrogamete nuclei; 7, ‘‘ reduction body.”

which has been studied by many workers. The essential features from
our present point of view are as follows. At the beginning of the asexual
cycle the young Arcella consists of a minute uninucleated cell. Soon
the nucleus divides into two, by mitosis. At about the same time a
great number of chromidia are emitted from the nuclei. These become

208 CYTOLOGY CHAP.

€

massed together into a “ chromidial net’ which forms a ring round
the edge of the cell. The Arcella therefore now consists of a cell with
two nuclei (primary nuclei) and a chromidial net.

At certain stages of the life history secondary nuclei are formed in
large numbers out of the chromidial net, by the aggregation of chromidia
into small masses. These secondary nuclei multiply by mitosis, and
ultimately may give rise either to asexual buds or to gametes.

An unusual type of syngamy is sometimes observed in this animal,
involving fusion or mingling of chromidia (chromidiogamy, Swarczewsky)
rather than of formed nuclei. Two Arcellas, in which the primary nuclei
have degenerated and all the chromatin is in the form of finely scattered
chromidia, come together. Their cytoplasms—and hence the chromidia—
mingle together, and then separate again into the two individuals, each

Fic. 88.

Arcella vulgaris. (A, B, after R. Hertwig, Festschr. Kupffer, 1899 ; C, after Swarczewsky, A.P.K., 1908.)
formation Dt secondary, naciey out of tue chromic ait + Gyfamto of'cecandary mie ane
c, chromidia ; p.., primary nucleus; s.%., secondary nuclei.
containing presumably a mixture of chromidia from both conjugants.
Out of these chromidia secondary nuclei are organized in a manner
similar to that described above.

In Coccidium schubergi also, according to Schaudinn, the micro-
gamete nuclei are produced from that of the microgametocyte by the
passage of the chromatin of the latter out of the nucleus into the cytoplasm
in the form of chromidia which rise to the surface of the cell, and there
become aggregated into the microgamete nuclei.

In Coccidium, Arcella, and still more definitely in Mastigella, therefore,
during certain periods of the life history the chromatin is aggregated
into a formed nucleus, and all the usual mechanism of mitosis is present
to ensure its accurate quantitative and qualitative partition among its
descendants. At other periods this methodical mode of nuclear division is
interrupted by a method of nuclear multiplication which affords no direct

ee

vil CHROMIDIA IN PROTISTA 209

evidence of qualitative chromatin distribution among the nuclei so formed.
Since, however, during nuclear multiplication by means of chromidia
the chromatin is resolved into small particles which are not improbably
its structural units, it is possible that there takes place a qualitative
sorting out of these units into the new nuclei which are formed by their
aggregation into masses. Moreover, in assessing the significance of
accounts of nuclear multiplication through the intermediation of chromidia
much caution must be used. It must be remembered on the one hand
that the study of the cytology of the Protista is often beset with much
greater difficulties of technique than in the case of the Metazoa, owing
to the minuteness of the elements concerned. Dobell and Jameson, as
the result of their study of Aggregata and Diplocystis, are inclined to doubt
accounts of chromidia formation in the Coccidia and Gregarines, such
as, for instance, that of Schaudinn for Coccidium schubergt mentioned
above. In the anaphase of the first division of the microgametocyte
nucleus of Aggregata (the mitosis shown in Fig. 86, C) the chromosomes
which were spheroidal in the metaphase become filamentar again. The
asters at the two poles of the spindle divide repeatedly, and at each
division the chromosomes divide longitudinally, becoming at last very
minute. They are finally sorted out in groups of six, each group forming
a nucleus at the periphery of the gametocyte cell, there to multiply by
mitosis to form the microgamete nuclei. It can hardly be doubted that
this process corresponds to the chromidial formation described by
Schaudinn as above,,a conclusion which suggests that there the term
‘“chromidia’’ might be translated into ‘‘ minute chromosomes ’’—a
change in terminology implying that the process of their formation
involves an exact division and partition of differentiated elements.

It must also be remembered that the Protistan nucleus may have a
composition very different from that of the Metazoa or Metaphyta. In
the latter groups the nucleus always, so far as we know, contains either
a single or a double series of differentiated elements (with the special
exception of the triploid, tetraploid, etc., nuclei considered on page 150).
In the Protista, however, it appears that the nucleus may be polyploid,
containing, not one or two, but a great number of series of elements.
Examples of such polyenergid nuclei (Hartmann, 1909) are afforded by
the great nuclei of the Radiolaria. The nuclear cycle of one of these,
Aulacantha (Borgert, 1901, 1909), is as follows :

Reproduction may take place asexually by binary fission of the cell,
or sexually through the intermediation of gametes. The division of
the nucleus in the first type of reproduction takes place by a form of
mitosis superficially very similar to a Metazoan mitosis (Fig. 89), but
accompanied by the formation of an enormous number of chromosomes.
The number of these is far more than a thousand, but varies greatly in

FP

210 CYTOLOGY CHAP.

different individuals. These chromosomes, however, are said to undergo
two longitudinal divisions in every mitosis. Each is said to divide
once during prophase, each daughter chromosome dividing again in the
metaphase. Another important point is that the spindle fibres do not
converge to a single centrosome, but run parallel with one another.

Gamete formation (Fig. 89, D) begins with the emission of chromatin
particles from the nucleus into the cytoplasm. Each of these particles
becomes enclosed in a vesicle to form a minute secondary nucleus.
Closer examination shows that the chromatin particles which are emitted
from the original or primary nucleus are the individual “‘ chromosomes”
which appear in the mitosis of this nucleus. The minute secondary
nuclei, each thus constituted out of a single “ chromosome” of the
primary nucleus, multiply by repeated mitosis to form the gametes.
The number of chromosomes appearing in these mitoses is, however,
not one, but ten to twelve.

Each of the enormous number of chromosomes of the primary nucleus
is therefore equivalent to an entire gamete nucleus containing ten to
twelve chromosomes. It may in fact be compared with the “ un-
segmented spireme’’ found in the prophase of certain Metazoan a
Metaphytan mitoses (p. 9).

The emission of the ‘‘ chromosomes ’”’ of the primary nucleus into
the cytoplasm obviously suggests ‘‘ chromidia formation,” though in
this case it is merely the breaking up of a compound polyploid nucleus
into its constituent haploid (or diploid ?) nuclei, and therefore raises no
special problem.

Amitosis, which, according to Borgert, occurs in Aulacantha in jnddried
to mitosis, also raises no difficulties in the case of a polyploid nucleus,
since each daughter nucleus may still have hundreds of representatives
of each individual chromatin element.

Summing up, further knowledge is required before we can decide
whether nuclear multiplication in the Protista by means of “ amitosis ”’
and “‘ chromidia”’ formation is to be conceived as an exception to, or
as a variant of, the orderly division of the chromatin elements which
appears to be universal in the Metazoa and Metaphyta and to be at
least common in the Protista.

,

B. ANIMALS AND PLANTS—HAPLOID AND DIPLOID
CONDITIONS

As will have been gathered from the occasional references to
plant cytology in the previous pages, the cytology of plants and animals
is on the whole so similar that detailed comparison is unnecessary.
Plants, however, exhibit a much more varied relation to the haploid

vil POEYPLOID NUCLEI IN PROTISTA 211

e 4»

9

nM

\.¢

‘
8

Fic. 89.

Nuclear multiplication in Aulacantha. (Borgert, Z.J.A.,1901, and A.P.K.,1909.) A-C, stages in the division
of the primary nucleus during binary fission of the animal; D-G, preliminary stages in gamete formation.
A, resting nucleus; B, mitosis, equatorial plate; C, portion of an anaphase shown on a larger scale; D,
formation of secondary nuclei (the little dark specks in the cytoplasm) from the primary nucleus; E, the
primary nucleus is almost used up in the production of the secondary nuclei; F, a few secondary nuclei
under a higher magnification, showing the single chromatin thread in each ; G, secondary nuclei in mitosis.

212 CYTOLOGY CHAP.

and diploid states than do animals. In the latter—at least in the
Metazoa, with certain rare exceptions such as the males of the Hymenoptera
—the individual is always diploid. In each life cycle occur one (post-
reduction) or probably two (pre-reduction) haploid cell generations,
namely, the gamete itself and generally the secondary oocyte or sper-
matocyte. In plants, however, the processes of meiosis and syngamy are
often separated by a long section of the life history giving rise to an
alternation of haploid and diploid generations. One of the best illus-
trations of such an alternation is in the ferns, where, as is well known,
the ordinary fern plant or sporophyte is diploid, and the prothallus or
gametophyte is haploid. The sporophyte produces, with reduction of
chromosomes, haploid spores from which grows the prothallus. This
produces gametes, without of course any further reduction of chromosomes,
and from the zygote cell develops the next sporophyte. In the fern
therefore the dominant phase in the life history is the diploid generation.
This is still more so in the case of the flowering plants, where the haploid
generation or gametophyte is reduced to a very few cell generations
(5 in the female and 4 in the male), and does not lead an independent
life, but is borne on and nourished by the sporophyte, which is the
plant body as we know it.

The cell generations involved in the haploid phase of the flowering
plants are shown diagrammatically in Fig. go. The diagrams start with
the pollen mother-cell in the male and the embryo-sac mother-cell in the
female—in each case the last cell generation of the diploid phase. These
cells divide twice in rapid succession, giving rise each to a group of four
cells. Reduction takes place in the first of these two divisions. The
process therefore is closely parallel to the meiotic phase in animals.
The four haploid cells forméd by these two divisions are spores, homologous
with the spores of ferns, mosses, etc. In the male, the spores are micro-
spores or pollen-grains. The nucleus of the pollen grain divides into
two, one being a vegetative nucleus and the other a nucleus which again
divides to give two gamete nuclei.

In the female, typically only one out of the four spores (megaspores)
derived from a single embryo-sac mother-cell develops, the other three
degenerating and thus again reminding us very strikingly of the ovum
and polar bodies in animals. The nucleus of that megaspore which is
destined to proceed with its development divides three times, thus pro-
ducing eight nuclei. Since cell division does not follow these mitoses,
these nuclei are all contained in one large vacuolated cell, the embryo-
sac. Of the eight nuclei, only one is a functional gamete nucleus (ovum) ;
three of the remainder become the antipodal nuclei, two the synergidae,
and the remaining two come together in the middle of the cell and fuse
to form the central fusion nucleus, which is therefore diploid.

VII MEIOTIC PHASE IN PLANTS 213

At fertilization the two male gamete nuclei which are formed in each
pollen grain are both introduced into the embryo-sac ; one fuses with
the ovum nucleus to produce the zygote, while the other fuses with the
central fusion nucleus, forming thus a ¢viploid nucleus. This afterwards
gives rise to the endosperm, or reserve food material of the seed.

For a more detailed account of the gametophyte and fertilization
in the flowering plants the reader is referred to any comprehensive
work on botany, such as that of Bower (1919).

Pollen Mother Cell Embryo-sac Mother Cell

Peiotife Division

Metotife Division

Microspores Megaspores
(Pollen-grains) ad

Degenerate

VegetativeNucleus 00

sy

=

S

fe H :

< Sh ie a a

5S g s _S 5

§ So) ote Ss Ns

se S, Ss Ss
cy § 8.8 &
Sp as 8 ®
YQ’ Ot x =
FIG. go.

Diagrams of the cell generations involved in the haploid phase of the flowering plants.

Mosses and liverworts present the reverse case to the flowering
plants, for the dominant phase (the ordinary moss plant, etc.) is the
haploid gametophyte. The zygote grows into a comparatively simple
sporophyte which is retained on and nourished by the gametophyte,
and produces spores with reduction of chromosomes. These are set free
to produce the new generation of gametophytes.

The relations of the haploid and diploid phases of the life cycle in
animals and plants is summarized in Fig. gt; the figures A-F form a
progressive series in the rise of the diploid and reduction of the haploid

214 CYTOLOGY

generations. Although the examples mentioned as illustrating the

a °

2, : I
D

Certain of the
Phaeophyceae
(Dictyota)
and ( Mevosis

B ryoph yta Sy peat
Meios Ree
B
A
Aggregata
Diplocystis
Certain of the Syngamy
Rhodophyceae. / / ;
(Nemalicnes) Mevosis
A
Lastrea
Pseudo-mas
varcristala
A

Fic. 91.

Pteridophyta

4
Meiosis
As} yngamy
Phanerogams
E
A
Meiosis
Syngamy

Metazoa and
certawn of the

Rhodophyceae.
(Fucus)

F

Parthenogenests
and budding in
Animals and Plants

Ff!

Diagrams illustrating the relations of the haploid and diploid phases in various organisms.
In each case the single line represents the haploid, and the double line the diploid, condition.

different stages manifestly do not in most cases form a phylogenetic
series, yet in the main it is probable that the condition shown in Fig.

CHAP.

vu HAPLOID AND DIPLOID PHASES 215

gt, A, is phylogenetically the oldest, from which the other conditions have
been derived, While all such discussions are of course highly speculative,
it is certainly probable that the earliest organisms, before sexual repro-
duction was evolved, were haploid. It is scarcely possible to avoid the
conclusion that the condition in a diploid organism with its duplex. set
of all hereditary factors is a secondary one and the direct consequence
of the introduction of syngamy, while the function of meiosis is to bring
back the organism to its original haploid condition.

At present we cannot say for certain whether any organisms exist
in which sexual reproduction has not yet been evolved, and which
therefore have no diploid stage ; obviously it would be a difficult matter
to identify such organisms, since in the absence of an alternation of
diploid and haploid generations there would be no certain criterion for
deciding whether the number of chromosomes (in the unlikely case that
this could be determined in such a primitive organism) were the haploid
or diploid number. Moreover, it would be necessary to prove a negative
—namely, the non-occurrence of occasional syngamy and diploid stages.

Nevertheless it is possible to cite at least one case of a life cycle which
has secondarily become completely haploid—namely, the fern Lastraea
_ pseudo-mas, var. cristata. In this fern the sporophyte and the game-
tophyte have the same number of chromosomes, and this (as can be
determined by comparison with its near allies) is the haploid number.
One generation passes into the other without syngamy in the one case
or meiosis in the other. This is a feature which has obviously been
acquired but recently from an evolutionary point of view, and therefore
it is shown as outside of the series in Fig. gt. The converse case, where
organisms have entirely eliminated the haploid phase from their life
histories, can be illustrated by many cases of parthenogenesis and asexual
reproduction in animals and plants.

It is interesting to note that the haploid and diploid conditions in
plants are not necessarily associated with a particular type of structure.
For instance, in ferns, the above-mentioned Lastraea pseudo-mas, var.
cristata, has a haploid sporophyte of the usual type of structure, though
in ferns generally the sporophyte is diploid and the haploid condition
is associated only with the prothalloid type of structure. The converse
is the case with another fern, Athyrium felix-foemina, in which the
prothallus is diploid as well as the sporophyte. Analogous cases
in animals are the haploid individuals developing in certain cases of
artificial parthenogenesis and the males of many Hymenoptera.

For a general discussion of the problems of alternation of generations
in plants, the reader is referred to Bower (1919).

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INDEX

Numbers in thick type refer to pages on which figures will be found

Achromatic figure, 21

Achromatin, 18

Acrosome, 70

Amitosis, in Metazoa, 25
in Protozoa, 203, 210

Amphiaster, 22

Amphinucleolus, 5, 63

Anaphase, II

Antipodal nuclei, 213

Archoplasm, 21

Aster, 21

Attraction sphere, 22

Bacteria, 201

Basichromatin, 18

Binuclearity in Protista, 202
Bivalent chromosomes, 34
Bouquet stage, 4, 33
Bud-variation and segregation, 172

Cell, 1
Cell membrane, 3
Central fusion nucleus, 213
Central spindle, 21
Centriole, 21
Centrosome, 4, 21
in artificial parthenogenesis, 95
in syngamy, 74
Centrosphere, 21
Chiasmatypie, 176
Chondrioconts, 195
Chondriomites, 195
Chondriosomes, 4, 195
in heredity and morphogenesis, 196, 198,
199
in spermatogenesis, 68
relation to chromidia, 200
Chromatin, 18
Chromidia, generative, 190
in Metazoa, 190, 192
in Protozoa, 207, 208
relation to chondriosomes, 200
vegetative, I9I
Chromidiogamy, 208
Chromioles, 18
Chromomeres, 10, 135, 137, 175

| Chromosomes, 6, 123

}

accessory, 98
composition of, 135, 136, 137, 138
conjugation. See Syndesis
continuity, 12, 60, 61, 124, 128, 129
division, 13
elimination, 158
functional differentiation, 162
heterotropic, 98
homologous, 13, 35, 125, 126, 138
of hybrids. See Hybrids
individuality. See Continuity
and Mendel’s law, 168
number, 12
variation due to fragmentation, 142
variation due to fusion, 151
variation due to irregular mitosis, 145
variation due to linkage, 139, 141
variation due to multiplication, 147
relation to resting nucleus, 14, 15, 16
sex, 98
transverse constriction, 40, 43, 136
vesicles, 130
Coupling of hereditary factors, 177
Crossing over, 176
Cytaster, 95
Cytoplasm, 1

Daughter plate, 11
Diakinesis, 10
Diminution of chromatin, 80
Diploid condition, 28
in A ggregata, 205
in plants, 214
Diplotene stage, 34
Dispermie eggs, development, 163, 165, 166
Dyad, 49

Echinoderm hybrids, 158
Embryo-sac mother cell, 213
Endosperm, 213

Equational division, 50
Equatorial plate, ro

Fertilization. See Syngamy

222

Gametogenesis, scheme of, 30
Gametophyte, 212

Gene, 169

Germinal vesicle, 55, 65

Germ track, 80, 82, 83, 85

Gonad differentiated from soma, 81
Gonomery, 78, 79

Growth period in gametogenesis, 31

Haploid condition, 28
in Aggregata, 205
in plants, 214
Hereditary factors, 168, 174
Heterochromosomes, 98
Heterotype division, 29
Homotype division, 2
Hybrids, Ascaris, 133
Drosera, 183
Echinoderms, 158
Echinus x Mytilus, 161
Lepidoptera, 184, 186
Memdia x Fundulus, 132
Mule, 186
Oenothera lata x O. gigas, 146, 184
Pheasant and Fowl, 186
Pigeon and Dove, 186
sterility of, 183

Idiochromatin, 155, 193
Idioplasm, 154

in relation to chromatin, 155
Idiosome, '70

Karyogamy, 72
Karyokinesis, 5
Karyolymph, 19
Karyomere, 131
Karyosome, 20

of Protista, 201
Kinetonucleus, 193, 202

Leptotene stage, 33
Linin, 18
Linkage of factors, 178

Macrogamete, 28
Macronucleus, 194, 202
Mantle fibres, 21
Megaspore, 213
Meiosis, 28, 35

in Aggregata, 206

in Ascaris, 51

in Copepods, 49

in the female, 56, 58, 72

in insects, 42

in Lepidosiren, 38

in plants, 212

in Tomopteris, 32

with tetrad formation, 49
Membrane, cell, 3

nuclear, 19
Mendel’s law, 168
Metaphase, 11
Metaplastic bodies, 4
Microgamete (see also Spermatozoon), 28

INDEX 223

Micronucleus, 194, 202
Microspore, 213
Mitochondria, 195
Mitosis, 6, 7, 8, 162
abnormal, 145, 163, 165
in Protista, 204, 205, 206, 207, 208, 211
Monad, 49
Monaster, 22
Multipolar mitosis, 163
Mutation, 180

Nebenkern, 69, 200
Nuclear membrane, 19
Nucleolus, 5

in oogenesis, 62
Nucleus, 4

in heredity, 154

in morphogenesis, 187

of Protista, 201

Octad chromosomes, 52
Oenothera gigas, 149

Oenothera lata, 146

Oocyte, 31

Oogonium, 29

Organ-forming substances, 188
Ovogonium, 29
Oxychromatin, 18

Pachytene stage, 33

Paraplastic bodies, 195

Parasynapsis, 43

Parasyndesis, 36, 43, 47

Parthenogenesis, 86, 94
artificial, 87, 94, 96
facultative, 87, 91, 93
Mendelian segregation in, 171
obligatory, 87, 88, go

Plasmosome, 5, 9

Plastin, 5

Plastochondria, 195

Plastocont, 195

Plastosome, 195

Polar body, 23, 29, 57, 72, '74

Pollen mother-cell, 213

Polyenergid nuclei, 209

Polymorphic nuclei, 26

Polyploid nuclei, 148, 209

Polyspermy, 77, 163

Post-reduction, 54

Pre-reduction, 54

Primordial germ-cell, 29

Primula kewensis, 151%

Primula sinensis, tetraploid variety, 170

Prochromosomes, 21

Pronucleus, 72

Prophase, 8, 14

Protoplasm, 1

Reduction division, 34
Repulsion of hereditary factors, 177

Segregation, Mendelian, and chromosomes,
168
in bud variation, 172
in parthenogenesis, 171

224 CYTOLOGY

Sex chromosomes in Aphis, 116
in Ascaris nigrovenosus, 114
in Birds, 111
in Echinoderms, 111
in hermaphroditism, 113

in Insects, 99, IOI, 103, 104, 105, 106, 107

in Lepidoptera, 112

in Mammals, r10

in Nematoda, 109

in parthenogenesis, 115

in Phylloxera, 118

in syndesis, 106

outside the meiotic phase, 106

relation to determination of sex, 121
Sex, determination of, 121
Sex-limited inheritance, 179
Sex-linked inheritance, 179
Soma, differentiated from gonad, 81
Spermatid, 30
Spermatocyte, 29
Spermatogonium, 29
Spermatozoon, 67, 70

development of, 68, '70
Spindle, 21
Spireme, 9
Sporophyte, 212
Strepsitene stage, 34
Synapsis, 38 ~

Syncytium, 3

Syndesis (see also Parasyndesis), 33, 48
function of, 175

Synergidae, 213

Syngamy, 72, 74, 75

Synizesis, 39, 54

Telophase, II, 14

Telosynapsis, 43

Telosyndesis, 43, 45

Tetrad, 49

Tetraploid nuclei, 148
plant, genetics of, 170

Tetraster, 163

Triaster, 164

Triploid nuclei, 150, 213

Trophochromatin, 155, 193

Trophonucleus, 202

Univalent chromosomes, 34 ‘e
X chromosome, 101

Y chromosome, IOI

Yolk, 63

Yolk nucleus, 64, 200

Zygotene stage, 33

THE END

966.

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